瘤内原位转导UPRT基因治疗小鼠胃癌的实验研究

背景与目的:5-FU是临床上重要的化疗药物,但总体疗效有限,如何提高其抗癌疗效一直是临床上亟待解决的难题。本研究采用基因治疗的原理与方法,探讨UPRT(uracilphosphoribosyltransferase)基因对5-FU杀伤效应的增敏作用。方法:采用PCR技术从大肠杆菌K12基因组中扩增UPRT基因,并构建pLXSN逆转录病毒表达载体,进一步转染小鼠胃癌细胞株MFC。采用MTT法检测转导UPRT基因前后MFC对5-FU敏感性的变化。建立615小鼠胃癌皮下移植瘤模型,瘤内反复多次注射携带UPRT基因的浓缩、纯化的逆转录病毒,同时腹腔内给予5-FU,观察抑瘤效果。结果:体外MTT法检测结果显示,转导UPRT基因后可使MFC对5-FU的敏感性提高约17.26倍。以逆转录病毒介导UPRT基因瘤内原位转导,同时联合应用5-FU进行治疗,结果肿瘤生长明显受抑(P<0.005),抑瘤率为87.18%。结论:UPRT基因修饰小鼠胃癌细胞株MFC,能明显提高MFC对5-FU杀伤的敏感性,以逆转录病毒载体介导UPRT基因肿瘤原位基因转导,同时联合应用5-FU,能获得良好的抑瘤效应。

In situ gene therapy for murine gastric carcinoma with UPRT/5-FU enzyme/prodrug system mediated by retrovirus

Background &Objective:5-Fluorouracil(5-FU),a widely used chemotherapeutic dru g,has a limited overall effect in the treatment of human soli d tumors due to resistance.This study was designed to investigate if anti tumor activation of5-FU could be enhanced by transfection of uracil phosphoribosyltransfe rase(UPRT)gene.Methods:The UPRT gene encoding uracil phosphoribosyltransferase was amplified from Escherichia Coli K12genome and subcloned into retrov irus expression vector pLXSN,Recombinant retrovir us was packaged and used further to in fect murine gastric cancer cell line MFC.The sensitivity of MFC transfected with UPRT gene to 5-FU was determined by MT T method.In situ gene therapy was performed by regional repeated injections of c oncentrated and purified recombinant retrovirus carrying UPRT gene int ratumorally and followed by administration of 5-FU i ntraperitoneally(i.p.).Results:The 5-FU sensitivity in MFC transfected with the UPRT gene increased 17.26-fold compared to the control cells.In situ transfe ction of the UPRT gene mediated by retrovirus vector followed by the administration of 5-FU(10mg /kg)significantly inhibited the tumor growth(P<0.005)with an inhibition rate of87.18%and prolonged the survival.Conclusion:Transfection of UPRT gene can render the murine gastr ic cancer cell line MFC be more sensitive to low concentration of 5-FU and significantly improve the antitumor effect of 5-FU both in vitro and in vivo.