表达miR-199*的脂肪MSC用于原发性肝癌靶向治疗的研究

The sensitivity of primary hepatocellular carcinoma (HCC) to the radiotherapy and chemotherapy is low, therefore, it is critical for us to explore the new effective target points for gene therapy. Recently, much attention to the relationship between miR -199a* and HCC has been paid. miR -199a* expresses at low level during the development of HCC, but the exogenous miR -199a* can inhibit the proliferation of hepatoma cells. Also miR -199a* can suppress the replication of HCV, which is accociated with HCC. So miR -199a* may be a promising candidate target for HCC treatment. Selection of optimal vector is another important issue for gene therapy. MSC, as new vector for gene therapy, has been recognized as a safe, highly efficient and tumor-specific vector. Several studies have confirmed that MSCs carrying anti-tumor genes could be used for treating cancers. Among various types of MSCs, human adipose tissue-derived MSCs (AMSC) serve as one of the most attractive vehicles for gene therapy because of their high throughput, lack of ethical concerns, and availability and ease of isolation.We found that the proliferation of hepatoma cells was markedly suppressed by introduction of AMSC carrying miR -199a*. Therefore, the aims of present study are to investigate the mechanisms underlying the anti-tumor and anti-viral effects of AMSC-carrying miR -199a* and to provide the theoretical base for this treatment of HCC.

原发性肝细胞肝癌(HCC)的放化疗敏感性差，探索有效的HCC新型靶点是其基因治疗的关键点。miR-199a*与HCC的关系日益受到关注, HCC的发生发展伴随其低表达，而外源导入miR-199a*则可抑制肝癌细胞增殖；另发现其还可抑制HCV复制，而HCV持续感染与肝癌相关，因此其有望作为肝癌防治的新靶点。基因治疗的另一关键问题是载体选择，间充质干细胞(MSCs)作为一类新型的基因治疗运载系统具有高效、安全、肿瘤靶向性等诸多优点。表达特定抗癌基因的骨髓MSC用于肿瘤靶向治疗已取得了一些进展。而成体脂肪MSC(AMSC)作为一类可重复获取、供者不适度低的干细胞将更具前景。我们已发现导入miR-199a*的AMSC可有效抑制肝癌细胞增殖，因此本项目拟进一步明确AMSC-miR-199*在体内外的抗肿瘤和抗病毒疗效和机制，从而为临床应用表达miR-199*的AMSC进行肝癌靶向治疗提供理论依据。

原发性肝癌(hepatocellular carcinoma, HCC)是人类常见的恶性肿瘤之一，WHO发表的《全球癌症報告2014》指出我国肝癌死亡占全球肝癌死亡人数的51%。其化疗敏感性差、侵袭性强，导致复发率极高，5年病死率更是超过90％，因此明确肝癌治疗新靶点，探索抗肿瘤或化疗增敏的新策略意义重大。而MiR-199*与HCC的发生发展关系密切，在临床肝癌病例中其往往表现为低表达，近年来的体外研究也显示外源性的导入miR-199*可有效抑制HCC的增殖。同时有报道显示其还可抑制HCV的复制，在因丙肝而导致的HCC中发挥重要的作用，因此其有望作为肝癌防治的新靶点。但是要实现miRNA的临床应用，安全高效的体内运载体系开发亦是重要因素。间充质干细胞(MSCs)作为一类新型的基因治疗运载系统具有高效、安全、肿瘤靶向性等诸多优点。其中成体脂肪MSC(AMSC)作为一类可重复获取、供者不适度低的干细胞将更具前景。本课题通过转染miR-199*的表达质粒至AMSC，结果显示其不仅不影响AMSC的分化潜能，并且其所分泌的exosome中miR-199*的含量较其它miRNA要高2-3倍，是较好的miR-199*载体。而且其分泌的exosome可实现体外的细胞间传输，对丙肝模型的受体细胞JC1-Huh7.5.1中的HCV可起到极好的抑制效果。另外在抗肝癌方面，携带miR-199*的exosome可经旁分泌的方式进入肝癌细胞，并通过抑制原癌基因Yap1而有效促进DNA损伤类药物5-FU的化疗增敏效应。在荷瘤小鼠的体内实验中，通过瘤内注miR-199*-Exo也可有效提升5-FU的化疗效果。项目有望为HCV相关肝肝癌治疗提供一个新思路、开辟一条新途径，建立一种临床可行的 、有效的治疗新方法。