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      《中国组织工程研究》
     杂志社
     ISSN 2095-4344  
     CN 21-1581/R
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中国组织工程研究  
 
 
2019 Vol.23 No.25
Published:2019-09-08

骨髓干细胞 bone marrow stem cells
脐带脐血干细胞 umbilical cord blood stem cells
干细胞培养与分化 stem cell culture and differentiation
干细胞移植 stem cell transplantation
干细胞基础实验 basic experiments of stem cells
干细胞综述 stem cell review
      骨髓干细胞 bone marrow stem cells
Galectin-1 secreted by bone marrow mesenchymal stem cells may suppress airway inflammation in an asthmatic mouse
Ge Xiahui, Zhang Guorui, Guan Wenbin, Bai Chong. Galectin-1 secreted by bone marrow mesenchymal stem cells may suppress airway inflammation in an asthmatic mouse[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3937-3943.

BACKGROUND: Our previous studies have demonstrated that bone marrow mesenchymal stem cells can ameliorate airway inflammation and regulate levels of inflammatory factors. Moreover, galectin-1 secreted by bone marrow mesenchymal stem cells can inhibit the immune function of dendritic cells in vitro. However, the effect of galectin-1 secreted by bone marrow mesenchymal stem cells on asthma is unknown. 
OBJECTIVE: To investigate the effect of galectin-1 secreted by bone marrow mesenchymal stem cells on airway inflammation of asthmatic mice. 
METHODS: A recombinant lentiviral vector, pLVX-gal-1-shRNA, was constructed for RNA interference of galectin-1 gene and then was transferred into bone marrow mesenchymal stem cells. Forty female BALB/c mice were equally randomized into normal control group, asthmatic group, bone marrow mesenchymal stem cells treatment group (cell treatment group), galectin-1 treatment group and galectin-1 interference group. The number of total inflammatory cells and differential cells in the mouse bronchoalveolar lavage fluid was determined. Furthermore, hematoxylin-eosin staining was used to compare airway inflammation among five groups.
RESULTS AND CONCLUSION: Four short-hairpin RNA sequences targeting mouse galectin-1 mRNA were designed. A real time-quantitative polymerase chain reaction demonstrated that bone marrow mesenchymal stem cells were inhibited most by interference site 249 RNA sequences which were later used in vivo study. Accumulation of inflammation cells, particularly eosinophils, around the airway and in the bronchoalveolar lavage fluid was evidence in asthmatic mice compared to the normal control group. However, bone marrow mesenchymal stem cells engraftment or protein of galectin-1 infusion significantly reduced inflammatory infiltration both in the airway and in the bronchoalveolar lavage fluid. Moreover, eosinophils in the bronchoalveolar lavage fluid and in the airway attenuated dramatically in the cell treatment group and galectin-1 treatment group. However, there was no effect on inflammation accumulation in the bronchoalveolar lavage fluid and airway by infusion of galectin-1-transfected bone marrow mesenchymal stem cells to asthmatic mice. These results indicate that galectin-1 secreted by bone marrow mesenchymal stem cells could alleviate airway inflammation in the asthmatic mouse.

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Human glioma-initiating cells stably transfected with red fluorescent protein gene can spontaneously fuse with host-derived bone marrow mesenchymal stem cells and induce malignant transformation
Han Hui, Dai Xingliang, Cheng Hongwei, Lan Qing. Human glioma-initiating cells stably transfected with red fluorescent protein gene can spontaneously fuse with host-derived bone marrow mesenchymal stem cells and induce malignant transformation[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3944-3950.
BACKGROUND: Malignant transformation of host-derived stromal cells in xenograft tumors has been reported, but little is known about the mechanisms of malignant transformation.
OBJECTIVE: To investigate the malignant transformation of host-derived tumor stromal cells and to explore the possible mechanisms in an in vivo model of two-color fluorescent tracing.
METHODS: Human glioma-initiating cells stably transfected with red fluorescent protein gene (GICs-RFP) were inoculated into nude mice expressing enhanced green fluorescent protein (EGFP). An in vivo model of two-color fluorescence tracer was established. The fluorescent expression of transplanted tumors was observed by confocal laser scanning. Flow cytometry was used to detect and classify various fluorescent cells, including EGFP cells, RFP cells and EGFP/RFP fusion cells. The fusion cells co-expressed EGFP/RFP and had malignant proliferation characteristics because of in vitro subcloning. Origin, surface markers and malignant features of the cells were identified in vitro. The tumorigenicity of fusion cells was verified by subcutaneous transplantation in nude mice. The study was approved by the Animal Ethics Committee of Soochow University (approval No. ECSU-201800090).
RESULTS AND CONCLUSION: The tumorigenic rates of GICs-RFP in EGFP nude mice were 100% (14/14). Fusion cells were observed in the transplanted tumors of all the animal models. The fusion cells not only co-expressed EGFP and RFP, but also co-expressed GICs-RFP marker Nestin and bone marrow mesenchymal stem cell markers CD44, CD105 and CD29. Fusion cells showed high proliferative activity and more invasive and migratory characteristics in vitro as compared with GICs-RFP. The tumorigenicity of fusion cells (1×105 cells per mouse) in thymus-free nude mice was 100% (4/4). These results indicate that GICs-RFP spontaneously fuses with host-derived bone marrow mesenchymal stem cells and induces malignant transformation, which provides new evidence for two-color fluorescence tracing of heterogeneous tumor cell sources.


中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程
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Co-culture model of bone marrow mesenchymal stem cell sheet and umbilical vein endothelial cells: establishment and identification
Zhou Zhen, Wang Yamin, Ren Fei, Zhang Zhaoqiang, Zeng Shuguang, Yang Xi.
Co-culture model of bone marrow mesenchymal stem cell sheet and umbilical vein endothelial cells: establishment and identification
[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3951-3955.
BACKGROUND: Cell sheet technology is a recent popular technique for tissue engineering. However, a failure is prone to occur because of poor nutrient supply in the middle part of the membrane and post-transplantation blood supply, so pre-vascularization determines the survival rate of tissue engineering materials after transplantation.
OBJECTIVE: To investigate the pre-vascularization of bone marrow mesenchymal stem cell sheets co-cultured with human umbilical vein endothelial cells under different oxygen tensions.
METHODS: Bone marrow mesenchymal stem cell sheets were prepared under physiological hypoxia (2% O2) and normoxia (20% O2), and then co-cultured with human umbilical vein endothelial cells under normoxia. At 7 days of co-culture the angiogenesis was observed and compared by immunofluorescence staining. The level of vascular endothelial growth factor was compared under different oxygen tensions using ELISA.
RESULTS AND CONCLUSION: Compared with the normoxia co-culture group, there were longer microvessels, higher vessel density and more microvessel networks in the hypoxia co-culture group (P < 0.05). The expression of vascular endothelial growth factor in the hypoxia co-culture group was higher than that in the normoxia co-culture group (P < 0.05). Therefore, the hypoxia co-culture is more beneficial to the growth of microvessels than the normoxia co-culture, and it is feasible to construct an optimized prevascularized cell sheets.
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Co-culture model of bone marrow mesenchymal stem cell sheet and umbilical vein endothelial cells: establishment and identification[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3951-3955.')" href="#">引用本文(Cite Me)
Lithium chloride promotes proliferation and differentiation of bone marrow mesenchymal stem cells via Wnt/beta-catenin signaling pathway
Fu Yin, Sun Guicai, Tu Yuanqing, Ling Xiaopeng.
Lithium chloride promotes proliferation and differentiation of bone marrow mesenchymal stem cells via Wnt/beta-catenin signaling pathway
[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3956-3960.

