Apoptin对前列腺癌细胞的促凋亡作用研究

Effect of apoptin on apoptosis of human prostate carcinoma cells

目的构建野生型及变异型apoptin的真核表达质粒pApoptin-EGFP、pM-apoptin-EGFP,并探讨其对人前列腺癌细胞系LNCaP、PC3的促凋亡作用。方法采用PCR的方法从变异型apoptin质粒p3×flag-m-apoptin- myc扩增出野生型apoptin片段及变异型apoptin片段m-apoptin,将其定向克隆于pEGFP-N1载体的EcoR I和BamH I酶切位点之间,构建EGFP标记的野生及变异型apoptin真核表达质粒pApoptin-EGFP及pM-apoptin-EGFP,酶切、测序鉴定,RT-PCR、荧光显微镜分析融合蛋白表达,脂质体介导瞬时转染人前列腺癌细胞系LNCaP、PC3,流式细胞仪检测凋亡及细胞周期变化。结果成功构建pApoptin-EGFP、pM-apoptin-EGFP重组质粒,并在被转染细胞检测到基因表达;瞬时转染后48h可检测到细胞凋亡,与空质粒组相比具有非常显著意义,细胞周期变化表现为G2/M期阻滞。结论apoptin对雄激素依赖及非依赖型前列腺癌均具有明显的促凋亡作用,末端突变影响其促凋亡作用,为进一步探讨apoptin应用于前列腺癌的治疗奠定基础。

Objective To construct the eukaryotic expression plasmids of pApoptin-EGFP and pM-apoptin-EGFP and evaluate it＇s role on apoptosis of prostatic carcinoma cell lines LNCaP and PC3. Methods Wild-type and mutatedtype gene of apoptin were amplified respectively by PCR from plasmid p3 X flag-apoptin-myc, the PCR products were cloned respectively into the vector pEGFP-N 1 between the restriction enzyme sites of EcoR I and BamH, and then the recombinant plasmids were identified by enzyme digesting and sequencing. The identified recombinant plasmids were transfected into LNCaP and PC3 cell lines with Lipofectamine 2000, the expression of the fusion protein was detected by RT-PCR and fluorescence microscope. Apoptosis and the cell cycle of transfected cells were analyzed by flow cytometry. Results The recombinant plasmids of pApoptin-EGFP, pM-apoptin-EGFP had been constructed success- fully and the expression of fusion protein in transfected cell lines could be detected. Apoptosis could be detected 48 hours after transient transfection, wild-type and the mutated-type gene of apoptin all induced apoptosis of two cell lines effectively compared with that of mock, and cell cycle G2/M arrest was found in the two transfected cell lines. Conclusion Apoptin could induce significantly apoptosis of androgen-depended or not human prostate cancer cell lines, and mutation of its C terminal might weaken cell apoptosis, which provided evidence for future therapy study using apoptin.