中文摘要：

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C0／G1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。

英文摘要：

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.