RNA干扰沉默蛋白激酶B基因表达对血管平滑肌细胞增殖活性的影响

Effect of RNA interference targeting PKB/Akt on the proliferation of vascular smooth muscle cells

目的构建靶向蛋白激酶 B(PKB/Akt)基因的逆转录病毒 RNA 干扰表达载体,观察其对血管平滑肌细胞(VSMC)增殖活性的影响。方法针对大鼠 PKB/Akt 基因序列(NM_033230)设计多个短发夹环 RNA(shRNA)序列,化学合成法合成单链寡核苷酸序列,pGEM-T 载体克隆后双酶切,将 cDNA 序列插入逆转录病毒载体 pLXIN,脂质体介导转染包装细胞 PT67后获得 PKB/Akt的重组逆转录病毒 RNA 干扰载体,感染血管平滑肌细胞(VSMC),Northern blot 和 Western blot 法检测 Akt 及其下游底物-核糖体蛋白 S6激酶(p70s6k)等表达的变化,流式细胞仪检测 VSMC 细胞周期的变化,噻唑盐(MTT)比色法检测 VSMC 增殖活性的改变。结果 shRNA 序列经 T 载体克隆,酶切确定该片段为 PKB/Akt 基因 shRNA 序列;感染 VSMC,证实其能够显著抑制 PKB/Akt 的 mR-NA 和蛋白产物表达,下游的 p70s6k 表达相应减少;被感染 VSMC 的分裂、增殖过程受阻,更多细胞停滞在 Go/G_1期。结论成功构建 PKB/Akt 基因逆转录病毒 shRNA 载体,感染 VSMC 能够明显抑制其分裂、分化和增殖。

Objective To construct RNA interference retroviral vector of PKB/Akt and study its effect on the proliferation of vascular smooth muscle cells （VSMC）. Methods The sequence of Akt gene was obtained from GenBank （ NM_033230）, and the sequence of short hairpin RNA （shRNA） was synthesized by chemical method and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the sequence was inserted into a retroviral vector pLXIN, and the vector was packaged in PT67 ceils by transfection with Lipofectamine. The efficiency of inhibition was verified by Northern blot and Western blot after transfection to VSMC. The change of p70s6k was also determined at the same time. The proliferation act/vity was determined by flow cytometry and MTT. Results After cloning by pGEM-T vector,the shRNA fragments of Akt gene were inserted into pLXIN vector and certified by DNA sequencing,the vector was successfully constructed by means of recombinant DNA technology and ceil transfection. Additionally, the vector could efficiently decrease mRNA and protein expression of Akt, while the expression of p70s6k was also decreased significantly. The ceil cycle of VSMC was also stunned in phase G0/ G1. Conclusion The PKB/Akt RNAi retroviral vector has been constructed successfully, which can significantly inhibit the proliferation of VSMC.