双调控双功效溶瘤腺病毒对肝癌的抑制作用  

Inhibitory effect of dual-regulated oncolytic adenovirus with dual-efficacy on hepatocellular carcinoma

作　　者：孙立臣[1] 潘旭波[1] 周先亭[1] 宋占文[1] 苏长青[2] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院肝胆外科,青岛264000 [2]第二军大学东方肝胆外科医院分子肿瘤研究室

出　　处：《中华实验外科杂志》2013年第3期505-508,共4页Chinese Journal of Experimental Surgery

基　　金：山东省烟台市科学技术发展计划资助项目

摘　　要：目的观察表达Survivin基因小发卡RNA（shRNA）和内皮抑素基因（mE）的双调控双功效的肿瘤特异性溶瘤腺病毒在体内外实验中对肝癌的抗癌活性。方法以重组的双调控双功效的肿瘤特异性溶瘤腺病毒感染肝癌细胞，Westernblot鉴定肝癌细胞基因的蛋白表达，噻唑蓝（MTI＇）实验检测癌细胞增殖活性；建立肝癌裸鼠移植瘤模型，给予溶瘤腺病毒治疗，观察对移植瘤生长的抑制作用。结果Westernblot实验显示，CNHKS00-shRNA—mE病毒增殖能够介导目的基因高效表达，有效抑制Survivin的表达；CNHKS00一shRNA—mE感染肝癌细胞后，对肝癌细胞有特异性杀伤作用，在感染强度（MOI）=2时，CNHKS00．shRNA—mE感染组癌细胞存活率下降到50％以下，而对照病毒CNHKS00．shRNA、CNHKS00一mE,Ad．mE感染组癌细胞存活率均在80％左右；裸鼠SMMC-7721移植瘤模型经病毒治疗后，与对照组比较，CNHKS00一shRNA—mE、CNHKS00-shRNA、CNHKS00一mE、Ad—mE的抑瘤率分别为77．41％（P〈0．01）、59．27％（P〈0．05）、35．96％（P〈0．01）和25．94％（P〈0．05），以CNHK500，shRNA—mE抑瘤作用最强；CNHKS00一shRNA—mE介导mE和shRNA高效表达，不但能够抑制间质肿瘤血管新生[对照组血管密度（MVD）：35．64-6．2，CNHKS00-shRNA-mE组MVD=11．54-3．8，P〈0．01]，而且大量诱导癌细胞凋亡[对照组凋亡率为（5．2±1．5）％，CNHKS00．shRNA-mE组凋亡率为（26．3±7．5）％，P〈0．01]。结论携带shRNA和mE的双功效溶瘤腺病毒CNHKS00一shRNA—mE比任一单功效腺病毒具有更强的肿瘤抑制作用。Objective To investigate the antitumor efficacy of dual-regulated oncolytic adenovirus carrying survivin-small hairpin RNA （shRNA）and mouse end0statin （mE） in hepatocellular carcinoma. Methods The dual-regulated recombinant oncolytic adenovirus was used to infect hepatocellular carcinoma cell lines, and the transgene expression was detected by using Western blotting. The proliferation activity of cancer cells was observed by using methyl thiazol tetrazolium （MTF） assay. The mouse models bearing hepatocellular carcinoma xenografts were established and treated with the oncolytic adenovirus, and the an- titumor efficacy was examined. Results The recombinant adenovirus CNHK500-shRNA-rttE mediated high expression of transgene selectively in cancer cells, inhibited survivin expression and suppressed cancer cetl proliferation. When MOI =2, cell viability was decreased to lower than 50% in the CNHKS00-shRNA-mE group, whereas about 80% in CNHK500-shRNA, CNHK500-mE and Ad-mE groups. As compared with the control group, the inhibitory rate of CNHK500-shRNA-mE, CNHK500-shRNA, CNHK500-mE and Ad-mE was 77. 41% （P 〈 0. 01 ）, 59. 27% （P 〈 0. 05）, 35. 96% （P 〈 0. 91 ） and 25.. 94% （P 〈 0. 05 ）, respec- tively, in mouse models bearing hepatocellular＋ carcinoma xenografts, especially in CNHKS00-shRNA-mE2 treated group. The mE and shRNA mediated by Viruses in tumors not only inhibited angiogenesis （ MVD = 35. 6 ±6. 2 versus 11.5 ± 3. 8 in the control and groups, P 〈 0. 01 ）, but also induced cancer cell apoptosis [ Apoptosis rate of （5.2±1.5） % versus （26. 3±7. 5） % in the control and CNHK500-shRNA-mE groups, P 〈0. 01 ]. Conclusion The dual-regulated oncolytic adenovirus carrying survivin-shRNA and mouse en- dostatin, CNHK500-shRNA-mE, can exert more antitumor efficacy than any mono-efficacy adenovirus.

关 键 词：SURVIVIN基因 小发卡RNA 内皮抑素基因 腺病毒 癌 肝细胞 

分 类 号：R735[医药卫生—肿瘤]