非酒精性脂肪性肝炎时YB-1调控肝祖细胞增殖及其在肝纤维化中的作用

In non-alcoholic steatohepatitis (NASH), the proliferation of hepatocytes is impaired due to ballooning degeneration and hepatic progenitor cells(HPCs) are stimulated to proliferate, accompanied by activation of hepatic stellate cells(HSCs) and liver fibrogenesis. However, the mechanism which regulates the proliferation of HPCs and the effect of HPCs on HSCs remain unclear. YB-1 is a transcriptional factor involving in embryogenesis and tissue repair. In this study, immunohistochemistry assays are performed to detect YB-1expression and the proliferation of HPCs and HSCs in liver sections from NASH patients. In vitro experiments, we will examine the proliferation of HPCs after up or down regulating YB-1expression and the effect of HPCs on HSCs behaviors. Further, chromatin immunoprecipitation assay coupled with next generation sequence (ChIP-Sequence) is performed to screen the potential target genes of YB-1 in HPCs and DNase foot printing as well as EMSA are adopted to confirm the detailed binding sites of YB-1 on HPCs chromatin. In addition, dual luciferase reporter assay is conducted to assess the transcriptional activity of YB-1 on the target gene. Besides, transgenic mice will be introduced to examine the effect of YB-1 on HPCs-mediated liver regeneration and NASH-associated fibrosis. This study would help us better understand the regulatory mechanism of HPCs activation and proliferation as well as clarify the influence of HPCs on HSCs proliferation and liver fibrosis.

非酒精性脂肪性肝炎（NASH）时肝祖细胞（HPC）增生，伴星状细胞（HSC）活化和肝纤维化。HPC增殖受多种因素调控，并影响HSC活化和肝纤维化。我们前期发现转录因子YB-1在NASH患者HPC中高表达。本课题拟在组织水平观察NASH时YB-1表达以及HPC和HSC增殖情况；在细胞水平，上调或下调YB-1评价其对HPC增殖和基因表达的影响，将HPC和HSC共培养观察HPC对HSC增殖和分泌细胞外基质影响；在分子水平，采用ChIP-Sequence筛选YB-1调控HPC增殖的靶基因，DNA酶足迹法和EMSA确定结合位点的核酸序列，荧光素酶报告基因检测YB-1对靶基因启动子转录活性影响;在动物水平，用转基因小鼠制备NASH模型观察YB-1对HPC增殖和肝再生的影响及其对HSC增殖和肝纤维化的影响。深入阐明HPC增殖调控机制及其对HSC的影响，为NASH相关肝纤维化的治疗提供实验室依据。

YB-1 能通过调控多种基因启动子的转录活性抑制或促进相应蛋白的表达，进而调控细胞增殖。我们前期研究发现健康人肝组织不表达YB-1，而慢性乙型肝炎患者肝穿刺标本YB-1阳性，并主要在胆管细胞和肝祖细胞(HPC)中表达。因此本课题研究了YB-1调控肝祖细胞增殖的机制。结果显示，低表达YB-1能显著抑制HPC增殖；RNA-sequence显示YB-1 能调控Hedgehog信号通路、Wnt信号通路以及MAPK信号通路；ChIP-sequence显示YB-1在HPC中的靶基因参与细胞粘附、Wnt信号通路、Hedgehog信号通路，且YB-1能结合于Gli2启动子区；ChIP-PCR和荧光素酶报告基因实验表明YB-1能与Gli2结合并正调控其转录；Real-time PCR和Western blot证实YB-1可以调控Hedgehog信号通路上多种信号分子的表达；Gli1/Gli2阻滞剂可显著抑制HPC的增殖。 据此我们认为， YB-1能与Gli2启动子区结合并正向调控其转录，进而调控HPC的增殖。该研究成果已在《肝脏》杂志发表论著1篇。.此外，在本研究中，我们构建了YB-1f/f Foxl1-Cre小鼠，并研究了在CDE和DDC小鼠模型中，YB-1在HPC中的作用。结果表明，在CDE和DDC模型中，肝损伤伴随着YB-1的表达升高、HPC增殖增多和自噬的激活。在CDE和DDC模型中，YB-1f/fCre+/-小鼠的肝损伤和纤维化程度低于YB-1f/fCre-/-小鼠。YB-1在体外和体内促进了HPC的增殖和自噬。转化生长因子-β（TGF-β）诱导YB-1的核转位，从而促进了HPC的增殖和自噬。YB-1核转位促进了Atg7的转录，并参与了TGF-β介导的HPC激活。总之，TGF-β在HPC中诱导的YB-1核转位促进了HPC的增殖和自噬，Atg7参与了YB-1介导的HPC扩张和肝纤维化。该研究成果已在Molecular Basis of Disease杂志发表论著1篇。