体外诱导小鼠心脏CD51^+细胞分化为血管内皮细胞的研究

In vitro induction of differentiation of cardiac CD51^+ cells into vascular endothelial cells in mouse models

目的探讨小鼠心脏CD51^+细胞的分离培养及体外定向诱导分化为血管内皮细胞的可行性,为其治疗心脏血管内皮损伤提供理论依据。方法取出日龄为7 d的C57 BL/6乳鼠心脏,用流式分选法分选原代细胞、悬浮培养法培养及纯化心脏CD51^+细胞。用内皮生长培养基(EGM-2)对纯化和扩增后的CD51^+细胞进行体外诱导内皮分化20 d。诱导后观察细胞形态变化;实时荧光定量PCR分析内皮相关基因CD31、CD34、v WF、VEGFR-2及VE-Cadherin的mRNA表达;细胞免疫荧光技术、流式表型分析术检测内皮相关标志CD31、VEGFR-2及v WF的表达;测定细胞一氧化氮(NO)含量、行体外血管形成实验以鉴定其功能。结果诱导内皮分化后细胞变为短梭形,呈铺路石样排列生长;荧光定量PCR结果显示诱导后细胞CD31、CD34、v WF、VEGFR-2、VE-Cadherin的mRNA相对表达量较诱导前增加(t值分别为24.529、6.383、25.872、5.256、5.255,P均<0.05);细胞免疫荧光染色及流式表型分析结果均显示诱导后细胞表达CD31、VEGFR-2及v WF;诱导后细胞NO含量较诱导前增加(t=9.549,P=0.011)并具有体外形成管腔样结构的能力。结论心脏CD51^+细胞具有在体外定向诱导分化为血管内皮细胞的能力。

Objective To investigate the feasibility of isolation,culture and in vitro induction of the differentiation of cardiac CD51＋cells into vascular endothelial cells,aiming to provide theoretical evidence for the treatment of cardiac vascular endothelial injury. Methods The heart tissues were collected from 7-day-old C57 BL/6 mice. Primary cells were isolated by flow cytometry and cell sorting. The cardiac CD51＋cells were cultured and purified by suspension cell culture method. After cell purification and proliferation,the cardiac CD51＋cells were subjected to in vitro induction of differentiation into endothelial cells in endothelial cell growth medium-2（ EGM-2） for 20 days. After in vitro induction,the changes in cell morphology were observed. The expression levels of CD31,CD34,v WF,VEGFR-2 and VE-Cadherin mRNA were quantitatively measured by real-time fluorescent quantitative PCR. The expression levels of CD31,VEGFR-2 and v WF were detected by immunofluorescent staining and flow cytometric immunophenotying. The level of nitric oxide（ NO）was measured and the function of the induced endothelial cells was evaluated by angiogenesis assay in vitro.Results The morphology of cardiac CD51＋cells was changed from the long-spindle to short-spindle shape and the induced cells grew in a cobblestone-like pattern. The results of fluorescent quantitative PCR demonstrated that the relative expression levels of CD31,CD34,v WF,VEGFR-2 and VE-Cadherin mRNA were significantly up-regulated after induction（ t = 24. 529,6. 383,25. 872,5. 256 and 5. 255,all P 〈0. 05）. Both immunofluorescent staining and flow cytometric immunophenotying revealed that the cells expressed CD31,VEGFR-2,and v WF after induction. The quantity of NO was considerably increased after induction（ t = 9. 549,P =0. 011） and had the ability to form lumen-like structures in vitro. Conclusion Cardiac CD51＋cells can be induced to differentiate into vascular endothelial cells in vitro.