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RNA干扰沉默低氧诱导因子--1#alpha#对低氧下大鼠肺动脉平滑肌细胞增殖的影响

张伟, 曹越, 张昱, 马祁生, 马兰, 格日力

青海大学高原医学研究中心.青海,西宁 810001

摘要

该研究应用基因克隆技术,将合成的发卡样特异性低氧诱导因子--1#alpha#(hypoxia inducible factor--1alpha,HIF--1#alpha#)干扰寡核苷酸(siRNA)序列插入真核表达载体中,构建出特异性HIF--1#alpha#基因RNA干扰(RNAi)真核表达载体。采用组织块种植法,原代培养大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs),将构建出的特异性HIF--1#alpha#RNAi真核表达载体转染到PASMCs;分别在常氧和低氧下进行细胞培养,采用RT--PCR检测PASMCsHIF--1#alpha#mRNA表达水平,用MTT和流式细胞仪检测细胞增殖水平,探讨低氧条件下HIF--1#alpha#RNAi真核表达载体对PASMCs增殖的影响。结果表明,低氧培养48h后,正常PASMCs和转染了HIF--1#alpha#siRNA阴性表达载体的细胞增殖显著,HIF--1#alpha#mRNA表达水平也显著升高;而转染了HIF--1#alpha#siRNA阳性表达质粒的细胞增殖不显著,HIF--1#alpha#mRNA表达水平较低。结果提示:HIF--1#alpha#RNAi真核表达载体能显著干扰培养的PASMCsHIF--1#alpha#mRNA表达,同时抑制低氧环境下PASMCs的增殖。

关键词： 肺动脉; 平滑肌细胞; 低氧诱导因子-1#alpha#; RNA干扰; 细胞增殖

Effects of RNAi on hypoxia inducible factor--1#alpha# activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rat

Zhang Wei, Cao Yue, Zhang Yu, Ma Qisheng, Ma Lan, Ge Rili

Research Center for High Altitude Medicine, Qinghai University.Xining 810001,Qinghai

Abstract

Pulmonary vascular remodeling is one of the major characteristics of hypoxia-induced pulmonary hypertension, mainly represented by over-proliferation of pulmonary artery smooth muscle cells (PASMCs). Hypoxia inducible factor-1#alpha# (HIF-1#alpha#) is a transcription factor which is produced by the cells exposed to hypoxia. HIF-1#alpha# up-regulates the expression of many hypoxia response genes (HRGs) for the body to adapt to hypoxia and maintain homeostasis. The expression of HIF-1#alpha# in the PASMCs is remarkably elevated under hypoxic condition and it stimulates the proliferation of PASMCs. In this experiment, we used gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1#alpha# mRNA. They were separately subcloned into the plasmid of pGenesil- 1 containing U6 promoter. The pGenesil- 1 vector of the RNA interference eukaryotic expression vector specific to HIF-1#alpha# gene was constructed. DNA sequencing of the plasmid verified the successful construction of the HIF-1#alpha# RNAi. We isolated and cultured the PASMCs of rat. The pGenesil-1 vector was transferred into the PASMCs with METAFECTENE in vitro. The positive cell clones transfected with pGenesil-1 were obtained after being screened with 400#mu#g/ml G418. These PASMCs were cultured in normoxia and hypoxia. After 48 h, the effects of RNAi on the expression of HIF-1#alpha# mRNA were detected by RT-PCR. The cellular growth activities were assayed by MTT colorimetry and flow cytometry in vitro. The results showed that for the PASMCs cultured in hypoxia for 48 h, the cell proliferation of blank group and control group were remarkably increased and the HIF-1#alpha#mRNA expressions were up-regulated, while the cell proliferation of the treatment groups did not increase and the HIF-1#alpha# mRNA expressions were not up-regulated. In conclusion, we successfully constructed the recombinant plasmid of RNAi and transfected them into the PASMCs in vitro. The RNAi inhibited the expression of HIF-1#alpha# mRNA in the PASMCs, and subsequently it remarkably suppressedthe proliferation of PASMCs in hypoxia. These results indicate that HIF-1#alpha# plays a pivotal role in PASMC proliferation.

Key words: Pulmonary artery;Smooth muscle cell;hypoxia inducible factor-1#alpha#;RNA interference;cell proliferation

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引用本文：

张伟, 曹越, 张昱, 马祁生, 马兰, 格日力. RNA干扰沉默低氧诱导因子--1#alpha#对低氧下大鼠肺动脉平滑肌细胞增殖的影响[J]. 生理学报 2006; 58 (1): .

Zhang Wei, Cao Yue, Zhang Yu, Ma Qisheng, Ma Lan, Ge Rili. Effects of RNAi on hypoxia inducible factor--1#alpha# activity and proliferation of hypoxic pulmonary artery smooth muscle cells in rat. Acta Physiol Sin 2006; 58 (1): (in Chinese with English abstract).