双基因共表达在血管再狭窄研究中的应用

目的 观察携带反义凝血酶受体(ATR)和p21的双基因载体共表达后对血管平滑肌细胞增殖的抑制作用，为再狭窄基因治疗寻求新途径。方法 以携带ATR或/和p21的单、双基因载体重组腺病毒伴随病毒(rAAV)感染培养的人主动脉平滑肌细胞(hASMC)，用半定量RT-PCR检测各基因的整合与表达，MTT法测定病毒感染后不同时间点的细胞存活率。将携带AP双基因的rAAV导入WKY大鼠拉伤侧的颈总动脉，免疫组织化学法分别检测凝血酶受体(TR)和p21两基因在动脉壁中的表达。结果 RT-PCR结果显示，TR单基因的mRNA表达降低，p2l单基因表达升高，AP双基因得到了共表达；MTT法测定的生长曲线显示，双基因对hASMC增殖的抑制作用大于两个单基因；免疫组织化学证实，AP双基因导入后，TR基因的表达受到完全抑制，p2l基因的表达明显增加，血管新生内膜与平滑肌细胞的增殖受到了抑制。结论 ATR和p21双基因共表达在体外可明显抑制ASMC的增殖，在体内可明显抑制血管内膜新生和中膜的增生。

THE APPLICATION OF DOUBLE GENE CO-EXPRESSION IN VESSEL RESTENOSIS STUDY

Objective To study the inhibiting effect of antisense thrombin receptor (ATR) and p21 double gene Co expression system on vessel smooth muscle cells (SMC) and searching a better way for restenosis gene therapy. Methods Cultured human aortic SMCs were infected with recombinant AAV(rAAV) containing ATR, p21 single gene or AP double gene respectively. The integration and expression of genes were confirmed by semiquantitative RT PCR. The cell survival rates were determined by MTT assay. According to the experiment outcome in vitro, rAAV carrying AP double gene was delivered into the balloon injured carotid arteries of WKY rats. The expressions of TR and p21 genes in aortic wall were examined respectively by immunohistochemical staining. Results RT PCR indicated that the exogenous genes had been integrated into aortic SMC. The curve of cell survival showed that the inhibiting proliferation effect of AP double gene on aortic SMC was stronger than that of ATR or p21 single gene. The expression of TR was inhibited and expression of p21 increased in carotid arteries wall, meanwhile, the proliferation of intimal and SMCs in medium can be inhibited markedly.Conclusion These date suggested that AP double gene Co expression system has most powerful effect for inhibiting SMC proliferation in vitro and in vivo.