BACKGROUND: Seeking proper methods to promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells can provide therapeutic ideas for the treatment of osteoporosis.
OBJECTIVE: To study the mechanism of lithium chloride on the proliferation and differentiation of rat bone marrow mesenchymal stem cells.
METHODS: Rat bone marrow mesenchymal stem cells were cultured with the whole bone marrow cell inoculation and adherent purification, followed by intervention with 0, 2, 5 nmol/L lithium chloride. Cell counting kit-8 was used to detect the effect of lithium chloride on the proliferation of bone marrow mesenchymal stem cells. Alizarin red staining was used to determine calcified nodules. PNPP method was applied to measure alkaline phosphatase activity. The contents of glycogen synthase kinase 3β, phosphorylated glycogen synthase kinase 3β and β-catenin in rat bone marrow mesenchymal stem cells were determined by western blot.
RESULTS AND CONCLUSION: Lithium chloride, 2 and 5 nmol/L, promoted the proliferation of bone marrow mesenchymal stem cells and
5 nmol/L lithium chloride had the strongest proliferative effect. Compared with 0 nmol/L group, more calcified nodules and higher alkaline phosphatase activity were observed in the 2 and 5 nmol/L groups, especially in the 5 nmol/L group. Both 2 and 5 nmol/L lithium chloride upregulated the expression of phosphorylated glycogen synthase kinase 3β and β-catenin proteins, but showed no difference in the expression of glycogen synthase kinase 3β. In conclusion, lithium chloride can promote the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells, and the possible mechanism is through the regulation of Wnt/β-catenin signaling pathway-related protein expression.

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Lithium chloride promotes proliferation and differentiation of bone marrow mesenchymal stem cells via Wnt/beta-catenin signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3956-3960.')" href="#">引用本文(Cite Me)

Simvastatin promotes osteogenic differentiation of bone marrow mesenchymal stem cells

Xing Lei, Gao Jingyuan, Wang Xiaoxu, Chen Yujie, Chi Bojing, Tian Faming.

Simvastatin promotes osteogenic differentiation of bone marrow mesenchymal stem cells [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3961-.

BACKGROUND: In vitro studies have shown that simvastatin can stimulate osteogenic differentiation of bone marrow mesenchymal stem cells, but the mechanism is unclear. Recent studies have shown that the Hedgehog signaling pathway is crucial for osteogenic differentiation of bone marrow mesenchymal stem cells.
OBJECTIVE: In combination with Hedgehog pathway blocker (cyclopamine), to observe the effect of simvastatin on osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro.
METHODS: Eight 4-week-old Sprague-Dawley female rats, SPF grade, were used in this study. The rats were killed by cervical dislocation and removed of bilateral femur and tibia under sterilization conditions. The second generation of bone marrow mesenchymal stem cells was randomly divided into four groups: control group cultured with osteogenic induction medium; simvastatin group cultured in the induction medium containing 10-7 mol/L simvastatin; simvastatin+cyclopamine group (combination group) cultured in complete culture medium containing 5 μmol/L cyclopamine for 2 hours and then cultured in the induction medium containing 10-7 mol/L simvastatin; cyclopamine group cultured in the osteogenic induction medium containing 5 μmol/L cyclopamine. After 7 days of culture, alkaline phosphatase staining was used. The levels of type I collagen and osteocalcin were evaluated by immunofluorescence and western blot assay. Alizarin red S staining was performed at 28 days of culture.
RESULTS AND CONCLUSION: Compared with the control group, the simvastatin group had more alkaline phosphatase positive cells and calcium nodules, and higher type I collagen and osteocalcin fluorescence intensities (P < 0.05). All the measurement indexes except for osteocalcin fluorescence intensity and protein expression were significantly lower in the combination group than the simvastatin group (P < 0.05). All the measurement indexes in the cyclopamine group were significantly lower than those in the control group (P < 0.05). To conclude, simvastatin can promote osteogenic differentiation of bone marrow mesenchymal stem cells via Hedgehog signaling pathway that cannot be completely blocked by cyclopamine.
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Simvastatin promotes osteogenic differentiation of bone marrow mesenchymal stem cells [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3961-.')" href="#">引用本文(Cite Me)
Bone marrow mesenchymal stem cells can effectively treat radiation-induced salivary gland injury by regulating Notch expression
Luo-Meng Yanan, Zhao Na, Ji Nannan, Wang Wufeng.
Bone marrow mesenchymal stem cells can effectively treat radiation-induced salivary gland injury by regulating Notch expression
[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3967-3972.

BACKGROUND: Cholinergic receptor agonists can promote the secretion of residual salivary cells in patients with radiation-induced salivary gland injury. However, there are serious adverse reactions associated with its long-term use and limited therapeutic effects on severely radioactive salivary gland damage. Bone marrow mesenchymal stem cells are a kind of cells with multipotential differentiation potential and almost unlimited proliferative capacity, which have the potential to treat radiation-induced salivary gland injury.
OBJECTIVE: To explore the mechanism of bone marrow mesenchymal stem cells repairing radiation-induced salivary gland injury.
METHODS: Bone marrow mesenchymal stem cells were extracted from mouse bone marrow, and co-cultured with the second-generation salivary gland acinar cells for 24 hours by a 3D co-culture system. One hundred and fifty C57 mice were randomly divided into normal control group, radiation-induced salivary gland injury group and bone marrow mesenchymal stem cells+radiation-induced salivary gland injury group (stem cell treatment group). A mouse model of radiation-induced salivary gland injury was made by an electron linear accelerator (15 Gy). One week after irradiation, the mice in the stem cell treatment group were subcutaneously injected with 2×109/L bone marrow mesenchymal stem cell suspension at multiple points of the salivary gland, and those in the other two groups were injected with the same amount of saline.
RESULTS AND CONCLUSION: After 24 hours of co-culture, bone marrow mesenchymal stem cells differentiated into salivary gland acinar cells, with a polygon-like shape and expressed α-amylase. Compared with the normal control group, the salivary flow, salivary gland mass, and salivary amylase level were significantly decreased, acinar cell structure was markedly damaged, and the Notch expression level in the salivary gland was decreased in the radiation-induced salivary gland injury group. Compared with the radiation-induced salivary gland injury group, the above indexes in the bone marrow mesenchymal stem cells+radiation-induced salivary gland injury group were significantly restored. These findings indicate that bone marrow mesenchymal stem cell transplantation can effectively treat radiation-induced salivary gland injury, and its effect may be achieved by regulating the Notch expression level in the salivary gland.

