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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。
OBJECTIVE: To investigate the effect of human Gax gene overexpression on the proliferation and apoptosis of rabbit VSMCs stimulated by adenovirus after transfection of rabbit VSMCs with adenovirus-mediated termination of specific homeobox gene (Gax) It provides an experimental basis for Gax gene to be an ideal gene candidate for venous bridge restenosis. Methods: Ad-hGax recombinant adenovirus vector was transfected into rabbit VSMCs, Western blot was used to detect the expression of hGax protein at different time points. The inhibition of Ad5-hGax on the proliferation of rabbit VSMCs stimulated with Ad5-hGax was detected by MTT assay. The apoptosis rate of VSMCs induced by Ad5-hGax-induced serum was detected by flow cytometry. Results: The hGax protein in Ad5-hGax group was obviously expressed at 1 d, 3 d and 5 d after transfection. The MTT assay showed that hGax gene overexpression significantly inhibited the proliferation of VSMCs stimulated by serum. Flow cytometry Histological examination showed that hGax overexpression significantly induced apoptosis of rabbit VSMCs after 72 h of transfection. CONCLUSION: Overexpression of hGax can effectively inhibit the proliferation of VSMCs stimulated by serum and promote its apoptosis, which provides an important experimental basis for adenosine-mediated Gax gene therapy of venous bridge restenosis.