人MUC4启动子驱动HSV-TK腺病毒靶向杀伤胃癌细胞的研究

Targeting cytotoxic effect of recombinant adenovirus carrying HSV-TK driven by MUC4 promoter on gastric cancer cells

目的:构建人MUC4启动子驱动下的HSV-TK重组腺病毒,研究其对胃癌细胞的靶向杀伤作用。方法:免疫荧光法检测MUC4在胃癌细胞系中的表达。克隆MUC4启动子区625bp活性序列,利用重组荧光素酶检测系统检测其在SGC-7901胃腺癌细胞及NUGC4胃印戒细胞癌细胞中的转录活性。以AdEasyTM腺病毒系统为载体,构建MUC4启动子驱动下的HSV-TK重组腺病毒,与前体药物GCV联合,检测其对上述两种胃癌细胞的细胞毒作用。结果:MUC4蛋白在两种胃癌细胞的胞浆和胞膜均有表达,而在成纤维细胞中无表达。克隆的MUC4启动子片段在SGC-7901和NUGC4细胞中具有强转录活性,转录活性高于强启动子SV40,而NIH3T3成纤维细胞系中几乎无转录活性。MUC4启动子驱动下的HSV-TK重组腺病毒与GCV联合能够诱导SGC-7901和NUGC4胃癌细胞凋亡,产生特异性靶向细胞毒作用。结论:人MUC4启动子驱动下的HSV-TK重组腺病毒联合GCV对SGC-7901和NUGC4胃癌细胞具有靶向杀伤作用,MUC4启动子可以作为胃癌靶向基因治疗的工具。

Objective：To construct the recombinant adenovirus carrying herpes simplex virus thymidine kinase（HSV-TK） driven by human mucin 4（MUC4） promoter and analyze its selective cytotoxic effect in vitro on human gastric cancer cells combined with ganciclovir（GCV）.Methods：The expression of MUC4 in gastric cancer cell lines and normal fibroblast cell line NIH3T3 was examined by immunofluorescence assay.To colon the fragment of MUC4 promoter from human genome DNA and construct pGL3-MUC4 recombinant luciferase report vector and analyze its transcriptional activities in SGC-7901,NUGC4 and NIH3T3 cell lines.To construct the recombinant adenovirus carrying HSV-TK driven by human MUC4 promoter（rAdeno-MUC4-TK） based on AdEasyTM adenoviral system and analyze the selective cytotoxic effect of the recombinant adenovirus on SGC-7901 and NUGC4 cells combined with prodrug GCV by MTT assays.Results：MUC4 protein was expressed in SGC-7901 and NUGC4 gastric cancer cell lines,but its expression in NIH3T3 was not detected.MUC4 protein was localized primarily in cytosol and membrane.We cloned 625bp MUC4 promoter fragment by PCR.pGL3-MUC4 showed high transcriptional activities in SGC-7901 and NUGC4 gastric cancer cells stronger than SV40 strong promoter but little in NIH3T3 fibroblast cell.rAdeno-MUC4-TK combined with GCV could evidently inhibit the proliferation of SGC-7901 and NUGC4 gastric cancer cells,but showed low activity in NIH3T3 fibroblast cell.Conclusion：Adenoviral-mediated suicide gene therapy controlled by MUC4 promoter could induce specific cytotoxic effect on SGC-7901 and NUGC4 gastric cancer cells,suggesting that MUC4 promoter can be used as a useful tool for transcriptional targeting of gastric cancer gene therapy.