论文部分内容阅读
目的:构建人粘蛋白(MUC4)启动子驱动下的单纯疱疹胸苷激酶基因(HSV-TK)重组腺病毒,研究其对SGC-7901胃癌细胞的靶向杀伤作用。方法:克隆MUC4启动子区625bp活性序列,构建重组荧光素酶报告基因载体pGL3-MUC4,检测其在SGC-7901胃癌细胞及NIH3T3成纤维细胞中的转录活性。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rAdeno-MUC4-TK,感染SGC-7901及NIH3T3,经更昔洛韦(GCV)处理,MTT法检测细胞活力,TUNEL法检测细胞凋亡。结果:成功扩增出大小为625bp的MUC4启动子序列。pGL3-MUC4在SGC-7901细胞中具有强转录活性,转录活性高于强启动子SV40 6.6倍,而NIH3T3成纤维细胞系中几乎无转录活性。构建重组腺病毒rAdeno-MUC4-TK,MTT法和TUNEL检测结果显示,其与GCV联合能够诱导SGC-7901细胞凋亡,产生靶向细胞毒作用。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 HSV-TK recombinant adenovirus driven by human mucin (MUC4) promoter and to study its targeted killing effect on SGC-7901 gastric cancer cells. Methods: The 625bp active sequence of MUC4 promoter was cloned. The recombinant luciferase reporter gene vector pGL3-MUC4 was constructed and its transcriptional activity in SGC-7901 gastric cancer cells and NIH3T3 fibroblasts was detected. Using AdEasyTM adenovirus system as a vector, HSV-TK recombinant adenovirus rAdeno-MUC4-TK driven by MUC4 promoter was constructed and transfected into SGC-7901 and NIH3T3 cells. The cells were treated with ganciclovir (GCV) TUNEL method was used to detect apoptosis. Results: The MUC4 promoter sequence of 625bp in size was successfully amplified. pGL3-MUC4 has strong transcriptional activity in SGC-7901 cells, which is 6.6 times higher than that of strong promoter SV40, while almost no transcriptional activity in NIH3T3 fibroblast cell line. Construction of recombinant adenovirus rAdeno-MUC4-TK, MTT assay and TUNEL assay showed that the combined with GCV can induce SGC-7901 cell apoptosis, resulting in targeted cytotoxicity. CONCLUSION: HSV-TK recombinant adenovirus combined with GCV driven by human MUC4 promoter has targeted killing effect on SGC-7901 gastric cancer cells, and MUC4 promoter can be used as a targeted gene therapy tool for gastric cancer.