携带人内皮抑素基因的重组腺相关病毒感染对人脐静脉内皮细胞增生的影响

Effect of recombinant adeno-associated virus-mediated human endostatin gene infection on human umbilical vein endothelial cells proliferation

目的评价携带人内皮抑素(endostatin,ES)基因的重组腺相关病毒(rAAV)感染对人脐静脉内皮细胞(ECV304细胞)增生的影响。方法以不同感染复数的rAAV-ES感染ECV304细胞,Western blotting检测细胞裂解液中ES蛋白的表达,ELISA法检测ES蛋白的分泌情况,流式细胞仪分析感染后ECV304的细胞周期,MTT法检测ECV304增殖情况。结果感染了rAAV-ES的ECV304细胞可表达并分泌ES蛋白,表达高峰在感染后96h,表达量为96.93μg·L-1;感染后的ECV304细胞增殖受到抑制,流式细胞仪分析其周期中G0与G1期细胞比例明显增加,S期细胞比例显著减少;MTT检测显示以感染复数为5×105vg.cell-1感染96h时对ECV304细胞增殖的抑制率最高(54.54±1.37)%。结论 rAAV-ES感染ECV304细胞后有高水平的ES蛋白表达并能显著抑制ECV304细胞的增殖。

Objective To evaluate the effect of recombinant adeno-associated virus-mediated endostatin gene（rAAV-ES） infection on human umbilical vein endothelial cells（ECV304 cells）proliferation.Methods rAAV-ES was used to infect ECV304 cells at different multiplicities of infection.ES expression in the cell lysate was analyzed by Western blotting.The concentration of ES protein in the supernatant was assessed with ELISA.Cell cycle and cell proliferation of infected ECV304 cells were assessed by flow cytometer and MTT method,respectively.Results The ES expression could be found in ECV304 cells infected by rAAV-ES,went to peak at 96 hours after infection and the expression level was 96.93 μg·L-1.The proliferation of ECV304 cells was inhibited after infection.Flow cytometer showed that cell ratio increased in G0 phase and G1 phase,decreased significantly in S phase.MTT method showed that the highest inhibitory rate on ECV304 cell proliferation was（54.54±1.37）% at 96 hours after being infected with rAAV-ES at multiplicity of infection of 5×105 vg per cell.Conclusion There are high level of ES expression and significant inhibition on ECV304 cells proliferation after rAAV-ES infection.