大鼠骨髓间充质干细胞向内皮细胞分化的体外研究

发布时间：2019-03-22 17:06

【摘要】：目的间充质干细胞(mesenehymal stem cells, MSC)来源于中胚层(Mesoderm),具有多向分化能力。大量研究表明,MSC可能在一定条件下向内皮细胞分化。本实验以建立大鼠骨髓MSC的培养体系为基础,研究MSC体外向内皮细胞分化的特性,体外观察模拟的心肌局部微环境及心肌缺血微环境对MSC向内皮细胞分化的影响。方法应用4周以内的大鼠骨髓分离培养MSC,进行体外扩增,对其形态学、增殖能力及向成骨和脂肪细胞分化的功能进行鉴定;在以上实验的基础上,建立大鼠的心肌梗死模型,制备不同时间心梗心肌的匀浆,参与到由成血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子β(bFGF)和胰岛素样生长因子-1(IGF-1)组成的成内皮诱导体系中,应用免疫组化方法检测vWF、CD31特异性抗原,RT-PCR检测vWF的表达,并进行吞噬实验,并观察心肌匀浆对MSC增殖和成内皮分化的影响。结果通过对培养细胞的形态学及功能鉴定,证明其为MSC,并具有向成骨、成脂肪细胞分化的能力;生长因子加或不加心肌匀浆诱导后的细胞,部分呈vWF、CD31阳性,Ac-LDL吞噬实验阳性,梗塞心肌匀浆与生长因子共同作用,能促进MSC向内皮细胞分化,vWF阳性率提高,P0.05,RT-PCR显示1-2周的梗塞心肌匀浆vWF的基因表达最多。
[Abstract]:Objective Mesenchymal stem cells (mesenehymal stem cells, MSC) derived from mesodermal (Mesoderm), have the ability of multidirectional differentiation. A large number of studies have shown that MSC may differentiate into endothelial cells under certain conditions. Based on the establishment of the culture system of rat bone marrow MSC, the characteristics of MSC differentiation into endothelial cells in vitro were studied. The effects of simulated myocardial microenvironment and myocardial ischemia microenvironment on the differentiation of MSC into endothelial cells were observed in vitro. Methods MSC, was isolated and cultured from rat bone marrow within 4 weeks for in vitro amplification, and its morphology, proliferation and differentiation into osteoblasts and adipocytes were identified. On the basis of the above experiments, the model of myocardial infarction in rats was established, and the homogenate of myocardial infarction at different time was prepared, which involved in the involvement of vascular endothelial growth factor (VEGF),) in myocardial infarction. In the endothelial induction system composed of basic fibroblast growth factor-尾 (bFGF) and insulin-like growth factor-1 (IGF-1), the specific antigen of vWF,CD31 was detected by immunohistochemical method, and the expression of vWF was detected by RT-PCR. The effects of myocardial homogenate on proliferation and endothelial differentiation of MSC were observed by phagocytosis test. Results through the morphological and functional identification of cultured cells, it was proved that the cultured cells were MSC, and had the ability to differentiate into osteoblasts and adipocytes. The cells induced by growth factor plus or without myocardial homogenate were partly positive for vWF,CD31 and positive for Ac-LDL phagocytosis test. Infarct myocardium homogenate combined with growth factor could promote the differentiation of MSC into endothelial cells, and the positive rate of vWF increased, P0.05, P 0.05, P < 0.05, P < 0.01, P < 0.05, respectively. RT-PCR showed that the gene expression of vWF in myocardial homogenate was the highest at 1 and 2 weeks after infarction.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

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