低氧促进人骨髓间充质干细胞成管、内皮分化及其在血管生成过程中的作用

发布时间：2019-06-04 03:57

【摘要】：本实验通过模拟骨髓内的低氧张力对人骨髓间充质干细胞(hMSCs)进行体外低氧培养,探讨低氧对hMSCs成管及内皮分化倾向的影响及其作用机制。体内实验中利用pLEGFP-N1逆转录病毒载体对hMSCs进行标记,通过EGFP来追踪hMSCs植入体内的变化;以BALB/C裸鼠为实验动物模型,通过Matrigel或肿瘤血管生成实验研究体内低氧环境对hMSCs内皮分化、血管生成作用的影响及其机制。结果如下:①成功地建立了一套hMSCs分离培养、纯化、鉴定及扩增的标准化技术平台;P12代内hMSCs生物学性状稳定并保持未分化状态,可以作为后续实验工作的细胞来源。②体外低氧培养能通过加速细胞的铺展、促进细胞外基质的降解和细胞的迁移而促进hMSCs成管及内皮分化,其机制为:a.低氧诱导hMSCs表达HIF-1α,进而上调VEGF与MT1-MMP的表达,从而促进hMSCs成管及内皮分化;b.低氧可能通过促进hMSCs细胞Ca~(2+)通道开放,而促进其向内皮细胞分化。③hMSCs能促进鸡胚绒毛尿囊膜血管新生。④pLEGFP-N1逆转录病毒载体能有效地转导hMSCs,转导后的细胞维持其原有的生物学特性。⑤Matrigel血管生成实验证实体内低氧环境能促进hMSCs血管生成及内皮分化,但大部分血管为宿主源性的新生血管,只有极少数血管的内皮细胞是由hMSCs分化而来的。⑥hMSCs能促进肿瘤的血管生成,这与肿瘤造成的低氧张力有关,其作用也主要是通过促进宿主源性的血管新生实现的。 综上所述,本实验为MSCs的内皮诱导分化提供了一个新的技术路线和实验方法,也为今后更好地应用MSCs治疗临床缺血性疾病或血管依赖性疾病,以及利用组织工程化MSCs抗肿瘤治疗提供了理论基础和实验依据。
[Abstract]:The aim of this study was to investigate the effect of hypoxia on the tendency of hMSCs tubule and endothelial differentiation and its mechanism by simulating hypoxia tension in bone marrow to culture human bone marrow mesenchymal stem cell (hMSCs) in vitro. In vivo experiment, pLEGFP-N1 retrovirus vector was used to label hMSCs, and EGFP was used to track the changes of hMSCs implantation in vivo. Using BALB/C nude mice as experimental animal model, the effects of hypoxia on endothelial differentiation and angiogenesis of hMSCs in vivo and its mechanism were studied by Matrigel or tumor angiogeny. The results are as follows: (1) A set of standardized technical platform for isolation, culture, purification, identification and amplification of hMSCs was successfully established. The biological characters of hMSCs in P12 generation are stable and remain undifferentiated, which can be used as the cell source of subsequent experimental work. 2 hypoxia culture in vitro can accelerate the spread of cells. The mechanism of promoting the degradation of extracellular matrix and the migration of cells to promote the tubule and endothelial differentiation of hMSCs is as follows: a. Hypoxia induced the expression of HIF-1 伪 in hMSCs, and then upregulated the expression of VEGF and MT1-MMP, thus promoting the tubule and endothelial differentiation of hMSCs. Hypoxia may promote the differentiation of Ca~ (2) channels into endothelial cells by promoting the opening of Ca~ (2) channels in hMSCs cells. 3hMSCs can promote the neovascularization of chorioallantoic membrane in chicken embryo. 4pLEGFP-N1 retrovirus vector can effectively transfer hMSCs,. The transferred cells maintained their original biological characteristics. 5 Matrigel angiogenesis assay confirmed that hypoxia in vivo can promote hMSCs angiogenesis and endothelial differentiation, but most of the blood vessels are host neovascularization. Only a very small number of vascular endothelial cells are differentiated from hMSCs. 6 h MSCs can promote tumor angiogenesis, which is related to the hypoxia tension caused by tumor, and its effect is mainly achieved by promoting host angiogeny. In summary, this study provides a new technical route and experimental method for endothelial differentiation of MSCs, and also provides a better application of MSCs in the treatment of clinical ischemic diseases or vascular dependent diseases in the future. And the use of tissue engineering MSCs anti-tumor therapy provides a theoretical and experimental basis.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

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