基因-病毒治疗系统CNHK300-murine endostatin治疗肝癌的实验研究  

Treatment of hepatocellular carcinoma with a novel gene-viral therapeutic system CNHK300-murine endostatin

作　　者：李根丛[1] 聂明明[2] 杨甲梅[3] 苏长青[1] 孙立臣[1] 钱炎珍[1] Jonathan Sham 方国恩[2] 吴孟超[3] 钱其军[1] 

机构地区：[1]第二军医大学附属东方肝胆外科医院病毒和基因治疗实验室,上海200438 [2]第二军医大学附属长海医院普外科 [3]第二军医大学附属东方肝胆外科医院肝胆外科,上海200438 [4]香港大学临床肿瘤系

出　　处：《中华医学杂志》2004年第11期943-948,共6页National Medical Journal of China

基　　金：国家自然科学基金国际合作重大项目资助( 3 0 12 0 160 82 4);国家"八六三"高新技术研究发展计划基金资助项目 ( 2 0 0 1AA2 170 3 1)

摘　　要：目的　探讨一种新的基因 病毒治疗系统CNHK30 0 murineendostatin(简称CNHK30 0 mE)对肝癌的治疗作用。方法　利用基因重组技术构建一种用人端粒酶逆转录酶 (hTERT)启动子控制腺病毒E1A基因表达并携带内皮抑素基因的基因 病毒治疗系统 ;通过 5 0 %组织培养感染剂量 (TCID5 0 )方法和细胞活性检测法四氮唑盐比色试验 (MTT法 )观察CNHK30 0 mE在 2种肝癌细胞株 (HepGII及Hep3B)及 1株正常的成纤维细胞株 (MRC 5 )中的病毒增殖能力和对细胞的杀灭能力 ;通过肝癌SMMC772 1细胞裸鼠皮下移植瘤模型观察该病毒对肝癌生长和对肿瘤血管生成的抑制作用 ;利用Western印迹法和酶联免疫吸附实验 (ELISA)法检测内皮抑素基因在体内和体外的表达。结果分别感染肝癌细胞株和正常细胞株 96h后病毒的增殖倍数相差 32 96倍 ,其达到半数杀伤量 (ED50 )时的感染复数 (MOI)值相差 2 5倍 ,差异有显著意义 ,且两者均明显优于ONYX 0 15 ;CNHK30 0 mE能明显抑制肝癌皮下移植瘤内血管的生成 ,对肿瘤生长的抑制作用与携带同一治疗基因的非增殖型腺病毒和不携带治疗基因的增殖型腺病毒ONYX 0 15相比均增强 (P均 <0 0 1) ;其所介导的内皮抑素基因在体内和体外的表达量均与携带该基因的非增殖型腺病毒载体相比均增高 (P <0 0Objective To investigate the anti tumor effects of a novel gene viral therapeutic system CNHK300 murine endostatin (CNHK300 mE) in hepatocellular carcinoma (HCC) Methods A novel gene viral therapeutic system named CNHK300 mE was constructed by employing the human telomerase reverse transcriptase (hTERT) promoter to drive the expression of adenovirus E1A gene and cloning the therapeutic gene murine endostatin (mE) into the adenovirus genome Hepatocellular cells of the HepGII and Hep3B lines and normal fibroblasts of the MRC 5 line were cultured and infected with the viruses CNHK300 mE, ONYX 015, replicative adenovirus without therapeutic gene, and Ad mE, non replicative adenovirus with the same therapeutic gene Ninety six hours after the infection, tissue culture infectious dose 50 method was used to detect the titer of virus in the supernatants MTT method was used to examine the cytolytic capability The expression of E1A and mE were examined by Western blotting ELISA assay was used to detect the transgene expression of mouse endostatin Healthy nude Balb/c mice were injected with hepatic cancer cells of the SMMC 7221 line Forty mice with tumors5～8 mm in diameter wererandomly divided into 4 groups of 20 mice: CNHK300 mE group (CNHK300 mE was injected into the tumor once every other day for 5 times),Ad mE group (Ad mE was injected), ONYX 015group (ONYX 015 was injected), and control group (diluent of virus was injected) 3, 7, 14, 21, and 28 days after the initial injection the size of tumor was examined 48 hours after the finish of the whole course of treatment, the mice were killed ELISA was used to detect the expression of mE in blood The growth of tumor was examined by HE staining, The angiogenesis in the tumor was observed by immunohistochemistry with von Willebrand factor and The proliferation of transplanted tumor was observed by immunohistochemistry with adenovirus envelop protein hexon Results Ninety six hours after the infection of the cells by CNHK300 mE

关 键 词：基因-病毒治疗系统 CNHK300-murine ENDOSTATIN 肝癌 端粒酶逆转录酶 HTERT 内皮抑素 

分 类 号：R735.7[医药卫生—肿瘤]