人VEGF基因转染大鼠骨髓间充质干细胞的实验研究

Experimental Study of the Transfection of Human VEGF165 Gene into Rat Bone Mes enchymal Stem Cells in Vitro

为建立hVEGF165基因转染大鼠间充质干细胞的方法.采用密度梯度离心-贴壁培养法获Wistar大鼠BMMSC,并测定其生长曲线和表面标志CD34、CD44、CD45及SH3,然后向成骨细胞及脂肪细胞诱导分化;用脂质体介导pcDNA3.1-hVEGF165转染BMMSC,观察转染后细胞形态和生长情况的变化,通过RT-PCR、Western和ELISA鉴定VEGF在细胞中的表达情况.经培养的大鼠BMMSC,CD44、SH3检测为阳性,CD45、CD34阴性,可诱导分化为成骨细胞和脂肪细胞;经RT-PCR、Western和ELISA检测证实阳离子脂质体能成功地将hVEGF165基因转染至大鼠BMMSC中,并获得有效的表达.真核表达载体pcDNA3.1-hVEGF165在BMMSC中获有效表达,为VEGF基因转染BMMSC移植对心梗后大鼠心功能及心室重构的影响提供了实验依据.

In order to develop a technique to transfect hVEGF165 gene into bone marrow mesenchymal stem cells in rats, BMMSC of Wistar rat was isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic. Detections of cell surface antigens, including CD34, CD45, CD44 and SH3, were performed using flow cytometry. BMMSC was tested the potentiality of differentiating into osteoblast and adipoblast in vitro. The vector pcDNA3.1-hVEGF165 was transfected into BMMSC with the method of liposome mediated. The expression of human VEGF165 in the transfected cells was detected by RT-PCR, ELISA and Western blotting analysis. The cultured BMMSCs were CD34^-, CD45^-, CD44^＋ and SH^＋. They can differentiate into osteoblast and adipocytes successfully. The expression of human VEGF165 in the transfected rat BMMSC was demonstrated by RT-PCR, ELISA and Western blotting analysis. The experiment demonstrated that hVEGF165 had successfully expressed in BMMSC.