一氧化氮合酶基因转染抑制缺氧大鼠肺动脉平滑肌细胞增殖的机制研究  

The inhibitory mechanism of nitric-oxide synthase gene transfection on hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

作　　者：曾琼[1] 冉丕鑫[1] 陈顺存[1] 刘景生[2] 

机构地区：[1]广州医学院-广州呼吸疾病研究所,510120 [2]中国医学科学院中国协和医科大学基础医学院药理室,北京100050

出　　处：《中华结核和呼吸杂志》2003年第6期358-361,共4页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：广东省自然科学基金资助项目 (95 0 3 72 )

摘　　要：目的　研究诱导型一氧化氮合酶 (iNOS)基因转染对缺氧条件下大鼠肺动脉平滑肌细胞 (PASMCs)增殖的影响 ,探讨细胞周期蛋白依赖性激酶抑制因子 p2 1、p2 7在细胞增殖过程中的调控作用。方法　经脂质体介导将iNOS重组逆转录病毒载体 (pLNCX/iNOS)转染大鼠PASMCs,检测外源性iNOS表达及其生物学活性 ;氚标记胸腺嘧啶脱氧核苷 (3 H TdR)掺入、流式细胞技术观察iNOS转染对缺氧条件下大鼠PASMCs增殖的影响 ;逆转录 聚合酶链反应 (RT PCR)和流式细胞技术检测p2 1、p2 7的变化。 结果　转染后检测证实 ,外源性iNOS基因可有效转录、表达 ,具有生物学活性 ;iNOS转染显著抑制缺氧条件下大鼠PASMCs3 H TdR的掺入 ,缺氧组 (D组 )大鼠每分钟衰变数值为 (180 11± 2 5 2 1)次 /min ,缺氧 +DETANONOate组 (E组 ,0 5、1mmol/L)每分钟衰变数值分别为(15 36 4± 1382 )次 /min、(13712± 1782 )次 /min、低氧 +iNOS转染组 (G组 )大鼠每分钟衰变数值为(15 14 5± 15 14 )次 /min ,3组比较差异有显著性 (P <0 0 1) ;iNOS转染导致停滞于G0 /G1期的细胞比例增加 ,G组G0 /G1期细胞百分比为 6 7 8% ,与D组 (46 8% )比较差异也有显著性 (P <0 0 1) ;iNOS转染可使低氧条件下PASMCs的P2 7蛋白表达下调受到抑制 ,P2Objective The underlying mechanism by which nitric-oxide synthase (iNOS) gene transfer inhibits hypoxia-induced PASMCs proliferation remains unknown. The aim of this study is to investigate if iNOS gene transfer to PASMCs during hypoxia has any effect on cell cycle progression. Methods Using the cationic liposome mediation method, we transfected a recombinant pLNCX/Inos vector into rat PASMCs. The instantaneous transgenic expression and the function of the recombinant protein were detected. Cell cycle analysis was performed by flow cytometry and cell proliferation assay by thymidine incorporation. The proteins involved in cell cycle control (P27 and P21) were determined by RT-PCR and flow cytometry. Results iNOS expression was detected in the transfected PASMCs. NO - 2 levels were increased in iNOS-transfected cells as compared to the untransfected cells. Expression of iNOS in rat PASMCs under hypoxia resulted in a delay in inhibition of DNA synthesis and cell cycle progression. The incorporation of thymidine in iNOS-transfected group (15 145±1 514)dpm was significantly lower than those in the hypoxia group (18 011±2 521)dpm( P <0.01). The G 0/G 1 cell cycle arrest rate in the iNOS-transfected group (67.8%) was significantly higher than those in the hypoxia group (46.8%) ( P <0.01). The protein level of P27 was down-regulated by hypoxia but not in iNOS-transfected cells under hypoxia, and the level of the latter was similar to that under normoxia. Conclusions Pre-transfer of iNOS gene to PASMCs under hypoxia inhibits cell proliferation via blocking P27 down-regulation, which is an important mechanism for the delay of cell cycle progression.

关 键 词：一氧化氮合酶 基因转染 缺氧 肺动脉平滑肌细胞 增殖 

分 类 号：R363[医药卫生—病理学]