转化生长因子β-结缔组织生长因子(TGFβ-CTGF)轴在肝脏祖细胞恶性转化中的作用与机制研究

Primary liver cancer can originate from the malignant transformation of hepatic progenitor cells (HPCs). Previous investigations reported that the malignant transformation of HPCs occurred under long-term induction of transforming growth factor β (TGF-β). However，the precise mechanism of TGF-β induced transformation of HPCs remains largely elusive. Our previous studies found that HPCs were activated in the cirrhotic liver and secreted TGF-β and connective tissue growth factor (CTGF), both of which showed oncogenic roles in liver cancer. Besides, we found that TGF-β induced secretion of CTGF in HPCs. Moreover, TGF-β signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. These results suggested that the activated TGFβ-CTGF axis existed in HPCs and was elevated by TGF-β in the cirrhotic liver. Besides, from our previous studies we supposed that in the cirrhotic liver, the TGFβ-CTGF axis may contribute to the malignant transformation of HPCs. To confirm this hypothesis, we plan to carried out in vitro and in vivo experiments to investigate the role of TGFβ-CTGF axis in the proliferation, self-renewal and differention of HPCs. In addition, experiments for investigating the TGFβ-CTGF axis in the cell growth, self-renewal capacity, migration, epithelial-mesenchymal transition, chemoresistance, and tumorigenicity of TGFβ-transformed HPCs are also plan to perform. The downstream cascades of TGFβ-CTGF axis involving in the transformation of HPCs will be screened by antibody array and be verified by in vitro and in vivo functional experiments of HPCs. The results of purposed experiments will elucidate the precise role of TGFβ-CTGF axis in HPCs, and could help us finding a novel strategy for HCC prevention and therapy targeting hepatic tumor-initiating cells.

肝脏祖细胞是原发性肝癌的起源细胞之一。研究表明在TGF-β长期作用下肝脏祖细胞可发生恶性转化而形成肝癌。但其具体机制尚待深入研究。我们前期研究发现：CTGF和TGF-β均在肝硬化组织中高表达，且肝硬化组织中有肝脏祖细胞的激活。肝脏祖细胞存在TGF-β的自分泌，CTGF可在肝脏祖细胞中被外源性和自分泌的TGF-β诱导。因此，TGFβ-CTGF轴在肝脏祖细胞中持续激活并在肝硬化的进展中持续增强。基于TGFβ-CTGF轴在肝硬化与肝脏祖细胞的表达特点及前期报道CTGF在原发性肝癌进展中的促进效应推测TGFβ-CTGF轴促进肝脏祖细胞恶性转化。本项目将通过体内外研究方案明确TGFβ-CTGF轴在肝脏祖细胞生物学功能和恶性转化中的作用及分子机制，为肝癌发生的机制以及防治奠定理论基础并提供新的靶点和思路。

越来越多的证据证实，肝脏祖细胞的恶性转化是肝癌发生的原因之一。然而，肝脏祖细胞恶性转化的分子机制尚待深入研究。本项目在前期研究的基础上提出持续激活的TGFβ-CTGF轴是诱导肝脏祖细胞恶性转化的原因之一这一假说，并设计体内外实验证实。目前本项目已如期完成。主要研究内容及成果概述如下：.1.在二乙基亚硝胺（diethylinitrosamine, DEN）诱导的肝癌大鼠模型中，已经发现在模型中随着肝纤维化的进展和肝癌的发生，肝脏祖细胞激活程度逐渐增多。以此模型为基础，我们发现CTGF合成抑制剂iloprost可明显减少DEN模型中肝脏祖细胞的激活和CTGF的表达，并减少了DEN诱导的肝癌形成。在iloprost的作用下，肝癌起始细胞的标记物如CD44，CD90，CD133，EpCAM的表达也明显降低；.2.在肝脏祖细胞系LE/6与WB-F344中敲减CTGF的表达后发现，肝脏祖细胞的增殖能力，定向分化能力，自我更新能力等干细胞特性均下降；.3.建立长期低剂量TGFβ-1处理的肝脏祖细胞系WB-F344-TβLT，发现其发生了上皮-间质转化，同时自我更新能力增强，同时获得了成瘤能力和软琼脂糖克隆形成能力，提示其已发生恶性转化。在WB-F344-TβLT中敲减CTGF后发现其自我更新能力，软琼脂糖克隆形成能力均下降，且肝癌起始细胞的标记物的表达也明显降低； .4.TGFβ-CTGF轴在肝脏祖细胞中的活性可能受多种因素调控。我们的研究发现，作为TGF-β超家族重要成员的activin A在肝脏祖细胞中同样可上调CTGF的表达，且在纤维化肝脏中同时存在activin A与CTGF的过表达。activin A在肝脏祖细胞中通过激活Smad2/3通路上调CTGF表达。此外，我们发现在肝脏祖细胞中存在activin A-Smad信号的自激活，这一自激活效应调控肝脏祖细胞内CTGF的表达；此外，广泛用于肝移植术后抗排斥药的rapamycin在肝脏祖细胞中可通过抑制mTOR信号进而通过活性氧途径上调有活性的TGF-β的浓度，进而上调CTGF的表达激活TGFβ-CTGF轴。.综上，我们的结果证明TGFβ-CTGF轴在肝脏祖细胞内长期激活诱导其恶性转化，且TGFβ-CTGF轴的活性受多种因素调控。上述结果为肝癌发生和肝纤维化到肝癌的演进阐明了新的分子机制。