新型基因病毒治疗系统CNHK300-内皮抑素对肝细胞癌的强效抗肿瘤作用

Potent antitumoral effects of a novel gene-viral therapeutic system CNHK300-mEndostatin in hepatocellular carcinoma.

作者信息

Li Gen-Cong, Yang Jia-Mei, Nie Ming-Ming, Su Chan-Ging, Sun Li-Chen, Qian Yan-Zhen, Fang Guo-En, Sham Jonathan, Wu Meng-Chao, Qian Qi-Jun

机构信息

Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai Rd, Shanghai 200438, China.

出版信息

Chin Med J (Engl). 2005 Feb 5;118(3):179-85.

PMID:15740644

Abstract

BACKGROUND

The expression of therapeutic gene and its anti-tumor effects will be augmented and a synergism of oncolytic virus with the therapeutic gene is speculated. This study was undertaken to assess the anti-tumor effects of a novel gene-viral therapeutic system CNHK300-mEndostatin (CNHK300-mE) in hepatocellular carcinoma (HCC).

METHODS

A novel gene-viral therapeutic system named CNHK300-mE was constructed using the human telomerase reverse transcriptase (hTERT) promoter to drive the expression of the adenovirus E1A gene and cloning the therapeutic gene mouse endostatin into the adenovirus genome. By the tissue culture infectious dose 50 (TCID50) method and cytoviability assay, the replicative and cytolytic capabilities of CNHK300-mE in two HCC lines (HepGII and Hep3B) and one normal cell line (MRC-5) were analyzed, and the transgene expressions of mouse endostatin in vitro and in vivo were detected by Western blotting and ELISA assay. Tumor growth suppression and anti-angiogenesis effects in vivo were investigated using nude mice xenografts model derived from SMMC-7721 HCC cells.

RESULTS

The 3296-fold replicating capacity of CNHK300-mE in HCC cell lines versus in the normal cell line at 96 hours post infection and the 25-fold effective dose for killing 50% cells (ED50) in the normal cell line versus HCC cell lines, which were both superior to ONYX-015, were observed. Tumor growth suppression of CNHK300-mE superior to either Ad-mE or ONYX-015 was demonstrated (P < 0.01) and the anti-angiogenic effects in vivo superior to Ad-mE were also observed with immunohistochemical staining of von Willebrand factor. In comparison with non-replicative adenovirus Ad-mE, the transgene expression of mE mediated by CNHK300-mE was significantly higher in vitro (P < 0.005) and in vivo (P < 0.05).

CONCLUSION

Being capable of replicating in and lysing the telomerase-positive HCC cells and mediating effective expression of the therapeutic gene in vitro and in vivo, the novel gene-viral therapeutic system CNHK300-mE is potentially effective in the treatment of HCC.

摘要

背景

治疗性基因的表达及其抗肿瘤作用将得到增强，推测溶瘤病毒与治疗性基因存在协同作用。本研究旨在评估新型基因病毒治疗系统CNHK300 - mEndostatin（CNHK300 - mE）对肝细胞癌（HCC）的抗肿瘤作用。

方法

构建一种名为CNHK300 - mE的新型基因病毒治疗系统，使用人端粒酶逆转录酶（hTERT）启动子驱动腺病毒E1A基因的表达，并将治疗性基因小鼠内皮抑素克隆到腺病毒基因组中。通过组织培养感染剂量50（TCID50）法和细胞活力测定，分析CNHK300 - mE在两种肝癌细胞系（HepGII和Hep3B）和一种正常细胞系（MRC - 5）中的复制和细胞溶解能力，并通过蛋白质免疫印迹法和酶联免疫吸附测定法检测小鼠内皮抑素在体外和体内的转基因表达。使用源自SMMC - 7721肝癌细胞的裸鼠异种移植模型研究体内肿瘤生长抑制和抗血管生成作用。

结果

感染后96小时，CNHK300 - mE在肝癌细胞系中的复制能力是正常细胞系的3296倍，在正常细胞系中杀死50%细胞的有效剂量（ED50）是肝癌细胞系的25倍，两者均优于ONYX - 015。证实CNHK300 - mE的肿瘤生长抑制作用优于Ad - mE或ONYX - 015（P < 0.01），并且通过血管性血友病因子免疫组织化学染色观察到其体内抗血管生成作用优于Ad - mE。与非复制性腺病毒Ad - mE相比，CNHK300 - mE介导的mE转基因表达在体外（P < 0.005）和体内（P < 0.05）均显著更高。