腺病毒介导Gax基因对血清刺激后兔血管平滑肌细胞增殖和凋亡的影响

目的:通过腺病毒介导人生长终止特异性同源异形盒基因(Gax)转染体外兔血管平滑肌细胞(VSMCs),观察人Gax基因过表达对血清刺激后兔VSMCs增殖和凋亡的影响,为Gax基因成为理想的静脉桥再狭窄基因治疗的候选基因提供实验基础。方法:Ad5-hGax重组腺病毒载体转染兔VSMCs后,采用Western blot检测不同时间细胞中hGax蛋白的表达;MTT法检测Ad5-hGax对血清刺激后兔VSMCs的体外增殖抑制情况;转染72 h后流式细胞术检测Ad5-hGax诱导血清刺激后VSMCs的凋亡率。结果:转染后1 d、3 d、5 d,Ad5-hGax组细胞中的hGax蛋白均有明显表达;MTT法检测显示hGax基因过表达能明显抑制血清刺激后兔VSMCs的增殖;流式细胞术检测显示转染72 h后,hGax过表达能显著诱导血清刺激后兔VSMCs的凋亡。结论:hGax基因过表达能有效抑制血清刺激后VSMCs的增殖,并促进其凋亡,为腺病毒介导Gax基因治疗静脉桥再狭窄提供重要的实验基础。

Effects on proliferation and apoptosis of serum-induced rabbit VSMCs by adenovirus-mediated transfer of the Gax gene

AIM:To investigate the effect of a recombinant replication-incompetent Ad5-hGax vector on the proliferation and apoptosis of serum-induced rabbit vascular smooth muscle cells(VSMCs) in vitro,and to provide an important support that Gax would be an optimal gene for gene therapy in vein graft failure.METHODS:The Ad5-hGax vector was infected into rabbit VSMCs,then the protein level of rabbit VSMCs was detected at 1 d,3 d,5 d by Western blot,respectively.MTT was applied to observe the inhibitory effects of overexpressed hGax on serum-induced rabbit VSMCs.the apoptosis of serum-induced rabbit VSMCs was measured by using flow cytometry in 72 h after transfection.RESULTS:The protein expression of human Gax in the Ad5-hGax transfected cells on 1st day,3rd day,5th day was determined;MTT showed that the proliferation of serum-induced rabbit VSMCs was significantly inhibited in Ad5-hGax group at 48 h,72 h and 96 h(P<0.05,respectively);After transfecting for 72 h,flow cytometry analysis showed that the number of the apoptosis cells was increased in serum-induced Rabbit VSMCs of Ad5-hGax group(P<0.05).CONCLUSION:Overexpressed hGax could inhibit the proliferation of serum-induced Rabbit VSMCs and induce their apoptosis.These findings carry significant implications for adenovirus vector-based Gax gene therapies for vein graft failure.