靶向STAT3的慢病毒表达载体的构建及其对血管平滑肌细胞增殖凋亡的影响

Construction of lentivirus vector carrying STAT3 gene and its effect on proliferation and apoptosis of vascular smooth muscle cells

目的构建大鼠STAT3基因的shRNA慢病毒表达载体，并观察其对大鼠血管平滑肌细胞增殖和凋亡的影响。方法针对STATS基因的不同部位设计4对shRNA的寡核苷酸片段，克隆到慢病毒载体PLKO-1中，构建靶向STA33基因的慢病毒载体PLKO．1-STA33-shRNA，检测并筛选最佳抑制效率的shRNA干扰载体。并将其转染大鼠血管平滑肌细胞，用噻唑蓝法和流式细胞仪检测沉默STAT3基因后对血管平滑肌细胞增殖和凋亡能力的影响。结果靶向STA33慢病毒表达载体构建成功。转染PLKO．1一STA33一shRNA后，STA33蛋白表达明显下降，其中以PLKO．1一STA33一S1最为明显，达到90％以上；转染PLKO．1-STA33-S1的细胞增殖能力（A值=0．25±0．05）明显低于未转染组（A值=0．62±0．12）和阴性对照组细胞（A值=0．59±0．11）（P〈0．05）；而早期细胞凋亡率（26．9±2．8）％和晚期细胞凋亡率（9．5±1．6）％均明显高于未转染组和阴性对照组（P〈0．01）。结论成功构建并筛选最佳抑制效率的靶向STAT3慢病毒表达载体PLKO．1-STAT3-S1，该载体能有效抑制大鼠血管平滑肌细胞增殖，并促进细胞凋亡。

Objective To construct a recombinant short hairpin RNA （shRNA） lentiviral vector carrying STAT3 gene in rats, and to investigate its effects on proliferation and apoptosis of vascular smooth muscle cells by silencing STAT3. Methods Four oligonucleotides targeting STAT3 gene were synthesized and cloned into lentivirus vector PLKO. 1. The shRNA lentiviral vector with best transfection efficiency was detected and identified, which was transfected into vascular smooth muscle cells in rats, and its effects on proliferation and apoptosis of vascular smooth muscle ceils were measured by MTT and flow cytometry after silencing STAT3. Results The recombinant lentivirus vector PLKO. 1-STAT3-shRNA was constructed successfully. PLKO. 1-STAT3-shRNA knocked down the expression of STAT3 protein dramatically, espe- cially PLKO. 1-STAT3-S1, whose transfection efficiency was more than 90%. The proliferation capacity of vascular smooth muscle ceils transfected with PLKO. 1-STAT3-S1 （A value = 0. 25 ± 0. 05 ） was significantly lower than no-transfected group （A value =0. 62 ＋0. 12） and negative control group （A value =0. 59±0. 11） （P 〈 0. 05 ）. Meantime the early apoptosis rate （ 26. 9 ± 2. 8 ） % and late apoptosis rate （ 9. 5 ± 1.6 ） % in PLKO. 1-STAT3-shRNA-transfected group were significantly higher than in no-transfected group and negative control group （P 〈 0. 01 ）. Conclusion The recombinant lentivirus shRNA vector targeting STAT3, PLKO. 1-STAT3-S1, with best transfection efficiency, is constructed successfully. PLKO. 1-STAT3-S1 can inhibit the proliferation of vascular smooth muscle cells, and promote the cell apoptosis. This study lays the foundation for further studying on targeting treatment of vascular restenosis.