Real-Time PCR检测间充质干细胞源性内皮细胞eNOS、Tie-2基因表达

Real-time quantitative RT-PCR for measurement of expression of eNOSmRNA and Tie-2mRNA in BMSCs-derived Endothelial Cells

[目的]检测骨髓间充质干细胞(BMSCs)源性内皮细胞功能基因表达水平,为骨组织工程血管化奠定基础。[方法]采用全骨髓贴壁法对BMSCs进行体外培养、纯化和扩增,用含有VEGF(10 ng.ml-1)和bFGF(2 ng.ml-1)的诱导培养液对其进行体外诱导分化3周,通过透射电镜观察内皮细胞特异性W-P小体鉴定细胞性质。以主动脉内皮细胞(VECs)为阳性对照,采用实时定量PCR(RT-qPCR)检测BMSCs源性内皮细胞一氧化氮合酶(eNOS)与酪氨酸激酶受体(Tie-2)mRNA的相对表达量。[结果]分化后的细胞在光镜下具有内皮细胞的形态学特征;透射电镜观察显示诱导后的细胞内出现内皮细胞特异性W-P小体。RT-qPCR检测显示:实验组Tie-2mR-NA的相对表达量为0.785,eNOS mRNA的相对表达量为0.747,二者与阳性对照组目的基因的表达量没有明显差异(P>0.05)。[结论]BMSCs能够成功体外分化为内皮细胞,且具有成熟内皮细胞功能基因的表达,是构建血管化组织工程骨的理想种子细胞。

[ Objective] To detect the functional gene expression of endothelial cells derived from the bone marrow mesenchymal stem cells （BMSCs） , so as to provide the promising strategies for the establishment of vaseularized tissue - engineered bone. [ Methods] BMSCs were cultured, purified, and expanded by bone marrow cell adherence in vitro. They were induced and differentiated in the medium of 10ng·ml^-1 VEGF and 2 ng·ml^-1 bFGF for 3 weeks. The differentiated cells were identi- fied for Weible -palade corpuscle in the cytoplasm by transmission electron microscopy. The relative gene expression of eNOS and Tie - 2in the BMSCs - derived endothelial cells were detected by real - time quantitative reverse transeriptase PCR （ RT - qPCR ） , with the vascular endothelial cells as the positive controls. [ Results ] The differentiated cells demonstrated the characters of endothelial cells under phase contrast microscope. W - P corpuscles could be seen under transmission electron microscope. RT -qPCR analysis showed that the differentiated cells expressed Tie -2 mRNA （0. 785 ） and eNOS mRNA （0. 747 ） in the trial group, but no difference was seen in BMSCs - derived endothelial or vascular endothelial cells （ P 〉 0. 05 ） . [ Conclusion] BMSCs could be successfully differentiated into endothelial ceils in vitro, and the functional genes were stably expressed in BMSCs - derived endothelial cells. They are ideal donor cells of tissue engineering vascularization.