CBP基因沉默抑制血管平滑肌细胞增殖研究.docVIP

CBP基因沉默抑制血管平滑肌细胞增殖的研究 　　作者：魏云杰,江洪,陈静,张静,徐昌武　　作者单位：武汉大学人民医院心血管内科，湖北 武汉　430060 　　【摘要】目的：探讨RNA干扰大鼠CREB结合蛋白(rCBP)基因对大鼠血管平滑肌细胞(VSMCs)增殖的影响。方法：根据CBP cDNA序列构建可同时表达4条针对rCBP mRNA特异的短发夹RNA(shRNA)、携带绿色荧光蛋白的腺病毒(CBP-shRNA/Ad)。以空腺病毒为转染对照组、含非特异性shRNA编码序列的腺病毒为阴性对照组、感染复数为12、16、25、50的CBP-shRNA/Ad为干预组，转染 0.1 U/mL凝血酶预处理的VSMCs 2 d。RT-PCR法、蛋白印记法分别检测rCBP mRNA和蛋白质的表达变化。流式细胞技术检测细胞周期，评价细胞增殖能力。倒置显微镜观察CBP-shRNA/Ad对细胞的毒副作用。结果：不同剂量的CBP-shRNA/Ad可在mRNA和蛋白质水平，明显抑制凝血酶诱导CBP高表达(P0.05)，具有量效依赖性。CBP-shRNA/Ad通过下调CBP含量，可使G0/G1期细胞比例显著升高(P0.05)，S期细胞比例下降(P0.05)，细胞周期被阻滞。倒置显微镜观察细胞贴壁状况良好。结论：RNA干扰技术可通过选择性下调rCBP的表达，显著抑制VSMCs的增殖，无明显细胞毒性。 　　【关键词】 血管平滑肌细胞;RNA干扰;环磷腺苷反应原件结合蛋白结合蛋白;基因治疗 　　[ABSTRACT] Objective： To explore the effect of CREB binding protein (rCBP) gene-silencing on vascular smooth muscle cells (VSMCs) proliferation by RNA interference. Methods： According to the CBP cDNA sequence， adenovirus with green fluorescent protein was constructed which could encode specific short hairpin RNA (shRNA) of rCBP mRNA (CBP-shRNA/Ad). Empty adenovirus was taken as transfection control group， adenovirus with non-specific shRNA coding sequence as negative control group， and CBP-shRNA/Ad with multiplicity of infection 12， 16， 25 and 50 as intervention groups， VSMCs 2d primed by 0.1U/mL thrombin was transfected. Then the change of CBP mRNA and protein were detected by RT-PCR and western blot; cell cycle was examined by flow cytometry to evaluate VSMCs proliferation. The adverse effect of CBP-shRNA/Ad on VSMCs was investigated by invert microscope. Results： CBP-shRNA/Ad could obviously inhibit CBP high-expression induced by thrombin at mRNA and protein level(P0.05) in dose-dependence. CBP-shRNA/Ad increased cells rate in G0/G1 phase (P0.05)and decreased cells rate in S phase(P0.05)via reduction of CBP content. Results of invert microscope showed cells had the good adherence ability. Conclusions： RNA interference could down regulate rCBP expression selectively， inhibit VSMCs proliferation apparently， without obvious