蛋白激酶B短发夹环RNA表达载体的构建及其对血管平滑肌细胞增殖活性的影响

目的构建靶向蛋白激酶B基因的短发夹环RNA表达载体,观察其对血管平滑肌细胞增殖活性的影响。方法设计多个针对大鼠蛋白激酶B基因的短发夹环RNA序列,化学合成方法合成并经pGEM-T载体克隆后双酶切,将cDNA序列插入逆转录病毒载体pLXIN,包装后获得蛋白激酶B的逆转录表达载体,感染血管平滑肌细胞,Northern blot和Western blot法检测蛋白激酶B及其下游底物的表达变化,流式细胞仪检测细胞周期变化,MTT法检测血管平滑肌细胞增殖活性的改变。结果成功构建蛋白激酶B基因的逆转录病毒载体并包装,感染血管平滑肌细胞,证实其能显著抑制蛋白激酶B的mRNA和蛋白产物表达,下游的p70s6k表达相应减少;被感染血管平滑肌细胞的分裂、增殖过程受阻,更多细胞停滞在G0/G1期。结论成功构建蛋白激酶B基因逆转录病毒RNA干扰表达载体,感染血管平滑肌细胞能够明显抑制其分裂、分化和增殖。

Construction of Short Hairpin RNA Vector Targeting PKB/Akt and Its Effect on the Proliferation of Vascular Smooth Muscle Cell

Aim To construct short hairpin RNA(shRNA)retroviral vector of PKB/Akt and study the effect on the proliferation of vascular smooth muscle cell(VSMC).Methods The sequence of shRNA of PKB/Akt was constructed by the software and synthesized by chemical method and then was cloned into pGEM-T vector,which was certified by DNA sequencing.The sequence was inserted into a retroviral vector pLXIN,then the vector was packaged in PT67 cells.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 activity was determined by flowcytometry and MTT.Results The PKB/Akt shRNA vector was successfully constructed by means of recombinant DNA technology and cell transfection.Additionally,the vector could efficiently decrease mRNA and protein expression of PKB/Akt,while the expression of p70s6k also decreased significantly.The cell cycle VSMC was also stunned in phase G0/G1.Conclusion The PKB/Akt shRNA retroviral vector has been constructed successfully,which can significantly inhibit the proliferation of VSMC.