移植肝免疫损伤过程中肠道微生态的失衡机制及干预研究

Episodes of warm and cold ischemia reperfusion injury to liver were essential procedures for harvesting and implanting organs. Immune injury caused by liver ischemia reperfusion attack was a leading cause to liver graft dysfunction. Of note, a series of researches revealed a close relationship between intestine microecology and hepatic immune response. We will focus on the scientific issue of intestine microecology imbalance response to immune injury after liver transplantation and associated impacts on post-transplantation immunosuppression. Liver transplantation and ischemia-reperfusion injury model of mouse will be our choice to conduct this study. DGGE fingerprint spectra, liquid mass spectrometry (LC-MS), protein immune imprinting, transfer rate of gel electrophoresis and quantitative polymerase chain reaction (PCR) technology will be available for us to determine quantitative and qualitative changes in intestine microecology associated with barrier function influence of gut after immune injury to liver graft. We will also discuss the characteristic pharmacokinetics of immunosuppressive agents under the intervention of different Microecologics (Antibiotics, Probiotic-preparation and Lactitol), clarify the question of interactions or regulations between intestine microecology and hepatic immune response after an ischemia reperfusion injury to liver, thus a clear understanding of pathophysiological process and mechanisms of metabolic spectrum change under intervention ensues. Furthermore, for the sake of providing theoretical and technical support, our work could enrich the biological theory referred to the interactions or regulations of intestine microecology and post-transplantation immunosuppressive therapy.

肝脏冷、热缺血再灌注是移植肝获取和植入必然经历的过程。其相关的免疫损伤是导致移植肝失功的主要原因之一。国内外报道及本项目组前期研究发现肠道微生态与肝脏病理生理及免疫反应存在密切关系。本项目基于小鼠肝移植模型，围绕肝移植术后免疫损伤过程中肠道微生态失衡及其对肝移植后免疫过程的相互影响这一科学问题，应用DGGE指纹谱、液相质谱（LC-MS）、蛋白免疫印迹、凝胶电泳迁移率和定量PCR技术等实验技术手段，确定肝移植对肠道微生态失衡及肠道屏障功能影响并揭示其机制，探讨不同微生态制剂（抗生素、益生菌制剂、拉克替醇）干预下移植肝病理和代谢的变化规律，阐明肝移植过程中肝脏免疫反应与肠道微生态的相互作用及调控，明确上述生理病理过程及干预方法下整体代谢谱变化及其机制。研究成果可丰富肝移植后肠道微生态与免疫损伤相互影响与调控的生物学理论，并为个体化免疫调控方案提供理论依据和技术支撑。

本课题严格按照计划书方案，开展了以下方面研究：1）通过建立小鼠原位肝移植冷缺血再灌注模型及热缺血再灌注模型，探索肝移植后肠道微生态的改变，分析肝功能的改善与肠道微生态的关系，运用RT-PCR、变性梯度凝胶电泳（DGGE）指纹图谱分析等技术，研究小鼠原位肝移植模型中关键菌株的变化，探索肝脏－微生态轴的反馈调节对于移植肝损伤及微生态失衡的作用机制；2）通过肠道菌群定量分析技术及DGGE聚类分析技术等多种分析方法，动态观察移植后不同时期移植肝功能，并重点分析肠道微生态变化对移植肝急性排斥反应的关系及作用机制；3）运用TUNEL法、流式细胞术、荧光定量PCR、细胞诱导分化等技术，研究肝移植后移植物排异相关的关键分子表达及作用机制；4）研究CD52单抗对肠黏膜屏障功能的影响及其机制，并研究小分子物质甘氨酰谷氨酰胺二肽对肠道微生态结构的影响及益生菌对营养不良小鼠肝移植后肠道屏障功能的恢复的影响；5）研究肠道细菌移位的关键机制，确定紧密连接微区域是细菌易位形成的关键部位，肠道细菌通过受损的TJ侵入机体，导致细菌易位的发生。本研究完成课题计划书任务，为肝移植后免疫抑制调控的研究提供新视野和新思路，对肝移植免疫基础研究及临床工作具有重要的启示意义。