腺病毒载体介导人BMP-2与VEGF-165对骨髓间充质干细胞形态特征及增殖能力影响的实验研究

The experimental research of bone marrow mesenchymal stem cell morphology and proliferation transfected by adenovirus vector co-expressing BMP-2and VEGF-165

目的探讨携带骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)与血管内皮生长因子165(vascular endothelial growth factor 165,VEGF-165)双基因的腺病毒载体转染骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)后对细胞形态特征及增殖能力的影响。方法选取4只新西兰大白兔抽取骨髓液,分离鉴定获得第3代BMSCs做为实验对象,实验分成4组:Ad-BMP-2-VEGF-165转染组(A组);Ad-BMP-2转染组(B组);AdVEGF-165转染组(C组);BMSCs对照组(D组),每组6个复孔,连测3次。首先,将腺病毒载体转染BMSCs,获得最佳感染复数(multiplicity of infection,MOI);其次,转染后1-3d观察转染各组细胞与D组形态和贴壁状况差异变化;再次,ELISA法检测转染各组目的蛋白表达量与D组的差异;最后,MTT法测定1-7d各组细胞增殖曲线,并进行组间比较。结果 BMSCs分离鉴定成功,第3代细胞高表达CD29(99.82%),CD44(94.14%)抗原。腺病毒转染组最佳MOI值分别为A组MOI=80,B组MOI=80,C组MOI=100。腺病毒转染后各组细胞形态和贴壁特性未见明显改变,边界清晰,细胞呈梭形,细胞核较大呈圆形或椭圆形,生长旺盛。转染组细胞高表达目的蛋白,在第5d目的蛋白浓度到高峰,A组BMP-2浓度为1525.47±58.16pg/ml,B组为1506.48±92.65pg/ml;A组VEGF-165浓度为1732.14±126.25pg/ml,C组为1733.59±127.07pg/ml,转染组在不同时间点的目的蛋白表达量与D组差异均具有统计学意义(P<0.05)。MTT法显示第1d、第2dBMSCs未见明显增长,第3d开始呈指数增长,第5d达高峰;第3d,A组、B组吸光度(optical density,OD)值分别为0.113±0.004、0.112±0.003,均高于C组、D组(P<0.05);第4d,A、B、C各组OD值分别为0.113±0.004、0.110±0.003、0.105±0.001,均高于D组0.102±0.004,且差异有统计学意义(P<0.05)。结论 BMP-2与VEGF-165对BMSCs形态和贴壁性无明显影响,但是均可以促进BMSCs增殖,且两者对细胞的增殖无明显叠加作用。

Objective To explore the effect of BMSCs morphology and proliferation transfected by adenovirus vectors co-expressing BMP-2 and VEGF-165. Methods Four New Zealand white rabbits were selected to extract marrow, and the third generation of BMSCs was been experimental subjects after separation and identification. The experiment subjects were divided into four groups, i. e., Ad-BMP-2VEGF-165 transfect group （group A）, AdBMP-2 transfect group （group B） ,Ad-VEGF-165 transfect group （group C） ,the control group of BMSCs （group D）. First,Appling adenovirus vector to transfect BMSCs,we ohserve the multiplicity of infection. Second,the morphology of each groups will be observed when they were transfected at 1-3 days. Third,the ELISA was selected to detect interest proteins expression and the differences in the group D. In the end, each cells proliferation curve determined by MTT method at 1 7 days and compared the difference of OD between groups, Results BMSCs were successfully isolated and identified and the third generation cells high expressed antigen of CD29 （99.82 %） and CD44 （94.14 %）. The good MOI of adenovirus transfection group is group A （ MOI = 80）,group B （MOI = 80）,group C（MOI= 100）,respectively. The morphology and adherence of transfect cells features were not seen obvious change, clear boundary, spindle morphology,larger nucleus,higher proliferation. The transfect groups expressed higher concentration of interest proteins and protein concentration goes to the peak at the 5 d. The BMP-2 concentration was 1525.47 ± 58. 16 pg/ml （group A）, 1506.48 ± 92. 65 pg/ml （group B）,respectively. The VEGF-165 concentration was 1732. 14± 126.25 pg/ml （group A）,1733.59±127.07 pg/ml （group C）, respectively. There were statistical difference between transfect groups and D group of interest proteins expression at different time （P〈0.05）. At 4 days, there were significant statistical difference between OD values of groups A （0. 113±0. 004） ,B （0. 110±0. 003）and C （0. 105±0. 001） and group D （0. 102±0. 004）（P〈 0. 05）. Conclusion The interest proteins of BMP-2 and VEGF-165 have not significantly affected on the morphology and adherence of BMSCs,but they are able to promote BMSCs proliferation and the effect of proliferation on cells does not accumulate in the dual-proteins culture medium.