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Bone marrow mesenchymal stem cells can effectively treat radiation-induced salivary gland injury by regulating Notch expression[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3967-3972.')" href="#">引用本文(Cite Me)
Lentiviral vector-mediated over-expression of CC chemokine receptor 7 promotes homing of rat bone marrow mesenchymal stem cells
Wang Zhihong, Chen Weimin, Lin Yun, Shang Jin, Wei Tiannan.
Lentiviral vector-mediated over-expression of CC chemokine receptor 7 promotes homing of rat bone marrow mesenchymal stem cells
[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3973-3977.
BACKGROUND: Bone marrow mesenchymal stem cells specifically express CC chemokine receptor 7 (CCR7). The interaction between secondary lymphoid-tissue chemokine and CCR7 contributes to the homing of bone marrow mesenchymal stem cells to the injured site.
OBJECTIVE: To explore the effects of CCR7 gene over-expression on the homing capacity of bone marrow mesenchymal stem cells in vivo.
METHODS: Rat bone marrow mesenchymal stem cells overexpressing CCR7 were constructed by lentiviral vector. We conducted a Transwell experiment to detect the effect of secondary lymphoid-tissue chemokine on the directional migration of bone marrow mesenchymal stem cells in vitro. Sprague-Dawley rats were divided into four groups (n=10/group): (1) Control group: non-irradiated rats were used as control group; (2) radiation group in which the rats were exposed to total body irradiation (60Co, 0.75 Gy/min, 7.5 Gy in total), and then were infused with 1 mL of normal saline; (3) BMSCs-GFP group in which the rats were infused with bone marrow mesenchymal stem cell suspension (1 mL, 5×105 cells) transfected by GFP via the tail vein after total body irradiation; (4) CCR7-BMSCs-GFP group in which the rats were infused with bone marrow mesenchymal stem cells (1 mL, 5×105 cells) simultaneously carrying GFP and CCR7 gene via the tail vein after total body irradiation. The rats were sacrificed at 24 hours after infusion, and the frozen sections of the spleen were prepared to detect the distribution of infused cells. Furthermore, the homing rate of bone marrow mesenchymal stem cells to the rat spleen was detected by flow cytometry. The level of secondary lymphoid-tissue chemokine was detected by ELISA.
RESULTS AND CONCLUSION: (1) Results of the Transwell experiment showed that CCR7 over-expression gene promoted the directional migration of bone marrow mesenchymal stem cells to secondary lymphoid-tissue chemokine. (2) The CCR7 over-expression could significantly enhance the homing rate of bone marrow mesenchymal stem cells into the spleen. (3) Flow cytometry results slowed that the number of bone marrow mesenchymal stem cells overexpressing CCR7 homing into the spleen was significantly higher than that in the BMSCs-GFP group (P < 0.05). (4) ELISA results showed that the level of secondary lymphoid-tissue chemokine in the peripheral blood after 24 hours of irradiation significantly increased (P < 0.05). These findings indicate that the over-expression of CCR7 gene mediated by lentiviral vector can promote the homing of bone marrow mesenchymal stem cells into the spleen.
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Lentiviral vector-mediated over-expression of CC chemokine receptor 7 promotes homing of rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3973-3977.')" href="#">引用本文(Cite Me)
Chrysin inhibits mouse osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand
Li Xianghe, Luo Wei, Hu Junxian, Yang Jing, Han Xinyun, Dong Shiwu, Yang Xianteng, Li Senlei, Yan Zhihui, Nie Yingjie, Tian Xiaobin, Sun Li.
Chrysin inhibits mouse osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand
[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3978-3986.
BACKGROUND: Chrysin (5,7-dihydroxyflavone) is a flavonol in many natural plant extracts. It has a wide range of therapeutic effects and is involved in inflammatory reactions in the body that can enhance osteoclast formation and lead to bone erosion.
OBJECTIVE: To investigate the effects of chrysin on osteoclast differentiation and its protective effect on bone erosion in inflammatory and non-inflammatory environments.
METHODS: RAW264.7 cells were selected as seed cells. First, the RAW264.7 cells were induced with receptor activator of nuclear factor kappa B ligand (50 μg/L) and macrophage colony-stimulating factor (25 μg/L) to generate osteoclasts. The cells were randomly divided into four groups according to chrysin concentration (0, 20, 40, and 60 μg/L). Second, lipopolysaccharide was used to simulate the inflammatory environment. RAW264.7 cells were induced by lipopolysaccharide to differentiate into osteoclasts, and the effect of different concentrations of chrysin (0, 20, 40, 60 μg/L) on osteoclast differentiation was observed in the same way.
RESULTS AND CONCLUSION: Chrysin effectively inhibited osteoclast differentiation, with the maximum effect at 60 μg/L. Chrysin significantly inhibited the bone absorption function of osteoclasts, suggesting that chrysin has a protective effect on bone erosion caused by osteoclasts. Chrysin suppressed the protein and gene expression related to osteoclast differentiation by nuclear factor kappa B signaling pathway. Therefore, chrysin has an anti-inflammatory effect and it is also powerful to inhibit osteoclast differentiation in an inflammatory environment.
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Chrysin inhibits mouse osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3978-3986.')" href="#">引用本文(Cite Me)
      脐带脐血干细胞 umbilical cord blood stem cells
Effect of human umbilical cord blood mesenchymal stem cells on the transformation of M2 macrophages from mouse bone marrow
Chen Bingquan, Peng Yi, Xiao Yi, Peng Zhiyong, Zhao Jiling, Yu Guolong. Effect of human umbilical cord blood mesenchymal stem cells on the transformation of M2 macrophages from mouse bone marrow[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3987-3992.
BACKGROUND: Mesenchymal stem cells have been used in the treatment of a variety of diseases. They may produce biological effects by promoting the conversion of macrophages to M2 subtypes, but it is unclear whether these effects are dose- and time-sensitive, as well as whether intercellular direct contact is involved in the cell transformation.
OBJECTIVE: To verify the effect of umbilical cord blood mesenchymal stem cells (hUC-MSCs) on the transformation of M2 subtypes of macrophages, and to investigate the dose- and time-dependent effects of hUC-MSCs on the transformation of M2 subtypes of macrophages. 
METHODS: Monocytes were extracted from mouse bone marrow and induced by macrophage colony-stimulating factor to produce M0 macrophages (F4/80+, CD11B+). M0 macrophages were implanted into Transwen plates or 6-well culture plates at a density of 1×106/well, and co-cultured directly or indirectly with hUC-MSCs at different concentrations (0, 2×105, 4×105) in complete medium containing lipopolysaccharide (100 μg/L) and interferon-γ (20 μg/L) for 24 and 48 hours. The morphological changes of macrophages were observed under direct microscope. The number of M2 macrophages (CD206+, CD11C-) was detected by flow cytometry. The levels of interleukin-10, interleukin-6 and interleukin-1β in the cell culture supernatant were measured by ELISA. 
RESULTS AND CONCLUSION: (1) After co-cultured with hUC-MSCs, the macrophages were elongated in cell antennae and the funicular cells, like M2 macrophages, appeared. (2) After 24 and 48 hours of direct or indirect co-culture with hUC-MSCs, the percentage of M2 macrophages was significantly increased (P < 0.05). (3) The percentage of M2 macrophages insignificantly differed after direct or indirect co-culture with low- and high-concentration hUC-MSCs. (4) The percentage of M2 macrophages was significantly higher after 48 hours co-culture than after 24 hours co-culture (P < 0.05). (5) After direct or indirect co-culture with hUC-MSCs, the levels of interleukin 6 and interleukin-1β in the cell supernatant were decreased significantly, while the interleukin-10 level increased significantly (P < 0.05). (6) The interleukin-10 level was significantly increased after 48 hours co-culture relative to that after 24 hours co-culture. In conclusion, hUC-MSCs can promote the transformation of M2 macrophages in a time-dependent but not concentration-dependent manner.
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Exosomes derived from human umbilical cord mesenchymal stem cells attenuate edema of spinal astrocytes after oxygen-glucose deprivation/reoxygenation injury in rats
Zhang Yong, Ma Xun, Sun Lin, Zhang Li, Guan Xiaoming, Lü Cong, Chen Xu. Exosomes derived from human umbilical cord mesenchymal stem cells attenuate edema of spinal astrocytes after oxygen-glucose deprivation/reoxygenation injury in rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4011-4017.
BACKGROUND: Exosomes can regulate and repair nerve cells, thus promoting the proliferation of nerve cells to alleviate spinal cord injury. Exosomes have been less reported to alleviate edema after spinal cord injury.
OBJECTIVE: To study the effect and mechanism by which human umbilical cord mesenchymal stem cell-derived exosomes (hucMSCs-exo) alleviate the edema of spinal astrocytes induced by oxygen-glucose deprivation/reoxygenation.
METHODS: Ultrahigh speed centrifugation method was used to isolate and extract hucMSCs-exo. Spinal astrocytes were extracted from newborn Sprague-Dawley rats by trypsin digestion. Part One: The cells were (1) cultured normally in normal control group, (2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group, or (3) cultured in medium containing 30, 60, and 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group. Western blot assay was performed to detect the expression of AQP4. Living cell workstation was used to detect cell volume. Transmission electron microscope was used to observe the ultrastructure of intracellular edema. Part Two: The cells were (1) cultured normally in normal control group, (2) subjected to 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in model group, (3) cultured in medium containing 90 μg of hucMSCs-exo for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in hucMSCs-exo group, or (4) cultured in medium containing 5 μmol/L JNK inhibitor sp600125 for 24 hours following 6 hours of oxygen-glucose deprivation and 24 hours of reoxygenation in JNK inhibitor sp600125 group. AQP4 expression and p-JNK protein expression were measured by western blot assay. Living cell workstation was used to detect cell volume
RESULTS AND CONCLUSION: (1) After 24-hour hucMSCs-exo intervention, mitochondrial and endoplasmic reticular edemas were alleviated, the number of lysosomes decreased, the volume of intracellular cell edema was significantly reduced, and the expression of AQP4 in the cells was significantly lowered as compared with the model group (P < 0. 05). (2) Intervention with hucMSCs-exo and JNK inhibitor SP600125 significantly reduced the expression of AQP4 and p-JNK (P < 0.05) and the volume of intracellular edema (P < 0.05) as compared with the model group. Therefore, hucMSCs-exo can reduce the expression of AQP4 protein in spinal astrocytes after oxygen-glucose deprivation/reoxygenation by inhibiting JNK signaling pathway, thereby alleviating cell edema.
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Umbilical cord mesenchymal stem cells support the in vitro growth of primary mouse hepatocytes by a paracrine mechanism
Wu Yuanquan, Wu Jianhua, Wang Liang, Lin Yang. Umbilical cord mesenchymal stem cells support the in vitro growth of primary mouse hepatocytes by a paracrine mechanism[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4031-4036.
BACKGROUND: Hepatocyte transplantation is a very effective alternative treatment for patients with advanced liver failure. It is of very important clinical significance to expand and culture hepatocytes in vitro.
OBJECTIVE: To explore the regulatory effect of human umbilical cord mesenchymal stem cells on the function of primary mouse hepatocytes under in vitro conditions. 
METHODS: Umbilical cord mesenchymal stem cells were co-cultured with primary mouse hepatocytes in a non-contacted way (co-culture group). Umbilical cord mesenchymal stem cell supernatant was collected and co-cultured with primary mouse hepatocytes (conditioned medium). Primary mouse hepatocytes were cultured in conventional culture medium containiing low-dose DMEM+10% fetal bovine serum as control group. Cell counting kit-8 method was used to detect the proliferation of three groups of hepatocytes. RT-PCR was used to detect the expression of hepatocyte-related genes. Indocyanine green detection was used to detect the function of hepatocytes. ELISA method was used to detect the secretion of albumin and urea. The implementation of the study was in line with the relevant ethical requirements of Kashgar Region First Hospital (ethical approval No. 01).
RESULTS AND CONCLUSION: Compared with the control group, umbilical cord mesenchymal stem cells could significantly retain the uptake ability of mouse hepatocytes (96 hours of culture) and promote their proliferation (48, 72, and 96 hours of culture) by non-contact co-culture (P < 0.05) or supernatant culture (P < 0.05). Compared with the control group, the primary mouse hepatocytes in the co-culture group and the conditioned medium group had significantly stronger albumin and urea secretion ability (P < 0.05). RT-PCR detection of related genes revealed higher expression of hepatocyte growth factor, basic fibroblast growth factor and album mRNAs in the co-culture group and conditioned medium group than the control group (P < 0.05). However, we observed similar results in the expression of the cytochrome gene Cyp2b9 in the three groups (P > 0.05). Overall, umbilical cord mesenchymal stem cells can preserve the function of primary mouse hepatocytes in vitro by the paracrine mechanism and promote their proliferation. It is a new application strategy for artificially expanding hepatocytes in vitro.
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      干细胞培养与分化 stem cell culture and differentiation
Tumor necrosis factor-alpha inhibits osteogenic differentiation of periodontal ligament stem cells through activating nuclear factor-kappa B signaling pathway
Zhang Yun, Chang Qun’an, Li Shengmei, Zhang Yuan.
Tumor necrosis factor-alpha inhibits osteogenic differentiation of periodontal ligament stem cells through activating nuclear factor-kappa B signaling pathway
[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3993-3997.

