中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (29): 4637-4642.doi: 10.3969/j.issn.2095-4344.0620

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

冻存人脐带间充质干细胞的分离培养

刘 亭,姜 翙,刘志华,孙 宁,李晓媚,翁锦生   

  1. 广州医科大学附属第五医院,广东省广州市 510700
  • 修回日期:2018-07-11 出版日期:2018-10-18 发布日期:2018-10-18
  • 通讯作者: 翁锦生,博士,研究员,广州医科大学附属第五医院,广东省广州市 510700
  • 作者简介:刘亭,女,1985年生,湖北省随州市人,汉族,2016年中国科学院毕业,博士,助理研究员,主要从事生物化学和分子生物学方面的研究。
  • 基金资助:

    国家自然科学基金面上项目(81570189,8177110782);广东省科技厅项目(2016ZC0145)

Cryopreserved human umbilical cord mesenchymal stem cells: isolation and culture

Liu Ting, Jiang Hui, Liu Zhi-hua, Sun Ning, Li Xiao-mei, Weng Jin-sheng   

  1. Department of Central Laboratory, the Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510700, Guangdong Province, China
  • Revised:2018-07-11 Online:2018-10-18 Published:2018-10-18
  • Contact: Weng Jin-sheng, MD, Researcher, Department of Central Laboratory, the Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510700, Guangdong Province, China
  • About author:Liu Ting, MD, Assistant researcher, Department of Central Laboratory, the Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510700, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81570189, 8177110782; the Project of Guangdong Provincial Science and Technology Department, No. 2016ZC0145

摘要:

文章快速阅读:

文题释义:
原代培养:
是指由体内取出组织或细胞进行的首次培养,也叫初代培养。原代培养离体时间短,遗传性状和体内细胞相似,适于做细胞形态、功能和分化等研究。较为严格地说是指成功传代之前的培养,此时的细胞保持原有细胞的基本性质,如果是正常细胞,仍然保留二倍体数。但实际上,通常把第1代至第10代以内的培养细胞统称为原代细胞培养。
分化标记基因:在干细胞向其他种类细胞分化的过程中,该基因mRNA表达量和表达蛋白产物不断升高,提示分化进程,其表达产物还可以促进特异性分化,该基因在未分化细胞和其他种类细胞中不表达或低表达。

 

摘要
背景:
目前大多数报道是从新鲜脐带中分离培养脐带间充质干细胞,而从冻存脐带组织中分离培养间充质干细胞鲜有报道。
目的:从冻存人脐带中分离培养间充质干细胞,并比较多种培养方法获得的脐带间充质干细胞的生物学特性。
方法:采用组织块贴壁法从冻存脐带组织中分离培养脐带间充质干细胞,分别用有血清和无血清培养基培养,观察其形态特征,绘制其生长曲线;流式细胞仪检测第3代脐带间充质干细胞(无血清培养基培养)和第14代脐带间充质干细胞(含血清培养基培养)表面抗原表达;第3代脐带间充质干细胞(无血清培养基培养)分别用成脂、成骨、成软骨诱导培养液诱导其分化,qPCR鉴定分化标记基因表达;检测第7代脐带间充质干细胞(无血清培养基培养)及其培养上清中的细胞因子水平。
结果与结论:①从冻存人脐带组织原代培养的脐带间充质干细胞,呈梭形纤维样细胞形态,生长力旺盛,脐带间充质干细胞可用无血清培养基传代培养,但增殖速率还远低于有血清培养基;②流式细胞术分析结果显示为脐带间充质干细胞CD90、CD73、CD105呈强阳性表达,CD45、CD34、CD14/CD11b、CD79a/CD19、HLA-DR呈阴性表达;③经成脂、成骨和成软骨诱导培养14-28 d后,脂肪细胞标记基因PPARγ、骨细胞标记基因Osteonectin、软骨细胞标记基因CollagenⅡ的表达量显著升高;④第7代脐带间充质干细胞及其培养上清可检测出粒细胞集落刺激因子、粒细胞-巨噬细胞集落刺激因子、干扰素α2、趋化因子 IP-10、白细胞介素4、白细胞介素6等细胞因子;⑤结果提示可从冻存人脐带中培养扩增脐带间充质干细胞。

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
ORCID:
0000-0001-5198-7528(刘亭)

关键词: 冻存人脐带, 组织块贴壁培养, 细胞鉴定, 无血清培养, 间充质干细胞, 细胞因子, 流式细胞术, 干细胞, 国家自然科学基金

Abstract:

BACKGROUND: Current studies have addressed the mesenchymal stem cells (MSCs) mostly from fresh human umbilical cord, but rarely from cryopreserved human umbilical cord.
OBJECTIVE: To isolate MSCs from cryopreserved human umbilical cord, and to compare the biological characteristics of human umbilical cord MSCs (hUC-MSCs) harvested using several culture methods.
METHODS: hUC-MSCs isolated from cryopreserved umbilical cord tissues by explants culture method were cultured in serum-containing and serum-free media. Cell morphology was observed to draw the growth curve of cells in different media. Passage 3 hUC-MSCs cultured in serum-free medium and passage 14 hUC-MSCs in serum-containing medium were selected to detect the expression of surface biomarkers by flow cytometry. Passage 3 cells in the serum-free medium were induced for adipogenic, osteogenic and chondrogenic differentiation, and differentiation marker genes were identified by qPCR. Expression of cytokines in the passage 7 cells cultured in the serum-free medium and in the culture supernatant was detected.
RESULTS AND CONCLUSION: The hUC-MSCs isolated from cryopreserved human umbilical cord exhibited a spindle-shaped appearance and exuberant growth, and moreover, the cells could be expanded in serum-free medium but the cell proliferation was certainly slower than that in the serum-containing medium. Flow cytometry analysis revealed that the hUC-MSCs were positive for CD73, CD90, CD103, but negative for CD45, CD34, CD14/CD11b, CD79a/CD19, and HLA-DR. Adipogenic (PPARγ), osteogenic (Osteonectin) and chondrogenic (Collagen II) biomarker genes were highly expressed at 14-28 days of induction. Passage 7 hUC-MSC and its supernatant secreted several cytokines including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon α2, chemokine IP-10, interleukin 4, and interleukin-6. In conclusion, we could successfully isolate and expand hUC-MSCs from cryopreserved human umbilical cord tissues.

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

Key words: Cryopreservation, Umbilical Cord, Mesenchymal Stem Cells, Culture Media, Serum-Free, Tissue Engineering

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