反义TR和p21双基因共表达对人主动脉平滑肌细胞增殖与凋亡的作用

Effect of Antisense Thrombin Receptor and p21 Double Gene Co-expression System on the Proliferation and Apoptosis in Human Aortic Smooth Muscle Cells

目的观察携带反义凝血酶受体(ATR)或/和p21的单、双基因重组腺病毒伴随病毒(rAAV)载体对人主动脉平滑肌细胞(ASMC)增殖与凋亡的影响。方法以携带ATR或/和p21的单、双基因rAAV感染人ASMC。半定量RT-PCR检测各基因的整合与表达;MTT法测定感染不同时间点的细胞存活率;流式细胞术检测细胞周期与凋亡细胞数目的变化;吖啶橙/溴化乙锭(AO/EB)染色检测凋亡细胞的比率。结果单、双基因在ASMC中均获得整合,且双基因发生了共表达;病毒感染4d时,ATR组、p21组和AP双基因组的细胞存活率与对照组相比分别降低了16.7%、21.6%和29.4%;三组的G0/G1期细胞数分别为(61.8±2.9)%、(82.5±4.0)%和(80.4±6.1)%;凋亡细胞数为(4.8±0.5)%、(5.7±0.1)%和(9.2±0.9)%;AO/EB染色显示的凋亡细胞比率分别为:对照组(1.5±0.8)%、ATR组(7.2±3.3)%、p21组(10.7±5.6)%、AP组(18.3±2.7)%。结论(1)双基因共表达对抑制细胞增殖和诱导凋亡的作用均较单基因具有较强的生物学效应。(2)为再狭窄的基因治疗提示了更优化的途径。

Objective To focus on the study of the effect on proliferation and apoptosis of human aortic smooth muscle cells (ASMC)by adeno-associated virus(AAV)vector carrying antisense thrombin receptor (ATR)and p21double gene co-expression system.Methods Cultured human AMSC was infected with recombinant AAV containing ATR,p21single gene and AP double gene respectively.The integration and expression of genes were confirmed by semi -quantitative RT-PCR.The antiprolif-eration effect was determined by MTT assay.Cell cycle and apoptotic cell counts were measured through Flow Cytometry.The rate of apoptotic cells was examined with acridine orange/ethidium bromide(AO/EB)stain.Results RT-PCR indicated that the exogenous genes had been integrated into ASMC.The rates of cell survival were decreased by16.67%,21.60%,and29.4%and the cell counts of G 0 /G 1 phase were(61.8±2.9)%,(82.5±4.0)%,(80.4±6.1)%in ATR,p21and AP group respectively after rAAV infected4days.The level and area of apoptotic peak were greater in AP double gene than ATR and p21single gene.Cell stain indicated that apoptotic cells were(7.2±3.3)%,(10.7±5.6)%,and (18.3±2.7)%in each transgene group compared with(1.5±0.8)%in control group.Conclusion AP double gene co-expression system has powerful effect for inhibiting proliferation and inducing apoptosis ASMC than ATR and p21single gene and that is a superior way for gene therapy to restenosis.