BACKGROUND: Previous studies have focused on the effects of inflammatory factors-activated nuclear factor-kappa B signaling pathway on bone cells. Little has been reported on the effects of micro-inflammatory environment on osteogenic differentiation and nuclear factor-kappa B signaling pathways of human periodontal ligament stem cells.
OBJECTIVE: To investigate the effect of tumor necrosis factor-alpha on osteogenic differentiation and nuclear factor-kappa B signaling pathway of periodontal ligament stem cells.
METHODS: Human periodontal ligament stem cells were cultured in vitro by enzymatic digestion combined with tissue explant method, and the cell source was identified by immunohistochemical staining. The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University, and informed consent was obtained from each patient prior to the enrollment in the study. The cells were cultured in simple osteogenic induction medium (control group) or osteogenic induction medium containing 10 μg/L tumor necrosis factor-alpha (tumor necrosis factor-alpha group). Alizarin red staining and alkaline phosphatase activity were used to detect the effect of tumor necrosis factor-alpha on osteogenic differentiation of periodontal ligament stem cells. Real-time quantitative RT-PCR was used to detect the effect of tumor necrosis factor-alpha on the expression of osteogenic differentiation genes, Osterix and Runx2. Western blot was used to detect the effect of tumor necrosis factor-alpha on nuclear factor-kappa B signaling pathway.
RESULTS AND CONCLUSION: Immunohistochemical staining showed that human periodontal ligament stem cells were positive for anti-vimentin and negative for anti-keratin. After alizarin red staining, mineralized nodules formed in the tumor necrosis factor-alpha group were fewer in number, smaller in scope and lighter in staining than those in the control group. Alkaline phosphatase activity in the tumor necrosis factor-alpha group was significantly lower than that in the control group (P < 0.05). Real-time quantitative RT-PCR results showed that the expression levels of Osterix and Runx2 mRNA in tumor necrosis factor-alpha group were significantly lower than those in the control group (P < 0.05). Western blot assay showed that, compared with the control group, the expression of p-IκBα protein increased significantly in the tumor necrosis factor-alpha group (P < 0.05), while the expression of p65 and IκBα proteins decreased (P < 0.05). These findings indicate that tumor necrosis factor-alpha can inhibit the osteogenic differentiation of human periodontal ligament stem cells by activating the nuclear factor-kappa B signaling pathway.

