腺病毒介导血管平滑肌细胞转染表达p27基因的研究

目的体外培养大鼠血管平滑肌细胞(VSMC)并以腺病毒介导转染表达p27。方法取大鼠主动脉血管,以常规组织块贴壁法培养VSMC。用位置特异性重组方法构建带p27基因的复制缺陷型腺病毒载体(AdCMV-p27),经鉴定、扩增,以获取高滴度AdCMV-p27转染液,体外转染VSMC。用RT-PCR方法检测p27 mRNA表达,免疫细胞化学法检测p27蛋白表达,流式细胞仪检测p27细胞表达率。结果构建的AdCMV-p27体外转染培养VSMC表达率为85.81%。以免疫细胞化学、流式细胞仪和RT-PCR检测p27表明,转染后p27表达明显增强,并随表达时间延长而增加,24h达峰值。结论腺病毒载体可较高效率介导p27在体外培养的VSMC转染表达。转染表达p27的VSMC可作为研究有丝分裂原对其增殖、迁移以及凋亡等生物学行为影响的良好细胞模型。

Study of transfer and expression of p27 gene in rat vascular smooth muscle cells mediated by recombinant adenovirus vector

Objective To construct the cell model transferred p27 in vascular smooth muscle cells(VSMCs).Methods The VSMCs,isolated from the aorta of rat,were cultured by routine method.Recombinant adenoviral vector,AdCMV-p27,containing rat p27 gene was constructed by site special recombination,and then it was used to transfer p27 gene to VSMCs in vitro.The rate of p27 expression in VSMCs was analysed by flow cytometry.The expression of mRNA and protein were detected by RT-PCR and immunocytochemistry,respectively.Results The expression rate of p27 in VSMCs was increased significantly after transferred by AdCMV-p27 with the time and the peak value detected by flow cytometry was about 85.81% at 24h.RT-PCR and immunocytochemistry showed that the expression of p27 mRNA and protein were increased obviously in transfected cells.Conclusion AdCMV-p27,generates high level expression of p27 in cultured VSMCs.The VSMCs transfected with p27 gene may be used as a good model to study the effect of p27 on their biological action such as proliferation,migration and apoptosis induced by mitogen.