靶向Pik3cb shRNA表达载体的构建及其对大鼠血管平滑肌细胞凋亡的影响

Construction of rat Pik3cb shRNA and its apoptosis effect on vascular smooth muscle cells

目的构建大鼠Pik3cb shRNA真核表达载体,并检测其下调大鼠血管平滑肌细胞Pik3cb mRNA表达的效应。方法根据Genbank中大鼠Pik3cb序列设计并合成两条shRNA寡核苷酸片段,退火形成双链并克隆导入载体pGenesil-1,酶切鉴定和测序无误后分别命名为pU6- Pik3cb-shRNA-1和pU6-Pik3cb-shRNA-2。通过脂质体转染大鼠胸主动脉平滑肌细胞,确定转染效率;通过荧光定量聚合酶链反应(PCR)检测Pik3cb mRNA的表达;TUNEL法检测细胞凋亡。结果pU6-Pik3cb-shRNA-1和pU6-Pik3cb-shRNA-2经测序和PCR扩增与原序列相同,转染大鼠平滑肌细胞48 h时转染率为15.7%,荧光定量PCR结果显示转染组Pik3cb mRNA表达明显减少,与对照组比较差异有统计学意义(P<0.05),错配质粒组与对照组比较Pik3eb mRNA表达差异无统计学意义(P>0.05),TUNEL结果显示转染组凋亡细胞明显增加,与对照组比较差异有统计学意义(P<0.05)。结论成功构建重组真核表达质粒pU6-Pik3cb-shRNA,并有效下调大鼠胸主动脉平滑肌细胞Pik3cb mRNA的表达,为靶向Pik3cb防治移植血管再狭窄的基因治疗奠定了基础。

Objective To construct rat phosphatidylinositol 3-kinase, catalytic,-β polypeptide （Pik3cb） shRNA eukaryon plasmid express vector and test its downregulating effect on Pik3cb mRNA expression in rat vascular smooth muscle cells （VSMC）. Methods Two shRNA of rat Pik3cb were designed and synthesized according to the sequence of Pik3cb in the Genbank, and then annealed to form double strands and cloned into pGenesil-1, named pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2, respectively. The sequences were examined. Both pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 were transfected into rat thoracic VSMC through METAFECTENETM. The transfection rate was identified and Pik3cb mRNA expression was detected by using fluorescent real time quantitative PCR. Results The sequence of synthesized pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 was exactly the same as the expected. The transfection rate was 15.7% 48 h after transfection of VSMC in rats. Pik3cb mRNA expression was significantly reduced in the transfected groups as compared with that in control group （P 〈 0.05 ）, while the scramble shRNA did not reduce the expression of Pik3cb mRNA （P 〉 0.05 ）. TUNEL revealed that the apoptosis cells were increased deeply in the transfected groups as compared with control group （P 〈 0.05）. Conclusion Rat Pik3cb shRNA eukaryon express plasmid vectors were successfully constructed and the Pik3cb mRNA expression in VSMC of rats were effectively downregulated.