雷帕霉素靶蛋白反义RNA真核表达载体的构建及其在血管平滑肌细胞中的表达

目的构建雷帕霉素靶蛋白（mTOR）的反义RNA真核表达载体，观察其对血管平滑肌细胞（VSMC）功能的影响。方法提取人VSMC总RNA，逆转录．聚合酶链反应（RT-PCR）扩增mTOR基因cDNA序列，经pGEM-T载体克隆后双酶切，将cDNA序列反向插入绿色荧光蛋白表达载体pEGP-C1，转染VSMC，Westernblot法检测反义表达载体对mTOR蛋白表达的影响，流式细胞仪检测细胞周期的变化。结果经RT-PCR获得664bp产物，T载体克隆后，酶切确定该片段为mTOR基因cDNA，进而构建反义RNA真核表达载体pEGFP-C1-mTOR，测序证明序列正确后转染VSMC，证实其能够显著抑制mTOR蛋白产物表达，转染72h的mTOR蛋白产物表达抑制率达82％，S期细胞由15％降低为7％，凋亡细胞增至9％。VSMC增殖过程在Gx／Go期→S期受阻。结论成功构建mTOR基因的反义RNA真核表达载体。

Construction of eukaryotic expression vector of mammalian target of rapamycin antisense RNA and its expression in vascular smooth muscle cell

Objective To construct pEGFP-C1-mTOR vector, a eukaryotic expression plasmid of mammalian target of rapamycin (mTOR) antisense RNA, and study the effect of the vector on vascular smooth muscle cells (VSMC). Methods The region of mTOR gene was amplified by RT-PCR after total RNA being extracted from human VSMC and then cloned into pGEM-T vector. After the recombinant plasmid was certified, the conservative region of mTOR gene was inserted into pEGFP-C1 vector reversely and pEGFP-C1-mTOR was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection to VSMC. The cell cycle was determined by flowcytometry. Results 664 bp fragment including conservative region of mTOR gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGP-C1-mTOR vector was successfully constructed. Additionally, pEGFP-C1-mTOR vector could efficiently reduce mTOR protein expression level by 82% after 72 h of transfection to VSMC. The VSMC in phase S were decreased from 15% to 7%, but the apoptotic cells were increased to 9% . The cell cycle was stunned. Conclusion pEGFP-C1-mTOR, the efficient antisense RNA expression vector of mTOR, has been constructed successfully.