血管内皮细胞生长因子在兔骨髓基质干细胞内的表达及影响

Expression and effect of vascular endothelial growth factor in rabbit mesenchymal stem cells

目的:构建血管内皮细胞生长因子pcDNA3/hVEGF165真核表达载体,并用脂质体法转染骨髓基质干细胞(mesenchymalstemcells,MSCs),观察血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)转染后对骨髓MSCs的生长、转化的影响。方法:采用贴壁法分离骨髓MSCs,脂质体法转染。100g/L胎牛血清DMEM培养MSCs,计数法绘制生长曲线,特殊染色观察MSCs的转化,免疫组织化学法观察转染后VEGF的表达。结果:转染了pcDNA3/hVEGF165的骨髓MSCs胞浆内出现VEGF阳性颗粒,而转染了pcDNA3组及未转染组中未出现阳性颗粒,生长曲线在3组中无明显差别。特殊染色出现的时间亦无明显差别。而且,注射有转染了pcDNA3/hVEGF165的MSCs的大白兔局部皮下血管数量增加,其他两组未发现。结论:本实验成功构建了pcDNA3/hVEGF165真核表达载体并能够在MSCs细胞内表达VEGF,其表达产物具有血管内皮增殖刺激活性。

AIM:To construct eukaryotic expression vector carrying pcDNA3/hVEGF vascular endothelial grouth factor 165 and transfect it into mesenchymal stem cells (MSCs). Then to observe the change of growth and proliferation of MSCs after transfection.METHODS:Rabbit bone morrow cells were cultured in Dulbecco's Modified Eagles Medium with low glucose containing 10%fetal bovine serum.Then the expression vectors of pcDNA3/h VEGF165 and pcDNA3 were transfected into MSCs.After about 14 days, the MSCs was divided into three teams, one was transfected by pcDNA3/h VEGF165, one by pcDNA3, one by nothing.On the 1 st,2 nd,4 th,7 th,14 th,21 st,28 th flow immunohistochemical analysis was performed to examine the expression of cell plasma and samples were assayed for alkaline phosphatase ALP histochemistry and for mineral deposition by Von Kossa staining respectively.RESULTS:The transfectant of pcDNA3/h VEGF165 expressed VEGF protein in cell plasma, the others didn't.ALP and depositing acalicified matrix showing positive on the 21 st and 28 th were not different significantly among the three groups after transfection.CONCLUSION:The transfectant doesn't affect the MSCs growth and proliferation, and the transfectant of MSCs expresses VEGF protein and induce vascular growth.