HO-1-磷酸化c-Jun-miR-221/222-LIFR轴调控小体积供肝再生的机制研究

Inhibition of liver regeneration is a key factor leading to small-for-size syndrome (SFSS) in small-for-size graft(SFSG). The research on promoting SFSG regeneration is an important breakthrough to overcome SFSS. Our previous studies have shown that HO-1 is involved in liver regeneration as a novel positive regulator, but its regulatory mechanism remains unclear. Our previous study found that HO-1 was closely related to SFSG regeneration; HO-1 could promote miR-221/222 expression by activating c-Jun; biosignal analysis suggested that LIFR was a downstream target of miR-221/222. This study intends to use the in vitro and in vivo models to confirm the role of HO-1 and its downstream p-c-Jun-miR-221/222-LIFR in SFS liver transplantation,and to investigate the nuclear import of HO-1 and its relationship with p-c-Jun, miR-221/222 and LIFR in the hypoxia/reoxygenation (H/R) environment by immunoprecipitation,laser confocal,dual luciferase reporter assay and other techniques. The results of the study aim to reveal a new mechanism for promoting SFSG regeneration and to clarify the role of HO-1 regulatory axis in it, which is expected to provide a new target for clinical treatment of SFSS.

肝脏再生抑制是导致小体积供肝（SFSG）常发生小体积综合征（SFSS）的关键因素，研究促进SFSG再生对SFSS至关重要。我们前期研究表明HO-1作为新型正调节因子参与了肝再生，但其调控机制仍不明确。我们前期研究发现：HO-1与SFSG再生密切相关；HO-1可通过激活c-Jun促进miR-221/222表达；生信分析提示LIFR是miR-221/222下游靶点。本项目拟利用体内外模型证实HO-1及下游磷酸化c-Jun-miR-221/222-LIFR轴在小体积（SFS）肝移植中的作用；结合免疫共沉淀、激光共聚焦、双荧光素酶报告基因分析等技术探讨HO-1在缺氧/复氧（H/R）环境下的入核反应及其与磷酸化c-Jun、miR-221/miR-222及LIFR之间的相互作用。研究结果旨在揭示一个全新的促进SFSG再生的机制，明确HO-1调控轴在其中的作用，可望为SFSS临床治疗提供新的靶点。

肝脏再生抑制是导致小体积供肝（SFSG）常发生小体积综合征（SFSS）的关键因素，研究促进SFSG再生对SFSS至关重要。我们前期研究发现：HO-1与SFSG再生密切相关；HO-1可通过激活c-Jun促进miR-221/222表达；生信分析提示LIFR是miR-221/222下游靶点。预实验是在人源HL7702细胞中,本项目在大鼠肝细胞clone9细胞株中再次验证。在clonce9细胞中发现H/R，WtHO-1，△hHO-1均引起HO-1入核效应。△hHO-1组活性较WtHO-1明显降低。沉默HO-1引起细胞凋亡升高和细胞周期阻滞，过表达△hHO-1作用相反。CO-IP、RT-pCR、Weston-blot、双荧光素酶报告基因、周期和凋亡实验进一步明确了HO-1-c-Jun与miR-221/222的相互作用及对肝细胞活性、增值能力的影响。在动物实验中，Ki67实验验证了HO-1对移植后肝细胞增值的正向作用。ALT,AST和大体标本再生率进一步验证了HO-1对移植肝的保护作用。RT-pCR和Weston-blot进一步明确了动物实验水平HO-1/磷酸化c-Jun、miR-221/222和LIFR之间的调控关系。因临床收集肝移植患者标本过程中遇到困难，我们更换计划为检测HO-1在肝再生中的作用。选取了临床研究常用的肝大部切除模型，来模拟SFSG。结果发现术前低水平HO-1 与术后LD发生率、发病率、肝脏功能再生率下降有关。证实了HO-1是不良临床结果的独立预测因子。我们首次验证HO-1水平可作为预测LD和临床预后的有效生物标记物。进而延伸出HO-1对SFS临床移植肝术后可能的正向作用，HO-1-磷酸化c-Jun-miR-221/222-LIFR轴可能在这个过程中起着关键作用，为我们将来的临床实验指明了方向，增加了信心。为将来成果大规模转化及临床应用提供了扎实的理论基础。