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Tumor necrosis factor-alpha inhibits osteogenic differentiation of periodontal ligament stem cells through activating nuclear factor-kappa B signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3993-3997.')" href="#">引用本文(Cite Me)
Combined use of exogenous nerve growth factor and basic fibroblast growth factor promotes proliferation of endogenous brain cells in a rat model of severe traumatic brain injury
Wang Tong, Liu Yang, Zhu Yetao. Combined use of exogenous nerve growth factor and basic fibroblast growth factor promotes proliferation of endogenous brain cells in a rat model of severe traumatic brain injury[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 3998-4003.
BACKGROUND: Either nerve growth factor or basic fibroblast growth factor can promote the self-renewal and differentiation of neural stem cells in vitro. Little has been reported on the effect of the combination of these two factors on endogenous brain cells after traumatic brain injury in adult rats.
OBJECTIVE: To explore the effects of exogenous nerve growth factor and basic fibroblast growth factor on the proliferation of endogenous brain cells in the rats with severe traumatic brain injury.
METHODS: Animal models of traumatic brain injury were made in 48 adult Sprague-Dawley rats using modified Feeney’s method, and then randomized into 4 groups: nerve growth factor, basic fibroblast growth factor, combination (injection of nerve growth factor and basic fibroblast growth factor), and control (normal saline injection) groups. Intraventricular injection of different factors was performed correspondingly at 24 hours after modeling. The restoration of limb function was assessed through behavior observation. BrdU-positive cells in the brain were counted and compared among four groups using immunohistochemical method.
RESULTS AND CONCLUSION: (1) Initially from the 5th day after modeling, the behavioral test scores in the three treatment groups were significantly lower than those in the control group (P < 0.05), and the behavioral test scores in the combination group were significantly lower than those in the nerve growth factor and basic fibroblast growth factor groups (P < 0.05). (2) There were more BrdU-labeled cells in the three treatment groups than the control group (P < 0.05). The number of BrdU-positive cells in the combination group was significantly higher than that in the nerve growth factor and basic fibroblast growth factor groups (P < 0.05). In summary, the proliferation of brain cells and restoration of limb function after traumatic brain injury can be promoted by nerve growth factor or basic fibroblast growth factor. Morever, this effect can be considerably improved by the combination of nerve growth factor and basic fibroblast growth factor.
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MicroRNA-210 promotes the proliferation and odontogenic differentiation of rat dental pulp stem cells
Wang Yanling, Zuo Chunran, Wang Jing, Yang Kun, Wang Shouru, Zhang Xiaoping. MicroRNA-210 promotes the proliferation and odontogenic differentiation of rat dental pulp stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4004-4010.
BACKGROUND: MicroRNAs profile predicts that microRNAs-210 may play a role in the differentiation of dental pulp stem cells into dentin and osteogenesis, but the specific role remains unclear.
OBJECTIVE: To investigate the role of microRNA-210 in the proliferation and odontogenic differentiation of rat dental pulp stem cells.
METHODS: The study was approved by the Ethics Committee of the Second Affiliated Hospital of Henan University of Chinese Medicine, China. The dental pulp tissues were collected from the mandibles of five Sprague-Dawley rats, and the pulp stem cells were isolated and identified. The dental pulp stem cells were divided into five groups. The normal control group was not treated. The cells in the miR-210 mimic negative control group were transfected with the miR-210 mimic negative control. The cells in the miR-210 mimic group were transfected with the miR-210 mimic. The cells in the miR-210 inhibitor negative control group were transfected with the miR-210 inhibitor negative control. The cells in the miR-210 inhibitor group were transfected with the miR-210 inhibitor negative control. Real-time quantitative PCR was used to detect the expression of microRNA-210, alkaline phosphatase, dentin sialophosphoprotein, osteopontin, osteocalcin, dentin matrix phosphoprotein-1 and GPCR-kinase interacting protein-2 in cells. Western blot was used to detect the expression of dentin sialophosphoprotein, osteopontin, osteocalcin and dentin matrix phosphoprotein-1 in cells. Cell counting kit-8 was used to detect cell proliferation. Cell alkaline phosphatase activity was detected by colorimetry. Alizarin red staining was used to detect the ability of mineral synthesis.
RESULTS AND CONCLUSION: (1) The proliferation activity of miR-210 mimic group was significantly higher than that of the other four groups (P < 0.01), while the proliferation activity of miR-210 inhibitor group was significantly lower than that of the other four groups (P < 0.01). (2) The activity of alkaline phosphatase in the miR-210 mimic group was significantly higher than that in the other four groups (P < 0.01), while the activity of alkaline phosphatase in the miR-210 inhibitor group was significantly lower than that in the other four groups (P < 0.01). (3) The results of alizarin red staining showed that the surfaces of cells in the miR-210 mimic group were covered with dark red nodules of different sizes owing to calcium salt deposition, and the number of dark red calcium nodules was higher than that in the other four groups. In the miR-210 inhibitor group, dark red calcium nodules were scattered on the cell surface, and the number of these nodules was lower than that in the other four groups. (4) The expressions of microRNA-210, alkaline phosphatase, dentin sialophosphoprotein, osteopontin, osteocalcin, dentin matrix phosphoprotein-1 and GPCR-kinase interacting protein-2 mRNAs and dentin sialophosphoprotein, osteopontin, osteocalcin and dentin matrix phosphoprotein-1 proteins were highest in the miR-210 mimic group and lowest in the miR-210 inhibitor group among the five groups, and there were significant differences among the five groups (P < 0.01). To conclude, microRNA-210 can promote the proliferation and odontogenic differentiation of rat dental pulp stem cells.
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Curcumin regulates odontogenic differentiation of human dental pulp stem cells through Wnt signaling pathway
Chen Dongmei, Xu Lili, Zhou Jianwei.
Curcumin regulates odontogenic differentiation of human dental pulp stem cells through Wnt signaling pathway
[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4018-4024.
BACKGROUND: Studies have shown that curcumin not only promotes osteogenic differentiation of adult stem cells, but also promotes proliferation and differentiation of neural stem cells by activating the Wnt signaling pathway. However, there is no report on whether curcumin can promote odontoblast the differentiation of human dental pulp stem cells by activating the Wnt signaling pathway. 
OBJECTIVE: To investigate the effect of curcumin on the proliferation and odontogenic differentiation of human dental pulp stem cells and its possible mechanism. 
METHODS: Human dental pulp stem cells (purchased from Shanghai Yansheng Industrial Co., Ltd., China) were divided into six groups according to different intervention methods. The normal control group was not intervened. Low-, medium- and high-concentration curcumin groups were treated with 50, 250, and 500 nmol/L curcumin, respectively. IWR-1 group was intervened with 1 μmol/L Wnt signaling pathway inhibitor IWR-1. IWR-1+curcumin group were treated with 1 μmol/L IWR-1 and 500 nmol/L curcumin. After 7 and 14 days of mineralization, the mRNA levels of Wnt5a, alkaline phosphatase and dentin sialophosphoprotein were detected by real-time quantitative PCR. The protein levels of Wnt5a, β-catenin, bone sialic acid protein, dentin sialophosphoprotein, osteocalcin and dentin matrix protein 1 were detected by western blot. The activity of alkaline phosphatase was detected by colorimetry, and the proliferation activity of the cells was detected by cell counting kit-8 method. 
RESULTS AND CONCLUSION: (1) Curcumin had a time-dependent and dose-dependent effect on the proliferation of dental pulp stem cells. (2) Curcumin had a time- and dose-dependent effect on the mRNA expressions of dentin sialophosphoprotein and alkaline phosphatase, as well as the protein expressions of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein and osteocalcin in dental pulp stem cells. (3) Curcumin up-regulated the expressions of Wnt5a and β-catenin proteins in dental pulp stem cells in a time- and dose-dependent manner. (4) Compared with the control and IWR-1 groups, the combination of curcumin and IWR-1 significantly increased the expressions of Wnt5a mRNA and protein, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein and osteocalcin proteins in human dental pulp stem cells, and significantly promoted the proliferative ability of the cells (P < 0.05). These results suggest that curcumin promotes the proliferation and odontogenic differentiation of human dental pulp stem cells by activating the Wnt signaling pathway.
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Curcumin regulates odontogenic differentiation of human dental pulp stem cells through Wnt signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4018-4024.')" href="#">引用本文(Cite Me)
Response to Hedgehog signaling in hair follicle epithelial cells is not associated with short-term hair regeneration
Ying Qinxin, Tao Yixin, Yang Qingchun, Ling Xuejian, Ma Gang. Response to Hedgehog signaling in hair follicle epithelial cells is not associated with short-term hair regeneration[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4044-4049.
BACKGROUND: Hedgehog signaling plays a vital role in development, homeostasis and tissue repair of multiple tissues. Previous studies underscore that Hedgehog signaling is essential for hair morphogenesis.
OBJECTIVE: To explore the role of Hedgehog signaling in adult epidermal homeostasis.
METHODS: Lgr5-EGFP-Ires-CreERT2, Smofl/fl and K14-CreER mice were purchased from The Jackson Laboratory. This study was approved by the Laboratory Animal Ethics and Use Committee of Shanghai Jiao Tong University with the approval No. 1602028. First, Lgr5-EGFP-Ires-CreERT2 mice were used to specifically label Lgr5+ cell subsets, confirming several Hedgehog signaling components were enriched in Lgr5+ hair follicle epidermal stem cells/precursor cells. Lgr5-EGFP-Ires-CreERT2 mice then copulated with Smofl/fl mice. Lgr5-EGFP-Ires-CreERT2;Smofl/fl mice were obtained and shaved of their fur at P49 days of the second telogen period, followed by tamoxifen-induced Hedgehog signaling pathway-mediated Smo knockout. We observed the effect of Smo knockout on hair regeneration rate in Lgr5+ cells. In the following experiments, tamoxifen was used to induce Smo knockout on P50-54 days, and the mice were shaved of their fur the mice at 1 week after Smo knockout. We further evaluated the functional changes of bulge epithelial stem cells in mutant mice. (2) K14-CreER mice copulated with Smofl/fl to produce K14-CreER;Smofl/fl mice. Tamoxifen, as described above, was used to delete Smo in hair follicle epithelial basal cells. We observed whether hair loss disorder and hair follicle Bulge stem cell dysfunction occur. 
RESULTS AND CONCLUSION: No hair loss disorder was detected in Lgr5-EGFP-Ires-CreERT2;Smofl/fl and K14-CreER;Smofl/fl mouse models, and hair follicle Bulge stem cells were not affected. It is indicated that the responsiveness to Hedgehog signaling in Lgr5+ cells and hair follicle epithelial cells is independent of hair follicle telogen-to-anagen transition and short-term hair regeneration as well as the maintenance of Bulge epithelial stem cells. This finding highlights the different molecular mechanisms of tissue development and homeostasis, and provides an important basis for a more accurate understanding of the mechanisms of hair follicle regeneration and tissue growth.
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      干细胞基础实验 basic experiments of stem cells
Optimization of culture and secretion of parathyroid cells via increasing the parathyroid hormone level in cell supernatant
Wang Hui . Optimization of culture and secretion of parathyroid cells via increasing the parathyroid hormone level in cell supernatant[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4025-4030.
BACKGROUND: Telomerase reverse transcriptase is an important enzyme that regulates cell differentiation and proliferation, and it has various biological activities. hTERT gene modification may improve the viability and proliferation of parathyroid cells, and can optimize the in vitro culture of parathyroid cells.
OBJECTIVE: To transfect rat parathyroid cells with retroviral PLXSN carrying hTERT gene, and to observe the biological characteristics and secretory function of transfected cells in order to optimize the culture of parathyroid cells. 
METHODS: Twenty-eight male Wistar rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., China. The study protocol was approved by the Animal Experiment Ethics Committee of Tianjin Medical University with the approval No. 2017-0652. Under anesthesia, the parathyroid glands of rats were surgically removed and pathologically confirmed. The parathyroid cells were digested by collagenase II. The protein expression level of calcium-sensing receptor, parathyroid hormone and glial cells missing-2 were detected by western blot. The parathyroid cells were divided into parathyroid cell group, empty virus group and hTERT transfection group. The expression levels of hTERT gene and protein in parathyroid cells were detected by RT-PCR and western blot after 3 days of transfection, respectively. Cell growth was observed by cell growth curve and cell counting kit-8. The cell cycle distribution was measured by flow cytometry. Enzyme-linked immunosorbent assay was used to detect the level of parathyroid hormone in the cell supernatant. 
RESULTS AND CONCLUSION: Western blot results showed that the parathyroid cells were positive for calcium-sensing receptor, parathyroid hormone and glial cells missing-2, indicating the cultured cells were parathyroid cells. After transfection of hTERT, hTERT gene and protein levels were significantly increased in the hTERT transfection group as compared with parathyroid cell group and empty virus group (P < 0.05). The cell growth rate in the hTERT transfection group noticeably increased (P < 0.05). The cell number in G0/G1 phase was significantly decreased and the number of cells in the S phase was increased (P < 0.05). The level of parathyroid hormone in the culture supernatant was significantly increased (P < 0.05). To conclude, transfection of the PLXSN carrying the hTERT gene can promote the proliferation of rat parathyroid cells in vitro and increase the level of parathyroid hormone (PTH) in the supernatant of parathyroid cells.
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In vitro isolation and culture of chondrocytes from human osteoarthritis patients
Wei Yu, Wei Min. In vitro isolation and culture of chondrocytes from human osteoarthritis patients[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4056-4061.

