靶向人GSK-3β基因的shRNA真核表达质粒的构建及其在人胰腺癌细胞株PANC-1中的稳定表达

摘要

目的 构建针对人糖原合成激酶-3β(GSK-3β)基因的shRNA真核表达质粒,并筛选基因沉默效果最明显的shRNA质粒表达载体;转染人胰腺癌细胞株PANC-1,建立稳定表达GSK-3β shRNA的细胞模型.方法 针对GSK-3β基因的mRNA序列设计并分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠埃希菌扩增,酶切、PCR、测序鉴定,转染胰腺癌PANC-1细胞,采用Real-time PCR检测GSK-3β mRNA被抑制情况.选取抑制效应最强的重组质粒和阴性质粒转染的PANC-1细胞,经G418筛选后,建立稳定表达GSK-3β-shRNA的PANC-1细胞株(实验组)和稳定表达control-shRNA的PANC-1细胞株(阴性对照组).分别采用荧光显微镜和流式细胞术观察细胞的转染情况;Real-time PCR和Western blot分析GSK-3β的表达.结果 ①经测序证实,成功构建GSK-3β-shRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致;②重组质粒瞬时转染PANC-1细胞后,GSK-3β mRNA明显下调(P＜0.05),其中以2号重组质粒效应最强;③重组质粒稳定转染后检测PANC-1细胞中GSK-3β的表达,Real-time PCR和Western blot结果均提示:与未转染的阴性对照组和载体对照组比较,实验组中GSK-3β mRNA和蛋白的表达均显著降低(均P＜0.05).结论 成功构建了携带以GSK-3β为靶向的shRNA的重组质粒.经脂质体途径稳定转染PANC-1细胞,该shRNA能够显著抑制GSK-3β的表达.%Objective To construct three short hairpin RNA(shRNA)interference expression plasmid vectors of human GSK-3β gene,detect the expression of GSK-3β in pancreatic carcinoma PANC-1 cells after transfection with recombinant plas mids,and transfect stably the most effective recombinant plasmid into PANC-1 cells. Methods Three plasmid expression vectors coding for shRNA targeting GSK-3β and a control vector were designed. The recombinant plasmids were amplified in E. coli DH5α,and then treated with restriction enzymes, PCR and sequencing. PANC-1 cells stably expressing GSK-3β shRNA and control-shRNA were screened with G418 ,as the experimental group and vector control group respectively. The cells were not transfected as the negative control group. The transfection efficiency of cells was observed by fluorescence microscope and flow cytometry( FCM). GSK-3β expression was detected by using realtime PCR and Western blot. Results ① DNA sequencing revealed that the recombinant plasmids were successfully constructed; ② GSK-3β expression was notably down-regulated after transfection of shRNA plasmids in PANC-1 cells. Recombinant plasmid 2 had the strongest effect; ③ Compared with the control group, the expression of GSK-3β was significantly down-regulated in experimental group. Conclusion Plasmid vector expressing shRNA against GSK-3β was successfully constructed and stably transfected into PANC-1 cells, which could facilitate further studies on GSK-3β function and its application in tumor gene therapy.