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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 the leading cause of cancer death. Some advances have been made in the traditional treatment of early-stage liver cancer, but the recurrence, metastasis and drug resistance of cancer have not yet been fundamentally solved. These phenomena Which can be explained by the cancer stem cell (CSC) .In this study, the three-dimensional sphere cells of MHCC-97H cells were obtained by suspension enrichment culture and their stem cell characteristics were detected. By deleting 5 The 24-bp bases of the E1A CR2 region of adenovirus type Adenovirus and the expression of E1A gene were regulated by Wnt active transcription element TCF / TEF. At the same time, the anti-oncogene TSLC1 was inserted and the double-targeting oncolytic adenovirus Ad.wnt-E1A (△ 24 bp ) -TSLC1, the transformation, killing, apoptosis and migration of EMT (epithelial-mesenchymal transition) cells were detected by MTT, crystal violet staining, Hoechst, cell scratch, Western blotting, Transwell and immunofluorescence The results showed that MHCC-97H sphere cells cultured in suspension enriched with self-renewal, differentiation, rest and drug resistance, high expression of liver cancer stem The cell surface markers (such as CD133 and so on), the recombinant virus treatment showed obvious killing effect and inhibit cell migration and invasion of the characteristics of targeting inhibit MHCC-97H sphere cells stronger (P <0.001), and the recombinant virus Effectively inducing apoptosis of hepatocarcinoma stem cells through caspase pathway.Therefore, recombinant adenovirus Ad.wnt-E1A (△ 24 bp) -TSLC1 may become a therapeutic agent targeting hepatoma stem cells and has certain clinical application prospects.