【摘要】 目的构建针对髓细胞白血病基因-1(Mcl-1)的shRNA的重组质粒,探讨该重组质粒对肝癌细胞化疗敏感性的影响。方法设计3对针对Mcl-1基因不同位点的shRNA片段的真核表达载体psiRNA-hH1neo-Mcl,用阳离子脂质体法将重组质粒转染至人肝癌细胞株HepG2中,采用RT-PCR和Western blot方法检测转染后Mcl-1mRNA及蛋白表达情况,筛选Mcl-1基因表达沉默效应最好的重组质粒。用MTT和流式细胞仪分别检测单独使用丝裂霉素(Mitomycin,MMC)、单独使用重组质粒以及联合使用重组质粒与MMC对HepG2细胞的增殖和凋亡的影响。结果成功构建含不同shRNA片段的重组质粒,依次命名为pMclsi-1、pMclsi-2、pMclsi-3。经测序证实,插入的DNA片段的序列与设计序列完全一致。重组质粒转染HepG2细胞后,Mcl-1基因的mRNA水平及蛋白水平明显下调,其中以pMclsi-1下调效应最强。MTT检测显示,pMclsi-1联合化疗药物MMC对HepG2细胞增殖的抑制活性显著高于单用MMC组或单用pMclsi-1组,流式细胞仪检测显示,pMclsi-1联合化疗药物MMC促进HepG2细胞凋亡的活性显著高于单用MMC组或pMclsi-1组。结论针对Mcl-1基因的siRNA可特异阻断Mcl-1基因的表达,从而明显增强肝癌细胞对化疗药物MMC的敏感性。更多还原

【Abstract】 Objective To construct recombinant plasmids containing short hairpin RNA(shRNA)against the myeloid cell leukemia-1(Mcl-1)gene,and to assay their activity to sensitize the human hepatoma cell line HepG2 to chemotherapeutics.Methods Three shRNAs directed against various fragments of the Mcl-1gene were designed and synthesized according to coding sequences of the gene,and cloned into the downstreams of the H1 promoter of psiRNA-hH1 neo.The recombinant plasmids constructed were then transfected into HepG2 cells with lipofectamine2000.The Mcl-1mRNA was measured with RT-PCR,and Mcl-1protein with Western blot,and the best effective silencing plasmid was screened.The proliferation capacity and the apoptosis state of cells treated with Mitomycin(MMC)or recombinant plasmid alone or combination were detected with MTT assay and flow cytometry respectively.Results Three recombinant plasmids were successfully constructed,confirmed through DNA sequencing and designated as pMclsi-1,pMclsi-2and pMclsi-3respectively.The expression of Mcl-1mRNA and protein in HepG2 cells transfected with the plasmids was obviously decreased in comparison with that not transfected with those plasmids,and pMclsi-1was detected to have the best silencing efficacy.MTT assay indicated that the inhibition of cellular proliferation by combination was obviously higher than that by MMC or pMclsi-1alone;and the same is true for the apoptosis rate with flow cytometry.Conclusion The constructed recombinant plasmids containing Mcl-1shRNA can specifically block the Mcl-1expression,and suppression of Mcl-1via RNA interference can enhance the sensitivity of human hepatoma cells to MMC chemotherapy.更多还原