靶向特定细胞PI3K基因shRNA表达载体的构建及鉴定

Construction and identification of shRNA expression vector targeting PI3K gene in specific cell

目的构建靶向血管平滑肌细胞和内皮细胞磷脂酰肌醇3激酶(phosphatidylinositol3-kinase,PI3K)基因的短发卡干扰RNA(shorthair-pinRNA,shRNA)表达载体,研究其对上述细胞PI3K基因的靶向沉默作用,探索抑制移植血管狭窄的基因防治手段。方法根据Genbank中大鼠PI3Kp110β亚单位编码基因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-10,经荧光定量PCR方法筛选一条较合适的shRNA转录模板;合成血管平滑肌细胞特异性SMHC增强子/SM22α启动子序列和血管内皮细胞特异性KDR增强子/启动子序列,将该增强子/启动子片段亚克隆至pGenesil-10-Pik3cb-shRNA上,构建并鉴定重组质粒载体SMHCe/SM22αp-pGenesil-10-Pik3cb-shRNA(简称SM22αe/pshRNA)和KDRe/p-pGenesil-10-Pik3cb-shRNA(简称KDRe/pshRNA);将重组质粒载体转染血管平滑肌细胞,分别于转染24h、48h、72h后,采用实时荧光定量PCR检测Pik3cb基因mRNA的相对表达量。结果荧光定量PCR检测转染Pik3cb-shRNA-1组与转染Pik3cb-shRNA-2组比较,前者细胞Pik3cbmRNA相对表达量降低更为明显,且两组均较对照组mRNA表达明显减少(P<0.05);酶切鉴定重组质粒载体SM22αe/pshRNA和KDRe/pshRNA构建成功;质粒转染细胞后,Pik3cb基因mRNA相对表达量SM22αe/p质粒组较阴性对照组均有所降低,且转染24h后,SM22αe/p质粒组与空白对照、CMV质粒组比较Pik3cb基因mRNA相对表达量明显降低,差异均有统计学意义(P<0.05)。结论选择Pik3cb-shRNA-1作为转录模板,能够成功构建SM22αe/pshRNA和KDRe/pshRNA质粒载体,且特异性启动子SM22α介导的靶向大鼠Pik3cb基因的shRNA质粒载体可有效沉默靶基因在血管平滑肌细胞中的表达。

Objective To construct expression vectors expressing short hairpin RNA （shRNA）sections targeting phosphatidylinositol 3-kinase（PI3K） gene expression in vascular smooth muscle cell（VSMC）and vascular endothelia cell （VEC）,to explore the silencing effect of PI3 K gene in these cells,and provide a gene therapy strategy to prevent angiostegnosis after bypass grafting.Methods Two of the sense and antisense RNA oligonucleotide strands targeting Pik3cb mRNA were designed and synthesized individually according to the sequence of the rat Pik3cb,then they were annealed to form double strands and then cloned into pGenesil-10,the sequences were examined all corrected as design.One effective shRNA was selected for this study by real time polymerase chain reaction （RT-PCR）.SMHC enhancer/SM22α promoter sequence and KDR enhancer/promoter sequence were synthesized respectively in VSMC and VEC and then cloned into pGenesil-10-Pik3cb-shRNA.These sequences were constructed and identified,named SMHCe/SM22α p-pGenesil-10-Pik3cb-shRNA（SM22α e/p shRNA）and KDRe/ppGenesil-10-Pik3cb-shRNA（KDR e/p shRNA）.The VSMC was transfected by recombinant plasmid vector,and the PI3K mRNA level was detected by RT-PCR at 24 h,48 h and 72 h,respectively.Results Expression of the Pik3cb in Pik3cb-shRNA-1 group was lower than Pik3cb-shRNA-2 group and these two groups were also lower than that of the control group（P 〈 0.05）.SM22α e/p shRNA and KDR e/p shRNA were successfully constructed and identified by restriction endonuclease digestion.Expression of the Pik3cb gene was lower after 24 h in SM22α e/p groups compared with vacuity contrast group and CMV group（P 〈 0.05）.Conclusions The Pik3cb-shRNA-1 sequence was selected to be transcription template and then SM22α e/p shRNA and KDR e/p shRNA were successfully constructed.The shRNA plasmid vector targeting on rat Pik3cb gene should be effectively downregulated the expression of Pik3cb mRNA by SM22α promoter in VSMC.