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

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 0

Objective 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 virus was replicated by 6329±1830 and 25 136±6890 times in the HepGII and Hep3B cells respectively, 3296 and 12 824 times higher than in the MRC 5 cells respectively The replication multiples of ONYX 015 virus in the HepGII and Hep3B cells were 2040±450 and 3980±740 times respectively, both significantly lower than those of CNHK300 mE virus (both P <0 05) However, no remarkable replication of Ad mE virus was seen in the Western blotting showed the expression of therapeutic gene mE in HepGⅡ and Hep3B cells infected with CNHK300 mE on Ad mE. Hep3B cells, the band of CNHK300 mE being thicker than that of Ad mE and the band of Ad mE being similar to that of CNHK300 mE in the MRC 5 cells ELISA showed that the expression of mE protein in the HepGII cells infected by CNHK300 mE virus increased time dependently during the period of 7 days after virus infection, significantly higher than the expression in the HepGII cells infected by Ad mE virus ( P <0 05) The tumors of the CNHK300 mE virus infected mice were significantly smaller than those of the Ad mE and ONYX 015 infected mice (both P <0 01) ELISA showed that the mE protein content in the blood of the CNHK300mE infected mice was significantly higher than that of the Ad mE group ( P <0 05) Hexon immunohistochemistry showed patchy and diffuse positive staining related to apoptosis and necrosis of tumor cells in the transplanted tumors of the CNHK300 mE virus infected mice, however, only sporadic positive staining was seen in the Ad mE virus infected mice Conclusion Being capable of specifically replicating in the telomerase positive HCC cells and mediating effective expression of therapeutic gene in vitro and in vivo, the novel gene viral therapeutic system CNHK300 mE holds potential for treatment of HCC