共培养诱导心外膜祖细胞向血管平滑肌细胞的分化

Co-culture with umbilical vein endothelial cells promotes differentiation of epicardial progenitor cells into vascular smooth muscle cells

目的探讨冠脉发育过程中,血管内皮细胞是否可以影响心外膜祖细胞(epicardial progenitor cells,EpiCs)的分化从而调节冠状动脉的发育。方法饲养7～8周龄成年C57BL/6小鼠60只(雌、雄各半,体质量25～30 g)。提取出高纯度的EpiCs,实验分为对照组与共培养组,通过Transwell共培养系统探讨血管内皮细胞和EpiCs间的相互作用,采用细胞免疫荧光技术和荧光定量PCR分别检测EpiCs标志物WT1和Tbx18、平滑肌细胞标志物α-SMA和Myh11的蛋白表达与mRNA表达;胶原凝胶收缩实验检测细胞收缩能力;ELISA实验检测培养基中生长因子的表达。结果与对照组比较,共培养系统下血管内皮细胞可促进EpiCs中α-SMA[(1.73±0.85)%vs(34.20±3.38)%,P<0.01,n=3]与Myh11[(3.70±1.49)%vs(45.63±4.85)%,P<0.01,n=3]的表达并增强EpiCs的收缩能力,并且对共培养条件培养基的检测可见在此过程中碱性成纤维细胞生长因子(basic fibroblast growth factors,bFGFs)的升高[(87.64±8.78)vs(304.96±52.91)pg/mL,P<0.01,n=3]。结论血管内皮细胞可通过分泌bFGFs调节EpiCs向血管平滑肌细胞的分化,从而调节冠状动脉的发育。

Objective To investigate whether vascular endothelial cells affect the differentiation of epicardial progenitor cells(EpiCs)during coronary development.Methods Highly purified EpiCs were extracted from 30 female and 30 male C57 BL/6 mice(7 to 8 weeks old,body weight 25-30 g)and cultured alone or in the presence of human umbilical vein endothelial cells(HUVECs)in a Transwell co-culture system.The expressions of EpiCs markers WT1 and Tbx18 and the smooth muscle cell markersα-SMA and Myh11 were detected using immunofluorescence assay and qRT-PCR.Collagen gel contraction assay was used to assess the contractility of the cultured EpiCs,and ELISA was used to detect the expression of growth factors in the cell culture medium.Results Compared with EpiCs cultured alone,the EpiCs co-cultured with HUVECs exhibited significantly increased expression levels ofα-SMA[(1.73±0.85)%vs(34.20±3.38)%,P<0.01,n=3]and Myh11[(3.70±1.49)%vs(45.63±4.85)%,P<0.01,n=3]with obviously enhanced contractile ability.The concentration of basic fibroblast growth factors(bFGFs)in the conditioned medium was also significantly higher in the co-cultured cells than in EpiCs cultured alone(304.96±52.91 vs 87.64±8.78 pg/mL,P<0.01,n=3).Conclusion In the co-culture system of EpiCs and HUVECs,the production of bFGFs by HUVECs promotes the differentiation of EpiCs into vascular smooth muscle cells,indicating a regulatory mechanism of the development of coronary arteries.