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[目的]探讨hTERT启动子驱动的HSV-TK基因对人胰腺癌细胞patu8988的杀伤作用。[方法]应用分子生物学方法构建hTERT启动子调控下的TK基因真核表达载体。经同源重组产生重组腺病毒PSG-TP-TK。同法制备PSU-CMV-TK。利用重组腺病毒PSG-TP-TK和PSU-CMV-TK感染人胰腺癌细胞株patu8988和正常人原代成纤维细胞,加入GCV。用MTT和流式细胞仪检测PSG-TP-TK对各种细胞的杀伤作用;应用RT-PCR检测转染腺病毒后肿瘤细胞和正常细胞中TK基因的表达情况。[结果]PSG-TP-TK对端粒酶阳性的人胰腺癌细胞有明显的杀伤作用,而对端粒酶阴性的正常细胞则无杀伤作用(P<0.05)。hTERT启动子诱导人胰腺癌细胞株patu8988凋亡的效能与CMV启动子相似,两者差异无显著性(P>0.05)。PSU-CMV-TK转染后,在patu8988细胞和正常人原代成纤维细胞中均可检测到TK基因;而PSG-TP-TK转染后只有patu8988细胞中有TK基因表达,正常人原代成纤维中则无。[结论]hTERT启动子驱动HSV-TK基因治疗是一种新的胰腺癌靶向治疗方法,具有更高的靶向性和高效性。
[Objective] To investigate the killing effect of hTERT promoter-driven HSV-TK gene on human pancreatic cancer cell line patu8988. [Method] The eukaryotic expression vector of TK gene regulated by hTERT promoter was constructed by molecular biology method. Recombinant adenovirus PSG-TP-TK was generated by homologous recombination. The same method to prepare PSU-CMV-TK. Human pancreatic cancer cell line patu8988 and normal human primary fibroblasts were infected with recombinant adenovirus PSG-TP-TK and PSU-CMV-TK, and GCV was added. The killing effect of PSG-TP-TK on various cells was detected by MTT and flow cytometry. The expression of TK gene in tumor cells and normal cells was detected by RT-PCR. [Result] PSG-TP-TK had obvious killing effect on telomerase positive human pancreatic cancer cells, but no anti-telomerase negative normal cells (P <0.05). The apoptosis of human pancreatic cancer cell line patu8988 induced by hTERT promoter was similar to that of CMV promoter. There was no significant difference between the two groups (P> 0.05). After transfection with PSU-CMV-TK, TK gene was detected in both patu8988 cells and normal human primary fibroblasts. Only TK gene expression was found in patu8988 cells after transfection with PSG-TP-TK, There is no fibrosis. [Conclusion] hTERT promoter-driven gene therapy of HSV-TK is a new targeted therapy for pancreatic cancer with higher targeting and efficiency.