腺病毒介导p27基因转染对离体血管平滑肌细胞增殖的影响

Effect of the transfer and expression of p27 gene on the proliferation of rat vascular smooth muscle cells mediated by recombinant adenovirus vector

目的探讨p27基因转染对血管平滑肌细胞（VSMC）增殖的影响及其机制。方法通过构建携带p27基因的腺病毒并转染培养大鼠VSMC，增强其p27的表达，以免疫细胞化学方法检测p27蛋白在VSMC上的表达；并用^3H标记的胸腺嘧啶核苷（^3H-TdR）掺入及流式细胞仪等方法检测p27过度表达，对有丝分裂原（血清和大鼠血小板衍化生长因子PDGF-BB）刺激VSMC增殖的影响以及细胞周期依赖性激酶2（CDK2）、细胞增殖核抗原（PCNA）和连接蛋白Cx43表达的变化。结果p27阳性表达率随AdCMV-p27转染VSMC时间延长而不断增加，24h达到峰值（85．81％）。AdCMV-p27转染可显著抑制有丝分裂原刺激引起的G0～G1期细胞减少和^3H-TdR掺入量的增加（P〈0．05）；可抑制PDGF-BB刺激引起的CDK2、PCNA和Cx43蛋白表达阳性率的升高（P〈0．05）；并使p27蛋白表达阳性率由PDGF-BB刺激后的4．23％上升至33．91％（P〈0．01）。结论p27过度表达可抑制有丝分裂原刺激引起的VSMC增殖，其机制与其增强p27表达及抑制CDK2、PCNA和连接蛋白Cx43表达有关。

Objective To investigate the effect of transfering and expression of p27 gene on cultured vascular smooth muscle cells （VSMCs） in rats and explore its mechanism. Methods Recombinant adenoviral vector, AdCMV-p27, containing human p27 gene was constructed by site special recombination,and it was used to transfer p27 gene into rat VSMCs. The ratio of p27 expression in VSMCs was analyzed by flow cytometry system（FCS）. The protein expressions of p27, proliferating cell nuclear antigen （PCNA）, cyclin-dependent kinase2 （CDK2） and connexin43 were detected by immunocytochemistry method 24h after the PDGF-BB or Serum added. The cell cycle and DNA synthesis of the VSMCs were used to determine the proliferation of VSMCs by FCS and [^3 H] thymidine incorporation,respectively. Results The expression ratio of p27 in VSMCs was significantly increasing after transfected by AdCMV-p27 with the time,and the peak value was about 85.81 % at 24th hour. The ratio of G0-G1 phase and [^3 H] thymidine incorporation stimulated by mitogen was significantly increased and decreased in transfected cells compared with non-transfected cells, respectively（P〈0.05）. Compared with the PDGF-BB stimulated group, the positive expressions of p27 protein in transfected cells were not only significantly increased from 4.23 % to 33.91 %, but also that of CDK2, PCNA and connexin43 induced by PDGF-BB were remarkably decreased （P〈0.01）. Conclusion The overexpression of p27 mediated by AdCMV-p27 can significantly inhibit the proliferation of rat VSMCs induced by mitogen, which is relate with the enhanced expression of p27 protein and de creased protein expression of CDK2,PCNA and Cx43.