大鼠Pik3cb短发卡状RNA对平滑肌细胞增殖的影响  

The construction of rat Pik3cb shRNA and its inhibitory effect on the proliferation of smooth muscle cell

作　　者：刘苏健[1] 刘宏[1] 赵京[1] 申庆民 常程[1] 邓勇志[2] 马捷[2] 

机构地区：[1]北京航天总医院胸外科,北京100076 [2]山西医科大学第二医院心胸外科,山西太原030001

出　　处：《南京医科大学学报（自然科学版）》2008年第10期1234-1239,共6页Journal of Nanjing Medical University(Natural Sciences)

摘　　要：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 rat Pik3cb （phosphatidylinositol 3-kinase, catalytic, beta polypeptide） shRNA eukaryon plasmid express vector,and test its downregulating effect on Pik3cb mRNA expression for the application of RNA interference in restraining vein graft restenosis. Methods：Two shRNA of rat Pik3cb were design and synthesize according to the sequence of Pik3cb in the Genbank,annealed to form double strands and then cloned into pGenesil-1, and then the sequences were examed. After shRNAs were transfected into rat thoracic VSMC through METAFECTENETM,the transfection rate was identified by EGFP. pAkt-473 protein expression was detected by Western blot, and cell apoptosis were obtained through flow-cytometry. Results：The two sequences of synthesized shRNA named pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 were exactly the same with the design. The 48 h and 72 h transfection rate were 15.7% and 10.1%,respectively. The pAkt-473 protein expression significantly reduced,white the apoptosis cells were more significantly increased in the shRNA groups than the control groups （P 〈 0.05）. The scramble shRNA did not interfere with the VSMC proliferation. Conclusion： Rat Pik3cb shRNA eukaryon express plasmid vectors were constructed successfully and effectively inhibited the proliferation of rat smooth mouse cells.

关 键 词：载体构建 Pik3cb SHRNA 血管再狭窄 RNAI 

分 类 号：R543.1[医药卫生—心血管疾病]