中文摘要：

本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-1α（hypoxia inducible factor-1alpha，HIF-1α）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-1α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-1αRNAi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-1αmRNA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-1αRNAi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-1αsiRNA阴性表达载体的细胞增殖显著，HIF-1αmRNA表达水平也显著升高；而转染了HIF-1αsiRNA阳性表达质粒的细胞增殖不显著，HIF-1αmRNA表达水平较低。结果提示：HIF-1αRNAi真核表达载体能显著干扰培养的PASMCsHIF-1αmRNA表达，同时抑制低氧环境下PASMCs的增殖。

英文摘要：

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-1α （HIF-1α is a transcription factor which is produced by the cells exposed to hypoxia. HIF-1α up-regulates the expression of many hypoxia response genes （HRGs） for the body to adapt to hypoxia and maintain homeostasis. The expression of HIF-1α 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-1α 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-1α gene was constructed. DNA sequencing of the plasmid verified the successful construction of the HIF-1α 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 400μg/ml G418. These PASMCs were cultured in normoxia and hypoxia. After 48 h, the effects of RNAi on the expression of HIF-1α 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-1αmRNA expressions were up-regulated, while the cell proliferation of the treatment groups did not increase and the HIF-1α 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-1α mRNA in the PASMCs, and subsequently it remarkably suppressedthe prolifera