【摘要】 目的:建立人血管内皮生长因子165(hVEGF165)基因转染大鼠骨髓间充质干细胞的方法。方法:采用密度梯度离心-贴壁培养法获Wistar大鼠骨髓间充质干细胞(MSCs),测定其生长曲线和表面标志CD34,CD44,CD45及SH3,检测其向成骨细胞及脂肪细胞诱导分化潜能后;用脂质体介导法将pcDNA3.1-hVEGF165导入MSCs,观察转染后细胞形态和生长状况,通过RT-PCR,Western blot和ELISA鉴定VEGF在MSCs中的表达情况。结果:大鼠MSCs经检测为CD44和SH3阳性,CD45和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转染至大鼠MSCs,并获得有效的表达。结论:真核表达载体pcDNA-VEGF在MSCs中获有效表达。更多还原

【Abstract】 Objective To create a method for transfecting human vascular endothelial growth factor 165 （hVEGF 165 ） gene into bone marrow mesenchymal stem cells （MSCs） in rats. Methods MSCs of Wistar rats were isolated by density gradient centrifugation and purified based on their ability of adhesion to plastic. Detections of cell surface antigens, including CD34, CD45, CD44, and SH3, were performed using flow cytometry. MSCs’ potential of differentiating into osteoblast and lipoblast in vitro was tested. The vector pcDNA 3.1 -hVEGF 165 was transfected into MSCs with the liposome mediated method. The expression of hVEGF 165 in the transfected cells was detected by enzyme linked immunosorbent assay （ELISA）, reverse transcription-polymerase chain reaction （RT-PCR）, and Western blot analysis. Results The cultured MSCs were CD34 - , CD45 - , CD44 + , and SH + , which were differentiated into osteoblasts and lipocytes successfully. The expressed hVEGF 165 in the transfected rat MSCs was demonstrated. Conclusion The vector pcDNA₃.1 -hVEGF₁65 is successfully expressed in MSCs.更多还原