【摘要】 目的利用AdEasy系统构建低氧射线双调控的TK表达载体——腺病毒Ad.HRE.CArG.HSV-TK,并观察其对人乳腺癌细胞系Bcap37和MDA-MB-435的放射增敏作用。方法用分子克隆技术构建插入HRE.CArG.HSV-TK片段的Ad.HRE.CArG.HSV-TK。CsCl梯度离心法纯化病毒,用AdEasy系统GFP标签测定病毒功能滴度。Ad.HRE.CArG.HSV-TK体外转导细胞48h,流式细胞仪测GFP阳性率,Real-time RT-PCR和Western blot检测TKmRNA和蛋白表达。MTT法检测腺病毒(Ad)转导联合放射(RT)对细胞的生长抑制作用和放射增敏比(SER)。结果 Ad.HRE.CArG.HSV-TK滴度为2.1×109TU/mL。50 MOI病毒转导Bcap37和MDA-MB-435细胞48 h,GFP阳性率分别为92%和93%,TK基因表达明显增加(P<0.05)。Ad与RT联用组(Ad+RT)细胞生长抑制率明显高于Ad组和相应同等剂量RT组(P<0.05),SER为1.55。结论应用AdEasy系统可制备同时表达GFP和TK的重组腺病毒。腺病毒对乳腺癌细胞具放射增敏作用,为进一步开展乳腺癌的基因放射治疗奠定基础。更多还原

【Abstract】 Objective To construct adenovirus vector Ad.HRE.CArG.HSV-TK regulated by hypoxia and radiation via AdEasy system and to observe its radiosensitization effect in human breast cancer cell lines Bcap37 and MDA-MB-435.Methods Using molecular clone technologies,Ad.HRE.CArG.HSV-TK inserted with HRE.CArG.HSV-TK fragment was constructed.The functional titer of adenovirus was detected by GFP tag expression.Cell lines of Bcap37 and MDA-MB-435 were transducted with adenovirus in vitro,48 h later,expression rate of GFP was detected by flow cytometry.TK gene mRNA and protein expression were detected with real-time RT-PCR and Western blot respectively.The growth inhibition effect and sensitizer enhancement ratio(SER)of adenovirus(Ad)transduction combined with radiation(RT) were detected with MTT assay.Results The final titers of adenovirus was 2.1×109TU/mL.Bcap37 and MDA-MB-435 cells transducted by 50 MOI adenovirus in vitro for 48 h,positive rate of GFP was 92% and 93%,respectively.TK expression increased dramatically(P<0.05).Growth inhibition rate in group of Ad+RT was significantly higher than that in Ad group and the same dose of RT group(P<0.05).SER were both 1.55.Conclusion The recombinant adenovirus expressing GFP and TK gene can be generated via AdEasy system.The adenovirus has radiosensitization effect on breast cancer cells.It may serve as a basis for later gene radiotherapy research in breast cancer.更多还原