抑制活化的肝星状细胞RSK1表达促进肝细胞增殖的机制研究

Hepatic fibrosis is the excessive wound-healing response to liver injury, and both activated HSC and hepatocyte proliferation are necessary for this response. There is a balance between hepatocyte proliferation and HSC activation, which is associated with the progression on hepatofibrogenesis. Ribosomal S6 kinase is the first downstream effector of of the extracellular signal-regulated kinase (ERK) 1/2 pathway, which has been involved in cell proliferation in chronic liver disease. In our previous study, we have confirmed that RSK is up-regulated in activated HSC, and inhibition of RSK1 in activated HSCs that lead to the suprression of HSC proliferation. However, the role of RSK1 in the hepatocyte proliferation in the fibrotic liver is elusive. It is interesting whether RSK1 regulates hepatocyte proliferation with or without HSC participation in hepatic fibrosis. In the current study, we will employ the RSK1 conditional knock-out of HSC of mouse and the co-culture of activated HSC and hepatocyte to explore the mechanism that links the inhibition of RSK1 in activated hepatic stellate cells and the promotion of hepatocyte proliferation by immunohistochemistry, western blot(WB), RT-qPCR, chromatin immunoprecipitation(ChIP) assay, RNA interference(RNAi), MTT in vivo and in vitro respectively. This study will clarity the relationship among RSK, activated HSC and hepatocyte proliferation, and provide a new insight and a new target to hepatic fibrosis.

肝纤维化是肝脏对慢性损伤的过度修复，HSC活化和肝细胞增殖是肝脏修复的两个必要条件。研究表明，肝细胞和HSC之间通过crosstalk，形成动态平衡，共同影响纤维化进程。RSK是ERK1/2通路下游的一级效应分子，是经典的促细胞增殖因子。我们前期研究表明，RSK1在活化的HSC中高表达，下调RSK1可抑制HSC增殖和胶原表达；而肝纤维化中，RSK1对肝细胞增殖的调控尚未见报导。RSK1是直接促进肝细胞增殖，还是通过HSC间接调控肝细胞增殖，是非常有意义的问题。本研究利用HSC的RSK1基因条件敲除小鼠肝纤维化动物模型和肝细胞与活化的HSC共培养细胞模型，采用免疫组化、WB、RT-qPCR、ChIP、RNAi、MTT等技术，通过体内和体外研究，明确抑制活化的HSC中RSK1表达促进肝细胞增殖的作用及其机制，阐明RSK1、活化的HSC和肝细胞增殖三者间关系，旨在为肝纤维化研究提供新思路、新靶点。

肝纤维化是肝脏对慢性损伤的过度修复，HSC活化和肝细胞增殖是肝脏修复的两个必要条件。目前研究表明，肝细胞和HSC之间通过crosstalk形成动态平衡，共同影响纤维化的进展。RSK是ERK1/2通路下游的一级效应分子，是经典的促细胞增殖因子。我们前期研究表明，在肝纤维化中，RSK在HSC中高表达，下调RSK1可以抑制HSC增殖和胶原表达；而在肝纤维化中，RSK1对肝细胞增殖的调控作用尚未见研究报导。本研究：1.利用RNAi腺病毒在体感染，下调大鼠体内RSK1表达， 联合C1-3–GT选择性清除肝纤维化大鼠体内活化的HSC，通过免疫组化和qRT-PCR的方法观察alph-SMA、COL A1、TGF-beta表达减少，而HGF以及肝细胞PCNA、AFP、ki67表达明显上调，提示HSC活化减少，肝细胞增殖增加，表明RSK1并不是直接调节肝细胞增殖，而是通过调节HSC的活化参与肝细胞增殖；抑制肝纤维化中RSK1表达，并不抑制肝细胞增殖，而是抑制 HSC增殖，并促进肝细胞增殖；2.采用染色质免疫共沉淀测序分析肝纤维化肝组织中HSC内 RSK1下游核转录因子C/EBPβ结合靶 DNA序列中HGF表达上调，TGF-β表达下调，进一步阐明RSK1/ C/EBPβ通过下调TGF-β表达抑制HSC增殖，上调HGF促进肝细胞增殖的分子机制；3.采用接触与非接触共培养的方法，将感染了AdshRSK1的活化状态的肝星状细胞株HSC-T6与大鼠原代肝细胞共培养。结果发现两种培养方式均能观察到HSC的增殖抑制、迁移降低以及分泌减少，同时接触共培养组肝细胞的增殖明显高于非接触共培养组，提示HSC通过分泌细胞因子以及细胞接触抑制两种方式共同调节肝细胞增殖。总结：本研究结果表明RSK1是一个重要的肝纤维化治疗新靶点，抑制RSK1表达具有抑制HSC增殖和促进肝细胞修复的双重效果，具有潜在临床应用价值。