BACKGROUND: Numerous studies addressing cells in osteoarthritis still focus on the in vitro culture of articular chondrocytes in animal models. Degeneration in the animal models is not completely consistent with that in human osteoarthritis. How to construct an in vitro cell model using chondrocytes from human osteoarthritis and to study changing trend of its characteristic proteins is the key to simulating the cartilage degeneration in human osteoarthritis in vivo.
OBJECTIVE: To investigate the in vitro isolation and culture of human chondrocytes from osteoarthritis patients, to observe the morphological characteristics of human osteoarthritis chondrocytes from primary to third generation, and to study the changes in biological characteristics of chondrocyte-related proteins in different generation.
METHODS: The study protocol was in line with the ethic requirements of Chinese PLA General Hospital with the approval No. S2017-23-7. Six cases undergoing arthroplasty for severe osteoarthritis (2 males and 4 females, age 62-72 years old with a mean age of (68.3±3.39) years) were enrolled. The chondrocytes from abandoned cartilage tissue in these patients were isolated and cultured by one-step enzymatic digestion (type II collagenase), and then subcultured, so as to construct an in vitro chondrocyte culture system for osteoarthritis. The morphology of cells at different generations was observed by inverted phase contrast microscope. Hematoxylin-eosin staining, toluidine blue staining and type II collagen immunofluorescence staining were used for cell identification. Western blot was used to detect the expressions of Col2a, Aggrecan and matrix metalloproteinase-13 in each generation of chondrocytes at the fusion rate of 70%.
RESULTS AND CONCLUSION: After digestion using type II collagenase, the cells scattered around the tissue mass could be observed at about 1 week of culture, and these cells could be subcultured for further study after about 3 weeks. Morphological observation, hematoxylin-eosin staining, toluidine blue staining and type II collagen immunofluorescence staining proved that human chondrocytes were successfully cultured. The relative expression of Col2a and Aggrecan in chondrocytes at the third generation was significantly decreased as compared with that in primary cells (P < 0.01), and the expression gradually decreased with the subculture times. Matrix metalloproteinase-13 expression gradually increased with the increase of subculture times (P < 0.01). In conclusion, chondrocytes can be successfully isolated from the osteoarthritis specimens by one-step digestion of type II collagenase and then subcultured. The dedifferentiation of osteoarthritis chondrocytes in vitro increases with the increase of subculture times and the expression of functional proteins decreases as a whole. The chondrocytes within three generations present with cartilage degeneration in osteoarthritis and may be the best choice for experimental studies.

