人内皮型一氧化氮合成酶基因转染抑制人血管平滑肌细胞增殖及其机制

Gene transfer of human endothelial nitric oxide synthase inhibits proliferation of human vascular smooth muscle cells and the mechanism

目的探讨人内皮型一氧化氮合成酶基因（heNOS）转染抑制人血管平滑肌细胞（HVSMCs）增殖的机制。方法以AdCMV—heNOS病毒感染复数分别为50、150、250、300、450MOI，转染HVSMCs，放射免疫法检测转染HVSMCs中的环一磷酸鸟苷（cGMP）的表达变化；Westernblot检测血管平滑肌细胞中p21、p27蛋白的变化，流式细胞术分析对细胞周期分布及凋亡的影响。结果（1）转染120h，A57f1值分别为1．410±0．081、1．357±0.150、1．303±0．311、0．995±0．248、0．731±0．101，其中感染复数300MOI明显稳定抑制血管平滑肌细胞的增殖。（2）转染72h，未转染组、Ad．LacZ转染组、Ad—heNOS转染组cGMP的含量分别为（7．91±0．39）、（8．36±0．34）、（12．89±2．06）μmol／L，差异有统计学意义（P〈0．01）。（3）转染48h，Westernblot检测转染组p27、p21表达明显上调，而未转染组虽也有p21、p27的表达，但两组差异有统计学意义（P〈0．05）。（4）无血清转染48h后血清刺激24h，未转染组、Ad—LacZ转染组、Ad—heNOS转染组G0／G1期分别为（64．23±1．58）％、（64．96±1．36）％、（76．03±2．27）％，差异有统计学意义（P〈0．01）。（5）转染3d，第1天，未转染组、Ad．LacZ转染组、Ad—heNOS转染组细胞凋亡率分别为（4．70±0．56）％、（5．53±0．74）％、（8．53±1．06）％，差异无统计学意义（P〉0．05）。第3天，细胞凋亡率分别为（5．40±0．62）％、（8．30±0．80）％、（9．30±0．90）％，差异无统计学意义（P〉0．05）。结论heNOS基因转染HVSMCs抑制细胞增殖，通过p21、p27上调导致细胞周期的阻滞，无诱导细胞凋亡。

Objective To invesigate the effect of human endothelial nitric oxide synthase （heNOS ） gene transfer on the proliferation of in vitro cultured human vascular smooth muscle cells （HVSMCs） and the mechanism. Methods The HVSMCs were transfeced with multiplicity of infection （MOI） of 50, 150, 250, 300,450. eGMP was measured by radioimmunoassay in HVSMCs. The expression levels of p21 and p27 were detected by Western blotting. Cell cycle and apoptosis were assayed by flow cytometry. Re- suits （1） At 120thh, the A570values were respectively 1. 410±0.081, 1.357±0.150, 1.303±0.311, 0. 995 ±0. 248 and 0. 731 ＋0. 101 at MOI of S0, 150, 250, 300, 450. The proliferation of HVSMCs was significantly and stably inhibited with MOI 300 of AdCMV-heNOS; （2） M 72nd h after the gene transfer, cGMP levels were increased in heNOS-transduced （ 12. 89 ± 2.06） compared to LacZ- （ 8.36±0. 34） and non-tranduced （7.91 ±0. 39） cells （P 〈0. 01 ） ; （3） At 48th h after the gene transfer, the expression of heNOS in HVSMCs up-regulated p21 and p27 （P 〈0. 05） ; （4） After 48 h tronsfeeted with sersuln-de- priveol and for 24 h serum stimulation, the cell cycle was significantly arrested in Go/G1 phase in heNOS- transduced group, and G0/G1 was respectively （ 64. 23 ± 1.58 ） %, （ 64. 96 ± 1.36 ） %, （ 76.03 ± 2. 27） % in non-, LacZ- and heNOS-transduced cells ; （5） At first and third day afer the gene transfer, there was no increase in apoptosis at the first day in all transduced cells （ P 〉 0. 05 ）. Conclusion Adeno- virus-mediated heNOS gene transfer to HVSMCs inhibits cell proliferation via up-regnlation of p21 and p27 resulting in a delay in cell progression not apoptosis.