VEGF诱导人骨髓间充质干细胞分化为内皮细胞的可行性

Induction and differentiation of human bone marrow mesenchymal stem cells into endothelial cells by VEGF

目的:研究内皮细胞生长因子(VEGF)诱导骨髓间充质干细胞(BMSCs)向内皮细胞定向分化的可行性并观察其生长增殖特性。方法:采用密度梯度离心法获取大量较纯的骨髓间充质干细胞,分为2组,实验组以含VEGF的M199培养液进行体外诱导分化,对照组用M199培养液培养。通过光镜、电镜及免疫组织化学染色方法分别观察所分化细胞的形态及表型,并绘制细胞生长曲线。结果:培养早期BMSCs多呈扁平、梭形、多角形形态,2周后实验组细胞渐呈集落状、椭圆形、铺路石样排列,而对照组细胞呈"毛发状"成纤维细胞外形。透射电镜下实验组细胞浆内有内皮细胞特征性单层质膜包裹结构——Weible-Palade小体。对照组无此结构。实验组第3代细胞的Ⅷ因子相关抗原阳性比例接近(78.20±5.90)%,与人血管来源内皮细胞((82.70±4.47)%)相比,差异无统计学意义,而对照组细胞Ⅷ因子相关抗原染色呈阴性。生长曲线呈指数增长,短期内无明显衰老现象。结论:人BM-SCs在VEGF的诱导下可定向分化为内皮细胞,并且保持了干细胞旺盛的增殖能力。

Aim ： To study the feasibility of human bone marrow mesenchymal stem cells（BMSCs） differentiating into endothelial cells,and to observe the cells′ proliferation characteristics. Methods：BMSCs were isolated by density gradient centrifugation. Afterwards, the obtained cells were allocated into two groups： cells in test group were propagated in M199 medium with vascular endothelial growth factor（VEGF） and those in control group were propagated in medium with M199 medium only. The differentiated cells were detected by light microscope, electron microscope and immunocytochemistry, and their growth and proliferation capabilities were determined by drawing cell growth curve simultaneously. Results： BMSCs were applanation, fusiform or polygon at the early stage,those in test group became into colony, ellipse and arranged like pebble 2 weeks later,but those in control group was like hairs, and the Weible-Palade body was observed by transmission electron microscope in the test group, the control group had not similar changes. Cytoehemistry staining evaluation revealed that close to （78.20 ± 5.90） % cells in test group of the third generation cells were positive for Ⅷ factor related antigen, which had no significant difference compared with vascular endothelial cells. The cells presented exponential growth and had no infinite senilism in short time. Conclusion ： human BMSCs can differentiate into endothelial cell under the induction of VEGF and keep active proliferation capability.