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Timosaponin B-II reduces necroptosis of VSC4.1 cells induced by oxygen-glucose deprivation 
Gao Junyan, Cao Yanfei, Zhang Yun, Liu Xuemin, Hou Yanhong, Li Jianbin, Wu Zhibing. Timosaponin B-II reduces necroptosis of VSC4.1 cells induced by oxygen-glucose deprivation [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4062-4067.
BACKGROUND: Timosaponin B-II has neuroprotective effects and reduces necroptosis of cells, but its mechanism is still unknown and needs further study.
OBJECTIVE: To study the mechanism by which the extract of timosaponin B-II of Anemarrhena asphodeloides on necroptosis of VSC4.1 cells induced by oxygen-glucose deprivation. 
METHODS: Firstly, VSC4.1 cells in good growth condition were cultured in six groups: group A as normal control; group B with hydrogen peroxide solution for 24 hours; groups C-F with 1, 10, 100, 1 000 μmol/L timosaponin B-II solution for 24 hours, respectively, and then each group was cultured with hydrogen peroxide solution for another 24 hours. MTT assay was used to detect the cell survival rate, and the drug concentration of the group with the highest cell survival rate was selected for the following experiments. Secondly, VSC4.1 cells were cultured in three groups: routine culture in normal control group; anaerobic hypoglycemic culture for 8 hours followed by reoxygenation and hyperglycemic culture for 6 or 12 hours in model group; necrostatin-1, a necroptosis inhibitor, was added in inhibitor group for 24 hours, and anaerobic hypoglycemic culture for 8 hours followed by reoxygenation and hyperglycemic culture for 6 or 12 hours. Flow cytometry was used to detect the number of necrotic cells after 6 hours of reoxygenation, and western blot assay was used to detect the expression of receptor-interacting protein 3 after 6 or 12 hours of reoxygenation. Thirdly, VSC4.1 cells were cultured in three groups: routine culture in normal control group; anaerobic hypoglycemic culture for 8 hours followed by reoxygenation and hyperglycemic culture for 6 hours in model group; drug group was treated with 100 μmol/L timosaponin B-II solution for 24 hours, followed by anaerobic hypoglycemic culture for 8 hours and anaerobic hyperglycemic culture for 6 hours. Flow cytometry was used to detect the number of necrotic cells after 6 hours of reoxygenation and western blot assay was used to detect the expression of receptor-interacting protein 3 after 6 hours of reoxygenation.
RESULTS AND CONCLUSION: (1) The MTT assay showed that compared with group B, group E had the highest cell survival rate. Hence, 100 μmol/L timosaponin B-II was selected for subsequent experiments. (2) The number of necrotic cells in the model group was higher than that in the normal control group (P < 0.05), and the number of necrotic cells in the inhibitor group was lower than that in the model group (P < 0.05). After 6 and 12 hours of reoxygenation culture the expression of receptor-interacting protein 3 in the inhibitor group was higher than that in the normal control group (P < 0.05), and the highest expression was observed at 6 hours after reoxygenation (P < 0.05). (3) The number of necrotic cells and the expression of receptor-interacting protein 3 in the model group were higher than those in the normal control group (P < 0.05), and the number of necrotic cells and the expression of receptor-interacting protein 3 in the drug group were lower than those in the model group (P < 0.05). All these results indicate that timosaponin B-II can effectively reduce the necroptosis of VSC4.1 cells deprived of oxygen-glucose, which may be related to the reduction of receptor-interacting protein 3.
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      干细胞综述 stem cell review
Research progress in stem cell-based tissue engineering technology assisting mandibular distraction osteogenesis
Han Zhiqi, Jiang Weidong, Zhou Nuo. Research progress in stem cell-based tissue engineering technology assisting mandibular distraction osteogenesis[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4031-4036.

BACKGROUND: Since mandibular distraction osteogenesis has achieved significant repercussions in clinical practice, many scholars have been working on studies that enhancing osteogenesis and shortening the course of treatment. Among them, stem cell transplantation has attracted much attention as a hotspot.
OBJECTIVE: To summarize the current progress and deficiency of stem cell-based tissue engineering technology assisting mandibular distraction osteogenesis.
METHODS: Following the PRISMA guidelines, a computer-based search of PubMed, WOS, CNKI and WanFang databases was performed by the first author for relevant articles with the keywords of “distraction osteogenesis,mandibular, mandible, stem cells” in English and Chinese, respectively. Animal models, types of stem cells and routes of transplantation in the selected literature were extracted and counted up. The results were tabulated and introduced briefly. 
RESULTS AND CONCLUSION: Fifty-one articles were strictly selected as per the eligibility criteria, analyzed and summarized. In the selected literature, the timing, methods and types of stem cell transplantation vary greatly. The majority of animal experimental studies have confirmed that stem cell-based tissue engineering technology can enhance bone and blood vessel regeneration in mandibular distraction osteogenesis. Explorations on the selection and application of stem cells in mandibular distraction osteogenesis assisted by stem cell-based tissue engineering technology cannot only provide ideas for future treatment, but also lay the foundation for clinical trials.

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Application potential of different origin stem cells in oral bone regeneration
Shen Mengjie, Yang Kun, Liu Qi. Application potential of different origin stem cells in oral bone regeneration[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4068-4074.

BACKGROUND: In recent years, with the development of stem cell and regeneration technologies, bone tissue engineering has been rapidly developed. Despite there are a variety of seed cells for bone tissue engineering, researchers need to decide which types of stem cells are the best suitable for their specific purposes. To date, there is no ideal treatment for oral bone tissue repair, although a plenty of therapeutic methods have been proposed. Therefore, it will provide a new idea for repairing oral bone defects by fully utilizing the potential of stem cells during bone regeneration.
OBJECTIVE: To review the osteogenic potential of stem cells from different sources on oral bone regeneration, thereby providing a basis for selecting seed cells for tissue engineering.
METHODS: Databases of CBM, CNKI, PubMed, Elsevier, and Web of Science were retrieved with the keywords of “stem cell, bone repair, alveolar bone regenerate, osteogenesis, bone tissue” in English and Chinese, respectively. After initial screening of titles and abstracts, irrelevant articles were excluded and 81 eligible articles were included in final analysis.
RESULTS AND CONCLUSION: Tissue engineering technology provides a new approach to oral bone regeneration, but the choice of stem cells has not been standardized. Many types of stem cells have been used for bone tissue regeneration, but their roles in the body vary due to different origins of stem cells. Therefore, choosing a suitable type of stem cells is important for the repair of specific tissue damage. In this review, we compared the advantages and disadvantages of embryonic stem cells, mesenchymal stem cells and induced pluripotent stem cells in the oral bone regeneration, and attempted to provide a reference for the selection of seed cells for the regeneration and repair of oral bone tissue.

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Response of bone marrow mesenchymal stem cells to mechanical microenvironment: viewpoint, actuality, thinking and future
Sun Bin, Duan Hao, Zhong Zongyu, Liu Zheng, Li Xiaozhuang, Tang Zhihong, He Fei. Response of bone marrow mesenchymal stem cells to mechanical microenvironment: viewpoint, actuality, thinking and future[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4075-4081.

