靶向Wnt信号的溶瘤腺病毒抑制肝癌干细胞研究  

Inhibition of Hepatocellular Stem Cells by Oncolytic Virus Targeting Wnt Signaling Pathway

作　　者：章健[1] 来维洁 李强[1] 金槿 肖伯端 郭婉[1] 王毅刚[1] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018

出　　处：《生物化学与生物物理进展》2017年第4期326-337,共12页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(81272687);浙江省自然科学基金(LZ13H160004;LY16H160056);浙江省大学生科技创新活动计划(2016R406007)资助项目~~

摘　　要：肝细胞癌(hepatocellular carcinoma,HCC)是全球第五大癌症并成为癌症死亡的主要原因,传统治疗早期肝癌取得了一定的进展,但是癌症的复发、转移和耐药仍未得到根本解决,这些现象可通过癌症干细胞理论(cancer stem cell,CSC)进行解释.本研究通过悬浮富集培养的方法,获得了MHCC-97H细胞的三维立体球细胞(sphere cell),并检测其干细胞特性,通过删除5型腺病毒的E1A CR2区域24 bp碱基,并用Wnt活性转录元件TCF/TEF调控E1A基因表达,同时插入抗癌基因TSLC1,得到了双靶向溶瘤腺病毒Ad.wnt-E1A(△24 bp)-TSLC1,通过MTT、结晶紫染色实验、Hoechst、细胞划痕、蛋白质印迹技术、Transwell及免疫荧光技术检测重组病毒对肝癌类干细胞的EMT(epithelial-mesenchymal transition)转化、杀伤、凋亡以及迁移的作用.结果表明,悬浮富集培养的MHCC-97H sphere细胞具有自我更新、分化能力、静息性以及耐药性,高表达肝癌干细胞表面标志物(如CD133等),重组病毒处理后表现出明显的杀伤效果及抑制细胞迁移与侵袭的特性,靶向抑制MHCC-97H sphere细胞能力更强(P<0.001),且重组病毒能有效诱导肝癌类干细胞通过caspase途径发生凋亡.因此,重组病毒Ad.wnt-E1A(△24 bp)-TSLC1有可能成为靶向肝癌干细胞的治疗药物,具有一定的临床应用前景.Hepatocellular carcinoma（HCC） is the fifth largest cancer in the world and is the main cause of cance death. Common treatment for early hepatocellular carcinoma has made some progress, but cancer recurrenc metastasis and drug resistance have not been fundamentally resolved, which can be explained by cancer stem ce theory（cancer stem cell, CSC）. In this study, we obtained MHCC-97 H sphere cells by suspension enrichmen culture method and detected their stem cell characteristics. We deleted the 24 bp of the E1A CR2 region of th adenovirus type 5 and used the Wnt transcription element TCF/TEF to regulate E1A and then inserted th anti-oncogene TSLC1 into the vector to obtain the double targeting oncolytic adenovirus Ad.wnt-E1A（△24 bp TSLC1. The effect of recombinant adenovirus Ad.wnt-E1A（△24 bp）-TSLC1 on the killing effect, cell apoptos and migration of hepatocarcinoma stem-like cells were detected by MTT, crystal violet, Hoechst staining, ce scratch, Western blot and immunofluorescence, respectively. The results show that MHCC-97 H sphere cells hav the capability of self-renewal and differentiation, and high expression of hepatocyte stem cell surface marker（suc as CD133）. After dealt with recombinant virus, it showed obvious killing effect and inhibition of cell migration an EMT（Epithelial-mesenchymal transition）. Therefore, ability of targeting inhibition of 97 H sphere cells wa significantly higher than that of 97 H cells（P〈0.001）. The recombinant virus could induce apoptosis o hepatocellular stem-like cells through caspase pathway. Therefore, the recombinant adenovirus Ad.wnt-E1A（△24 bp TSLC1 may be a promising therapeutic agent for targeting hepatoma stem cells, which has certain applicatio value to clinical treatment.

关 键 词：肝癌类干细胞 溶瘤腺病毒 WNT信号通路 TSLC1 

分 类 号：Q813[生物学—生物工程]