中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (1): 1-6.doi: 10.3969/j.issn.2095-4344.2001

• 骨髓干细胞 bone marrow stem cells •    下一篇

联合应用血管内皮生长因子及血小板衍生生长因子BB干预骨髓间充质干细胞的血管化及增殖能力

孙  安,毕翔宇,韩祥祯,周琦琪,何惠宇   

  1. 新疆医科大学第一附属医院口腔修复科,新疆维吾尔自治区乌鲁木齐市  830054
  • 收稿日期:2019-06-15 修回日期:2019-06-18 接受日期:2019-07-18 出版日期:2020-01-08 发布日期:2019-12-10
  • 通讯作者: 何惠宇,博士,主任医师,教授,硕士、博士生导师,新疆医科大学第一附属医院口腔修复科,新疆维吾尔自治区乌鲁木齐市 830054
  • 作者简介:孙安,女,1990年生,新疆维吾尔自治区巴州人,汉族,新疆医科大学在读硕士,目前主要从事口腔修复学、颌面骨组织工程研究。
  • 基金资助:
    国家自然科学基金项目(81660177)

Effects of vascular endothelial growth factor combined with platelet-derived growth factor-BB on the angiogenic and proliferative abilities of bone marrow mesenchymal stem cells

Sun An, Bi Xiangyu, Han Xiangzhen, Zhou Qiqi, He Huiyu   

  1. Department of Prosthodontics, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • Received:2019-06-15 Revised:2019-06-18 Accepted:2019-07-18 Online:2020-01-08 Published:2019-12-10
  • Contact: He Huiyu, MD, Chief physician, Professor, Master’s supervisors, Doctoral supervisor, Department of Prosthodontics, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • About author:Sun An, Master candidate, Department of Prosthodontics, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81660177

摘要:

血管内皮生长因子:是血管新生家族的重要成员,成血管作用最为明确,主要作用于血管形成早期。血管内皮生长因子促进血管形成的机制主要在于:①能够促进蛋白酶分泌使基底膜通透性增加,便于内皮细胞的增殖及迁移;②激活并趋化呈静止状态的内皮祖细胞迁移。血管内皮生长因子165在人体内存在数目最多、生物效能也是最强的这一观点在学术界早已达成共识。基于以上,此实验选择促血管生成效果最强大的血管内皮生长因子165作为研究对象,为了方便起见,简称血管内皮生长因子。

血小板衍生生长因子:是重要的血管生成因子,在血管形成及稳定过程中发挥重要作用。近年来因为其促细胞增殖剂效果强劲以及能够有效影响管周细胞活动而进入广大学者视野,该实验选取血小板衍生生长因子也是为了更好地发挥双因子间的协同作用,最大程度地促进形成完善的功能性血管网,达到真正促进血流灌注的效果。

背景:组织工程骨为修复极限骨缺损提供了新的选择,但只有先形成完善的功能性血管网,保证稳定的成骨和骨整合,才能取得良好的治疗效果。因此,血管化可以说是组织工程骨面临的最大挑战与难题。

目的:探讨体外联合应用血管内皮生长因子(vascular endothelial growth factor,VEGF)和血小板衍生生长因子BB(platelet-derived growth factor-BB,PDGF-BB)对骨髓间充质干细胞增殖和成血管能力的影响。

方法:体外分离培养SD大鼠骨髓间充质干细胞,分别用不同质量浓度VEGF(20,40,60,80,100,120 μg/L)、PDGF-BB(20,40,60,80,100,120 μg/L)联合干预以及100 μg/L VEGF单独干预、100 μg/L PDGF-BB单独干预,CCK-8实验检测2种细胞因子促进细胞增殖的最佳质量浓度,然后在第7天和第14天通过RT-PCR方法检测血管生成素1、缺氧诱导因子1α、肝细胞生长因子、胰岛素样生长因子等相关成血管基因的表达量。

结果与结论:①加入生长因子后,细胞增殖能力明显提高,联合作用效果更优,最佳组合为80 μg/L VEGF+   80 μg/L PDGF-BB;②VEGF、PDGF-BB都可以促进血管生成素1、缺氧诱导因子1α、肝细胞生长因子和胰岛素样生长因子的mRNA表达,联合应用时效果最佳;③缺氧诱导因子1α、肝细胞生长因子 mRNA表达随时间延长有所升高,差异有显著性意义(P < 0.05);而血管生成素1、胰岛素样生长因子 mRNA表达量随时间延长有所降低,差异有显著性意义(P < 0.05);④体外实验结果证明,当VEGF和PDGF-BB质量浓度均为80 μg/L时,能够持续稳定促进整个血管形成过程,且促进作用优于单独一种生长因子。

ORCID: 0000-0003-1918-579X(何惠宇)

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

关键词: 骨髓间充质干细胞, 血管内皮生长因子, 血小板衍生生长因子BB, 组织工程骨, 血管化

Abstract:

BACKGROUND: Bone tissue engineering provides a new path to treat critical-sized bone defects. However, a stable osteogenesis and osseointegration can only be insured via formation of an intact vascular network, which can achieve satisfactory therapeutic effects. Thereafter, angiogenesis is a challenge and difficulty in bone tissue engineering.

OBJECTIVE: To investigate the effect of combined application of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB) on proliferation and angiogenesis of bone marrow mesenchymal stem cells.

METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured and isolated in vitro, and interfered with different concentrations of VEGF (20, 40, 60, 80, 100, 120 μg/L) and PDGF-BB (20, 40, 60, 80, 100, 120 μg/L) in combination or 100 μg/L VEGF, and 100 μg/L PDGF-BB alone. The optimum concentration of promoting cell proliferation was detected by cell counting kit-8 assay. The expression levels of angiopoietin-1, hypoxia-inducible factor-1α, hepatocyte growth factor and insulin-like growth factor were detected by RT-PCR at 7 and 14 days after intervention.

RESULTS AND CONCLUSION: (1) After growth factors were added, the cell proliferative ability was significantly improved, and combined application revealed better effect. The optimal concentration grou was 80 μg/L VEGF+80 μg/L PDGF-BB. (2) Both VEGF and PDGF-BB could promote the mRNA expression levels of angiopoietin-1, hypoxia-inducible factor-1α, hepatocyte growth factor and insulin-like growth factor, and the effect was more obvious in combined application. (3) The mRNA expression levels of hypoxia-inducible factor-1α and hepatocyte growth factor were significantly increased with time (P < 0.05). The mRNA expression levels of angiopoietin-1 and insulin-like growth factor were significantly decreased with time (P < 0.05). (4) In vitro experimental results suggest that VEGF and PDGF-BB at the concentration of  80 μg/L can consistently promote angiogenesis, and the effect of combined application is better than that of single application.

Key words: bone marrow mesenchymal stem cells, vascular endothelial growth factor, platelet-derived growth factor-BB, tissue-engineered bone, angiogenesis

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