分泌型PTD4-Apoptin融合蛋白诱导HepG2细胞凋亡

目的研究分泌型的含蛋白质转导结构域的凋亡素(PTD4-Apoptin)融合基因经人脐静脉血管内皮细胞(HUVEC)表达和分泌后,对人肝癌HepG2细胞凋亡的影响,探讨其用于肝癌治疗的可能性。方法构建PTD4-Apop-tin融合基因的pSecTag2分泌型真核表达载体,并采用脂质体介导将其转染入HUVEC细胞,Western blot检测PTD4-Apoptin融合蛋白的表达及分泌,转染48h后收集培养上清作为条件培养液,用于HepG2、L02细胞培养,共培养24h后,采用Western blot检测细胞内和核内Apoptin的含量,流式细胞术检测细胞凋亡。结果瞬时转染pSecTag2-PTD4-Apoptin的HUVEC细胞可表达及分泌PTD4-Apoptin融合蛋白,其培养上清中的PTD4-Apoptin融合蛋白可有效进入HepG2和L02细胞,并可在HepG2细胞核内聚积,显著诱导HepG2细胞凋亡达47.4%(P<0.001),而对L02细胞无此效应。结论 HUVEC细胞表达分泌的PTD4-Apoptin融合蛋白可显著诱导邻近HepG2细胞凋亡,而对L02正常肝细胞无损伤。

Effect of Secretive PTD4-Apoptin Fusion Protein on Apoptosis of HepG2 Cells

Objective To investigate the effect of PTD4-Apoptin secreted by human umbilical venous endothelial cells(HUVECs)on apoptosis of HepG2 cells and the possible application for hepatocellular carcinoma gene therapy.Methods Recombinant plasmid of pSecTag2-PTD4-Apoptin was constructed and identified by restriction enzyme digestion analysis and DNA sequencing.HUVECs were transiently transfected with pSecTag2-PTD4-Apoptin by lipofectamine and the culture supernatant was collected at 48 h after transfection.The expression and secretion of the PTD4-Apoptin fusion protein were detected by Western blot.HepG2 and L02 cells were co-cultured with the supernatant respectively.At 24 h after culture,the distribution of PTD4-Apoptin fusion protein in cells was detected by Western blot and apoptosis rate was measured by flow cytometry.ResultsThe PTD4-Apoptin fusion protein secreted by pSecTag2-PTD4-Apoptin-transfected HUVECs could reenter adjacent untransfected HepG2 and L02 cells.The PTD4-Apoptin fusion protein could assemble in HepG2 nucleus and induce apoptosis of HepG2 cells(47.4%,P<0.001).This effect was not detected in L02 cells.Conclusion The PTD4-Apoptin fusion protein secreted by pSecTag2-PTD4-Apoptin-transfected HUVECs can induce the apoptosis of adjacent untransfected HepG2 cells but not in L02 cells.