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目的:构建大鼠Pik3cb短发卡状RNA(shRNA)真核表达载体,为利用RNA干扰(RNA interference,RNAi)技术从转录后水平抑制血管移植术后移植静脉再狭窄的研究做准备。方法:经预实验设计并合成6条shRNA寡核苷酸片段,酶切鉴定和测序正确后挑选2条质粒转染入大鼠胸主动脉平滑肌细胞内,48、72h用荧光显微镜观察转染效率,Western blot检测Phospho-Akt(Ser473)蛋白的表达,流式细胞仪分别在24、48、72、96h检测细胞凋亡数目。结果:构建的两条大鼠Pik3cbshRNA经限制性内切酶酶切和DNA测序正确后分别命名为pU6-Pik3cb-shRNA-1和pU6-Pik3cb-shRNA-2,转染平滑肌细胞48h和72h转染率分别为15.7%和10.1%;Westernblot结果显示转染组Phospho-Akt(Ser473)蛋白表达明显下降;流式结果表明pU6-Pik3cb-shRNA-1和pU6-Pik3cb-shRNA-2组凋亡细胞数目明显增加,与对照组相比有统计学意义(P<0.05),各时间点错配质粒组与正常对照组相比差异无统计学意义。结论:成功构建了2条大鼠Pik3cbshRNA真核表达载体,有效促进凋亡,抑制平滑肌细胞增殖,为靶向Pik3cb的RNAi防治血管再狭窄奠定了基础。
OBJECTIVE: To construct a eukaryotic expression vector for short hairpin RNA (shRNA) of rat Pik3cb in preparation for the study of suppressing postoperative venous restenosis after transplantation with RNA interference (RNAi) technique. METHODS: Six shRNA oligonucleotide fragments were designed and synthesized pre-experimentally. Two plasmids were selected and transfected into rat thoracic aortic smooth muscle cells by restriction enzyme digestion and sequencing. The transfection efficiency was observed by fluorescence microscopy at 48 and 72 hours The protein expression of Phospho-Akt (Ser473) was detected by Western blot. The number of apoptotic cells was detected by flow cytometry at 24, 48, 72 and 96 hours respectively. RESULTS: Two rat Pik3cb shRNAs were successfully transfected into smooth muscle cells transfected with pU3-shRNA-1 and pU6-Pik3cb-shRNA-2 by restriction endonuclease digestion and DNA sequencing respectively The results of flow cytometry showed that the number of apoptotic cells in pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 groups was significantly lower than that in pU6-Pik3cb-shRNA- (P <0.05). There was no significant difference between the mismatched plasmid group and the normal control group at each time point. Conclusion: Two eukaryotic expression vectors of rat Pik3cb shRNA have been successfully constructed, which can effectively promote apoptosis and inhibit the proliferation of smooth muscle cells, which lays the foundation for the prevention and treatment of vascular restenosis by targeting Pik3cb RNAi.