肝星状细胞参与肝硬化时肝细胞向肝祖细胞转化的实验研究

In the setting of liver cirrhosis, massive necrosis of hepatocytes occurred, and the ability of hepatocytes to proliferate was impaired; subsequently, the liver progenitor cells (oval cells) were activated and then differentiated into hepatocytes, liver function was restored through regenerative hepatocytes derived from liver progenitor cells(LPC). Liver function is the most important prognostic factors for survival in patients with liver cirrhosis. Previous studies showed liver progenitor cells derived from biliary epithelial cells or hepatic stellate cells. Recent breakthrough studies demonstrated that hepatocytes were major source of liver progenitor cells in chronic liver injury and liver cirrhosis. Our preliminary data showed that the hepatocytes located in portal area were converted into LPCs, it revealed that niche of liver progenitor cells played a key role in ductal metaplasia of hepatocytes into liver progenitor cells. Activated hepatic stellate cells (HSCs )and extracellular matrix (ECM) were located in LPC niche．The HSCs activation and ECM deposition produced by HSC activation occurred as an important event, prior to LPC activation. Our preliminary data showed the expression of LPC markers was induced in hepatocytes after co-cultured with activated HSCs. Thus, we hypothesized that HSC is a crucial factor in ductal metaplasia of hepatocytes into liver progenitor cells. The aim of our project is to evaluate the involvement of HSCs in ductal metaplasia of hepatocytes into liver progenitor cells using transgenic and knockout models of mice, and explore the mechanism of this process through in vitro experiment of LPC and the experiment of coculture of hepatocytes and HSCs. It will provide theoretical principle for improving liver regeneration through altering the biological characteristic of HSCs, and then it will benefit the patients of liver cirrhosis.

肝硬化时，肝细胞大量坏死、增殖能力受限，进而启动肝祖细胞的激活，肝祖细胞进一步分化形成肝细胞,代偿肝功能；肝功能是决定肝硬化预后的关键因素。既往研究认为肝祖细胞来源胆管上皮和肝星状细胞；最近的研究证实，在慢性肝损伤、肝硬化时，肝祖细胞来源于肝细胞。我们前期研究发现，只有汇管区的肝细胞才可以转化为肝祖细胞，这就提示局部微环境是其转化的关键要素。肝星状细胞活化及其形成的细胞外基质存在于肝祖细胞龛（微环境）中，其是肝祖细胞激活前的重要的生物学事件。我们的前期研究证实，肝星状细胞可促进损伤的肝细胞表达肝祖细胞标记物。因此，我们推测肝星状细胞是肝硬化时肝细胞向肝祖细胞转化的关键要素，本研究以模式动物为研究工具在整体水平验证肝星状细胞参与肝细胞向肝祖细胞转化，并通过肝祖细胞的体外实验、肝细胞和肝星状细胞的共培养实验阐述其机制；从而有望通过改变肝星状细胞的生物学特性而促进肝脏再生，改善肝硬化病人的预后。

肝硬化时，肝细胞大量坏死、增殖能力受限，进而肝细胞转化为肝祖细胞，肝祖细胞进一步分化形成肝细胞,代偿肝功能；肝功能是决定肝硬化预后的关键因素。局部微环境（肝祖细胞龛）是肝细胞转化为肝祖细胞的关键要素。活化的肝星状细胞及其形成的细胞外基质存在于肝祖细胞龛中，其是肝祖细胞激活前的重要的生物学事件。因此，我们推测活化型肝星状细胞是肝硬化时肝细胞向肝祖细胞转化的关键要素，在肝硬化的小鼠模型中，可以观察到肝祖细胞的大量激活和肝星状细胞的活化，利用DAPT去除活化型的肝星状细胞，其可以抑制肝祖细胞的激活。利用R26R-EYFP小鼠，标记肝细胞；在此基础上，制备肝硬化模型，并使用DAPT去除活化型肝星状细胞，证实活化型肝星状细胞参与肝细胞向肝祖细胞的转化作用。最后我们探索肝星状细胞参与肝细胞向肝祖细胞转化的机制，发现活化型肝星状细胞分泌层粘蛋白，层粘蛋白通过整合素ɑvβ6受体参与肝细胞向肝祖细胞转化；通过上述研究，进而为将来利用此机制改变局部微环境，促进肝脏再生，改善肝硬化病人的预后。