Zeng Qiong, Ran Pi-xin, Chen Shun-cun, Liu Jing-sheng
Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2003 Jun;26(6):358-61.
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.
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 [(3)H] thymidine incorporation. The proteins involved in cell cycle control (P27 and P21) were determined by RT-PCR and flow cytometry.
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 [(3)H] 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.
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.
一氧化氮合酶（iNOS）基因转移抑制缺氧诱导的肺动脉平滑肌细胞（PASMCs）增殖的潜在机制尚不清楚。本研究旨在探讨缺氧时iNOS基因转移至PASMCs对细胞周期进程是否有影响。
采用阳离子脂质体介导法，将重组pLNCX/Inos载体转染至大鼠PASMCs。检测瞬时转基因表达及重组蛋白的功能。通过流式细胞术进行细胞周期分析，采用[³H]胸苷掺入法进行细胞增殖测定。通过逆转录聚合酶链反应（RT-PCR）和流式细胞术测定细胞周期调控相关蛋白（P27和P21）。
在转染的PASMCs中检测到iNOS表达。与未转染细胞相比，iNOS转染细胞中的NO₂⁻水平升高。缺氧条件下大鼠PASMCs中iNOS的表达导致DNA合成抑制和细胞周期进程延迟。iNOS转染组[³H]胸苷掺入量（15,145±1,514）dpm显著低于缺氧组（18,011±2,521）dpm（P<0.01）。iNOS转染组的G₀/G₁期细胞周期阻滞率（67.8%）显著高于缺氧组（46.8%）（P<0.01）。缺氧可下调P27蛋白水平，但在缺氧条件下iNOS转染细胞中未出现下调，后者水平与常氧条件下相似。
缺氧时将iNOS基因预先转移至PASMCs可通过阻断P27下调来抑制细胞增殖，这是细胞周期进程延迟的重要机制。
