摘要
目的通过观察人骨髓间充质干细胞(hMSCs)与成熟内皮细胞非接触共培养表达特异性内皮细胞标志物的情况,探讨微环境依赖性的、体外非接触共培养方法对MSCs向内皮样细胞分化的影响。方法密度梯度离心法分离纯化培养的hMSCs与人脐静脉内皮细胞(HUVECs)在Transwell内共培养,通过流式细胞术检测hMSCs表型,RT-PCR鉴定诱导后hMSCs的CD31、VWF、VE-Cadherin mRNA的表达,免疫组化鉴定诱导后hMSCs特异性内皮细胞标志物VCAM1和CD31的表达,透射电镜观察诱导后hMSCs的超微结构。结果细胞培养第2周,开始出现形态学改变,胞体回缩,呈多角形改变。细胞培养第3周,细胞增生速度明显加快,形态学上呈卵圆形或"铺路石"样改变;在基因水平上,RT-PCR显示经典内皮细胞标志物CD31、VWF、VE-cadherin mRNA的高表达;在蛋白水平上,免疫荧光染色CD31、和VCAM1表达阳性;透射电镜下诱导后细胞显示内皮细胞特有的Weibel-Palade小体。结论 hMSCs在共培养的微环境中,可以分化成内皮细胞表型,其机制是通过内皮细胞的旁分泌作用,而不需要hMSCs与内皮细胞的直接接触。转化的内皮细胞在形态上、基因、蛋白表达、超微结构等方面均显示了内皮细胞的特征。
Objective To investigate the microenviroment - dependent endothelial differentiation of hMSCs in vitro. Methods hMSCs were isolated from bone marrow undergoing by density gradient centrifugation. Human umbilical vein endothelial cells （HUVECs） were harvested from fresh umbilical cords by collagenase treatment. We established the endothelial differentiation environment by co - culturing hMSCs with mature endothelial cells （HUVECs） indirectly in vitro. CD31 , VWF, VE - Cadherin mRNA expression were detected after induction by RT -PCR analysis and VCAM1 and CD31 expression by fluorescence immunoeytochemistry. The endothelial characteristic Weibel - Palade bodies of the differentiated hMSCs were observed by electron microscopy. Results In Transwell co - culture system, the induced hMSCs showed little change after 1 week, but their bodies contracted and showed polygonal - shaped morphology after 2 weeks. The differentiated hMSCs showed positive expression of CD31 ,VWF, and VE - cadherin mRNA by RT - PCR analysis and showed positive CD31 and VCAM1 expression by fluorescence immunoeytochemistry. Electron microscopy analysis of the differentiated hMSCs showed the typical endothelial Weibel - Palade body. Conclusion The expression of mature endothelial cell - specific markers both at mRNA and protein levels, and endothelial characteristic Weibel - Palade body from electron microscopy confirm that hMSCs has the milieu -dependent differentiation potential along endothelial lineage.
出处
《医学研究杂志》
2010年第8期34-39,142,共7页
Journal of Medical Research
基金
"973"中医专项课题(2005CB523305)
广东省科技计划立项资助课题(0711222700104)
广东省中医药局立项课题(2007380)
