摘要：
Objective To observe the influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2 (BMP2)-vascular endothelial growth factor 165 (VEGF165) in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods The isolated rabbit BMSCs were used as the research objects,and the recombinant plasmid pIRES-BMP2-VEGF165 was transfected into cells in the presence of liposomes.Then,G418 was used to screen stable cell lines in vitro and mRNA and protein levels were detected for transfection efficiency.Stable cell lines were then cultured and the cells were divided into pIRES group,pIRES-BMP2-VEGF165 transfection group and osteogenic induction group.Western blotting was used to detect the expression of Collagen Ⅰ (Col Ⅰ) and osteocalcin (OCN) protein at 7th and 21st day after cultivation,respectively.After 14 days of cultivation,gomori modified calcium and cobalt staining was used to detect alkaline phosphatase activity.After 21 days of cultivation,calcified nodules of cells were detected by alizarin red method calcium staining.Results Compared with the empty plasmid pIRES group,the expression of BMP2 and VEGF165 was detected at the mRNA and protein levels of pIRES-BMP2-VEGF165 plasmid group.Compared with empty plasmid group and osteogenic induction group,the expression levels of Col Ⅰ and OCN protein in recombinant plasmid group were significantly increased (Col Ⅰ:0.597 ±0.040 vs.0.230 ± 0.020,P =0.000;0.597 ± 0.040 vs.0.457 ± 0.035,P =0.005;OCN:0.433 ± 0.038 vs.0.083 ± 0.021,P =0.000;0.433 ±0.038 vs.0.350 ±0.036,P =0.046).Positive relative area (％) of alkaline phosphatase staining increased significantly (69.000 ± 5.568 vs.18.333 ± 3.056,P =0.000;69.000 ± 5.568 vs.43.333 ± 5.508,P =0.002).Positive relative area (％) of calcified nodules increased markedly (37.747 ±3.763 vs.1.587 ±0.972,P =0.000;37.747 ±3.763 vs.19.330 ±4.033,P =0.008).Conclusion Human recombinant eukaryotic expression plasmid pIRES-BMP2-VEGF165 was successfully transfected into BMSCs.As compared with osteogenic induction group,the recombinant plasmid pIRES-BMP2-VEGF165 was more effective in osteogenic differentiation of the cells.%目的 观察人重组真核表达质粒pIRES-骨形态发生蛋白2(BMP2)-血管内皮生长因子165 (VEGF165)的体外转染对兔骨髓间充质于细胞成骨分化的影响.方法 以原代分离的兔骨髓间充质干细胞为研究对象,在脂质体的介导下将重组质粒pIRES-BMP2-VEGF165转染至细胞中,采用G418体外筛选稳转细胞株,且在mRNA和蛋白水平上分别检测转染效果.继续培养稳转细胞株,细胞分:空质粒pIRES组、pIRES-BMP2-VEGF165转染组以及成骨诱导剂组;采用Western blot分别检测培养7d和21 d后各组细胞Ⅰ型胶原蛋白(Col Ⅰ)和骨钙素(OCN)蛋白表达水平;培养14 d后,采用Gomori改良钙钴法染色检测碱性磷酸酶活性;培养21 d后,采用茜素红法钙质染色检测各组细胞钙化结节情况.结果 与空质粒pIRES组比较,pIRES-BMP2-VEGF165质粒组细胞在mRNA和蛋白水平上均检测到骨形态发生蛋白2和VEGF165的表达;与空质粒组及成骨诱导剂组比较,重组质粒pIRES-BMP2-VEGF165组细胞Col Ⅰ和OCN蛋白的表达水平均显著提升(Col Ⅰ:0.597±0.040比0.230 0.020,P =0.000;0.597 ±0.040比0.457 ±0.035,P =0.005;OCN:0.433 0.038比0.083 ±0.021,P=0.000;0.433±0.038比0.350±0.036,P=0.046);碱性磷酸酶染色阳性区域占比均显著增加[(69.000±5.568)％比(18.333±3.056)％,P=0.000;(69.000±5.568)％比(43.333 ±5.508)％,P=0.002],钙化结节阳性区域占比均明显增加[(37.747±3.763)％比(1.587±0.972)％,P=0.000;(37.747±3.763)％比(19.330 ±4.033)％,P=0.008].结论 人重组真核表达质粒pIRES-BMP2-VEGF165成功转染至兔骨髓间充质干细胞,且与成骨诱导剂比较,重组质粒pIRES-BMP2-VEGF165对该细胞的成骨分化能力有更明显的促进作用.
