AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepat...AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepatocellular carcinoma （HCC）. METHODS： CNHK500-p53 was constructed by using human telomerase reverse transcriptase （hTERT） promoter to drive adenovirus E1a gene and hypoxia response element （HRE） promoter to drive adenovirus E1b gene. p53 gene expressing cassette was inserted into the genome of replicative virus. Viral replication experiments, cytopathic effect （CPE） and methyl thiazolyl tetrazolium （MTT） assay were performed to test the selective replication and oncolytic efficacy of CNHK500-p53. RESULTS： Immunohistochemistry verified that infection with CNHK500-p53 was associated with selective replication of adenovirus and production of p53 protein in telomerase-positive and hypoxia-inducible factordependent HCC cells, p53 protein secreted from HepG2, infected with CNHK500-p53 was significantly higher than that infected with nonreplicative adenovirus Ad-p53 in vitro （388 ± 34.6 μg/L vs 76.3 ± 13.17 μg/L）. Viral replication experiments showed that replication of CNHK500-p53 and CNHK500 or WtAd5, was much stronger than that of Ad-p53 in tested HCC cell lines. CPE and H1-F assay indicated that CNHK500-p53 selectively replicated in and killed HCC cells while leaving normal cells unaffected. CONCLUSION： A more efficient gene-viral system is developed by combining selective oncolysis with exogenous expression of p53 against HCC cells.展开更多
目的探讨以HRE和hTERT修饰条件复制型腺病毒携带Egr-1介导的Smac(CARd.pE-Smac)对人食管癌细胞Eca109周期和凋亡的影响。方法利用氯化钴进行化学乏氧,病毒感染滴度为5MOI[Multiplicity of infection(virus/cell)]感染人食管癌Eca109细胞...目的探讨以HRE和hTERT修饰条件复制型腺病毒携带Egr-1介导的Smac(CARd.pE-Smac)对人食管癌细胞Eca109周期和凋亡的影响。方法利用氯化钴进行化学乏氧,病毒感染滴度为5MOI[Multiplicity of infection(virus/cell)]感染人食管癌Eca109细胞24h,并进行2Gy照射,12h后分别利用Western blotting检测Smac蛋白的表达,PI染色和AnnexinⅤ-FITC双染,流式细胞术检测细胞周期和细胞凋亡变化。实验分为control、CARd.pESmac、hypoxia、2Gy、H+CARd.pE-Smac、CARd.pE-Smac+2Gy和H+CARd.pE-Smac+2Gy组。结果 CARd.pE-Smac感染常氧和乏氧的Eca109细胞后均未见Smac蛋白表达增加,而2Gy照射后,常氧、感染CARd.pE-Smac和乏氧再感染CARd.pE-Smac均能使Smac蛋白表达增加,尤其以三者联用后Smac表达增加最大;感染CARd.pESmac未对细胞周期有明显改变,而乏氧、2Gy和感染CARd.pE-Smac任二者(乏氧与2Gy除外)或者三者联用均能显著增加S期和G2/M期细胞百分比增加(P<0.05,P<0.01),尤其以三者联用作用更强;而且,对于细胞凋亡的诱导作用与周期进程变化的规律相类似。结论 HRE和hTERT修饰条件复制型腺病毒携带Egr-1介导的Smac在人食管癌细胞Eca109中显著过表达,且能够导致细胞发生S期延迟和G2/M期阻滞,并诱导细胞发生凋亡。展开更多
基金Supported by the Major State Basic Research Development Program (973 Program) of China, No. 2003CB515507
文摘AIM： To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepatocellular carcinoma （HCC）. METHODS： CNHK500-p53 was constructed by using human telomerase reverse transcriptase （hTERT） promoter to drive adenovirus E1a gene and hypoxia response element （HRE） promoter to drive adenovirus E1b gene. p53 gene expressing cassette was inserted into the genome of replicative virus. Viral replication experiments, cytopathic effect （CPE） and methyl thiazolyl tetrazolium （MTT） assay were performed to test the selective replication and oncolytic efficacy of CNHK500-p53. RESULTS： Immunohistochemistry verified that infection with CNHK500-p53 was associated with selective replication of adenovirus and production of p53 protein in telomerase-positive and hypoxia-inducible factordependent HCC cells, p53 protein secreted from HepG2, infected with CNHK500-p53 was significantly higher than that infected with nonreplicative adenovirus Ad-p53 in vitro （388 ± 34.6 μg/L vs 76.3 ± 13.17 μg/L）. Viral replication experiments showed that replication of CNHK500-p53 and CNHK500 or WtAd5, was much stronger than that of Ad-p53 in tested HCC cell lines. CPE and H1-F assay indicated that CNHK500-p53 selectively replicated in and killed HCC cells while leaving normal cells unaffected. CONCLUSION： A more efficient gene-viral system is developed by combining selective oncolysis with exogenous expression of p53 against HCC cells.
基金国家自然科学基金杰出青年资助项目(No.30325043)国家重点基础研究发展(973)计划资助项目(No.2004CB518804)+5 种基金上海市卫生局医学领军人才基金上海市科委攻关计划(No.064119539)Supported by the National Natural Science Foundation of China(No.30325043)The Major State Basic Research Development Program(973)of China(No.2004CB518804)the Shanghai Outstanding Field Leaders ProgramKey Science and Technology Research Project of Shanghai(No.064119539)