BACKGROUND: Bone marrow mesenchymal stem cells are important seed cells in bone tissue engineering repair. However, it is difficult to control the cell proliferation and differentiation. Although previous studies have focused on biochemical regulation, little is reported on the mechanical microenvironment for controlling the proliferation and differentiation of bone marrow mesenchymal stem cells.
OBJECTIVE: To summarize the research status and recent progress in the effect of mechanical microenvironment on the proliferation and differentiation of bone marrow mesenchymal stem cells.
METHODS: A search with the keywords of “mechanical microenvironment; mesenchymal stem cells; stiffness; proliferation and differentiation” in Chinese and English was conducted in the CNKI, WanFang and PubMed, respectively. Initially 117 articles were retrieved, and 65 eligible articles were finally summarized.
RESULTS AND CONCLUSION: Different mechanical forces cause different effects on the proliferation and differentiation of bone marrow mesenchymal stem cells. Under mechanical tension and fluid shear forces, the cells mostly differentiate into osteoblasts. Under compressive and hydrostatic pressure, the majority of the cells differentiate into chondrocytes, while a small amount of the cells are induced to differentiate into osteoblasts. Mechanical signals from different basal mechanics have an effect on neurogenesis adipogenesis, myogenesis and osteogenesis in the mesenchymal stem cells. There are different optimal conditions for the cells differentiating into different tissues. Different self-components of the cells also play an important role in their response to mechanical signals. To explore the relationship between mechanical dynamics and proliferation and differentiation of mesenchymal stem cells, we establish a simulated matrix and scaffold system similar to the mechanical microenvironment of bone marrow mesenchymal stem cells.

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Stem cells in the treatment of skin defects: a focus of future research
Liu Boyu, Pu Lei, Zhang Yingjie, Yang Yingnan, Li Yaxiong. Stem cells in the treatment of skin defects: a focus of future research[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4082-4088.
BACKGROUND: As a kind of special cells capable of self-renewal and multidirectional differentiation, stem cells have the potential in the treatment of skin defects. Owing to their rich sources, low immunogenicity, and multipotent differentiation ability, stem cells are expected to overcome the limitations of existing therapies and develop into new therapies.
OBJECTIVE: To overview the mechanisms and research focus of different stem cells in the treatment of skin defects, and to seek a kind of stem cells that meet clinical needs.
METHODS: The first author retrieved the databases of PubMed for relevant articles about stem cell-based therapies for skin defects published from 1998 to 2018. The key words were “stem cell, skin defect, wound healing” in English. In accordance with the inclusion and exclusion criteria, 63 articles were finally reviewed.?
RESULTS AND CONCLUSION: Stem cells have tremendous potential in the treatment of skin defects. These cells cannot only regenerate damaged tissue, but also promote the wound healing by immunomodulation and angiogenesis. Compared with other types of stem cells, mesenchymal stem cells have more advantages. Studies concerning mesenchymal stem cells have achieved satisfactory results in recent years. Mesenchymal stem cells have become the most ideal cells for the treatment of skin defects, and they are also the focus of future research.
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Application and prospect of adipose-derived stem cells in wound inflammation
Zhang Liming, Cai Jieyun, Pan Fuqiang, Wang Jing. Application and prospect of adipose-derived stem cells in wound inflammation[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4089-4093.

BACKGROUND: Since human embryonic stem cells were successfully obtained in 1998, clinicians have been trying to use stem cells in wound healing. Importantly, stem cells can regulate wound inflammation.
OBJECTIVE: To investigate the application and prospect of adipose-derived stem cells in wound inflammation.
METHODS: The first author searched CNKI and PubMed databases (from 1999 to October 2018) with the retrieval words of “adipose-derived stem cells, wound, inflammation” in Chinese and English, respectively. Literatures regarding adipose-derived stem cells, wound and inflammation were selected. The articles published lately or authoritatively in the same field were preferred.
RESULTS AND CONCLUSION: Wound healing is a complex and orderly process, and closely related to wound inflammation. With the development of regenerative medicine, the application of stem cells brings a new approach to wound healing. Adipose-derived stem cells have a promising prospect as characterized by the strong abilities of proliferation and multidirectional differentiation, easy to obtain, and abundant sources. To date, adipose-derived stem cells are still one of the most popular candidates for regulating wound inflammation and promoting wound healing.

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Clinical progress in the treatment of non-hematopoietic diseases by human umbilical cord blood cell transplantation
Liu Yang, Zheng Yuanyuan, Chen Yizhu, Zhao Jing, Li Jianyou, Wu Mingyuan, Sun Quan. Clinical progress in the treatment of non-hematopoietic diseases by human umbilical cord blood cell transplantation[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4094-4100.

BACKGROUND: Human umbilical cord blood contains a large number of stem cells that are capable of differentiating into hematopoietic, epithelial, endothelial and neural tissues both in vitro and in vivo. Thus, human umbilical cord blood has a therapeutic potential to a wide variety of diseases.
OBJECTIVE: To summarize the clinical application of human umbilical cord blood cells in the treatment of non-hematopoietic diseases.
METHODS: A computer-based retrieval of PubMed and CNKI databases was performed in order to search relevant articles published from 2001 till now, using the keywords of “umbilical cord stem cells, umbilical cord blood mesenchymal stem cells, umbilical cord blood” in English and Chinese, respectively. After removal of repetitive or irrelevant articles, 64 articles were finally reviewed.
RESULTS AND CONCLUSION: The clinical research of umbilical cord blood stem cells involves many fields such as the circulatory system, endocrine system and nervous system, in most of which good experimental results have been achieved. Findings from in-depth basic and clinical studies on biological and immunological characteristics of cord blood indicate an extensive application of umbilical cord blood stem cells that will be a new therapeutic choice for patients.

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      干细胞移植 stem cell transplantation
Effect of human amniotic mesenchymal stem cell transplantation on airway goblet cell proliferation and mucin 5ac expression in asthmatic mice
Yang Hongxia, Shi Qinghong, Weng Minhua, Zhang Jianyong, Zhao Jianjun, Chen Ling. Effect of human amniotic mesenchymal stem cell transplantation on airway goblet cell proliferation and mucin 5ac expression in asthmatic mice[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4050-4055.
BACKGROUND: Our preliminary studies indicated that human amniotic mesenchymal stem cells transplantation can reduce airway inflammatory responses in asthmatic mice, but the effect of human amniotic mesenchymal stem cells on airway mucus hypersecretion is unclear.
OBJECTIVE: To investigate the effect of human amniotic mesenchymal stem cells transplantation on airway goblet cell proliferation and mucin 5ac expression in asthmatic mice.
METHODS: Twenty-eight BALB/C mice were randomly divided into sham operation group (sensitized with normal saline at the 1st and 13th days, and motivated with nebulized normal saline at the 19th to 23th days), model group (sensitized with egg albumin and motivated with nebulized egg albumin to construct a mouse model of asthma), experimental group (transplanted with human amniotic mesenchymal stem cells via the tail vein of the mouse model of asthma at the 18th day), and blank group (sensitized with normal saline, motivated with nebulized normal saline, and transplanted with human amniotic mesenchymal stem cells via the tail vein at the 18th day). There were seven mice in each group. All the mice were sacrificed at the 24th day. We observed the pathological changes in the airway and the proliferation of epithelial goblet cells in each group. We detected the expression of interleukin-5 in the bronchoalveolar lavage fluid and the mRNA and protein levels of mucin 5ac in the lung tissue.
RESULTS AND CONCLUSION: (1) In the model group, there were a large amount of inflammatory cells infiltrated in the bronchus, perivascular and lung interstitial tissues; the goblet cells of the airway epithelium proliferated obviously, the tracheal wall was thickened to varying degrees, and there were a large amount of mucus secretion in some lumens with the lumen being narrow or even occluded. In the experimental group, airway inflammatory cell infiltration and goblet cell hyperplasia were alleviated. (2) Compared with the sham operation and blank groups, the model group showed increases in the proportion of airway epithelial goblet cells, number of goblet cells, interleukin-5 level in the bronchoalveolar lavage fluid, and expression of mucin 5ac protein and mRNA in the airway (P < 0.01). Compared with the model group, the above-mentioned indexes of the experimental group were significantly decreased (P < 0.01). All the findings indicate that transplantation of human amniotic mesenchymal stem cells can inhibit airway inflammatory response and epithelial goblet cell proliferation, downregulate mucin 5ac expression, and alleviate airway mucus hypersecretion.
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