通过干预老年肝脏微环境（Niche）抑制Wnt通路，逆转外源干细胞早衰的机制研究

One solution to scarce of the donors for liver transplantation is to initiate the use of marginal donors.In the severe ageing social background,the use of elderly donor is an important ongoing concern. Our study found the exogenous stem cells being presenility in elderly organs when we tried to use exogenous stem cells to improve the function of elderly donor livers. We further blocked the stem cells’ Wnt signaling pathway and introduced the TERT gene to avoid the presenility. But the effect is not obvious or not durable. Thus we believe that there are underlying factors in the ageing microenvironment of elderly organs leading to the presenility of stem cells. On the basis of our previous work, we plan to find out the differential protein factors along the elderly, middle-aged and young-aged livers by using the proteomic methods(SELDI-TOF MS、Tricine-SDS-PAGE、LC-MS/MS) and protein microarray technology and clear the interaction of protein by using the pSOS yeast two-hybrid system. To find out the factor sets which can reverse the presenility of the exogenous stem cells in elderly organs, we mimick the internal environment by stromal cells culture medium and interfere the protein regulators with viral infections and siRNA. And then to identify the prevention and reversal effects on elderly organ microenvironment intervention to the presenility of stem cells, we interfere the donor liver with the use of vascular perfusion in vitro and detect the ischemia-reperfusion injury, liver function, the ageing of stem cells and the Wnt pathway in mice liver transplantation model. The purpose of our study is to provide the fundamental research and the experimental study for increasing the clinical using rate of elderly donor.

肝脏移植供体匮乏的解决方案是启用边缘供肝，在老龄化严重的社会背景下老年供肝备受关注。本课题组在外源干细胞改善老年供肝功能的研究中发现外源干细胞的早衰现象，并通过阻断外源干细胞Wnt通路和导入TERT基因进行改善，但效果不明显。因此相信老年器官的微环境中存在导致干细胞早衰的根源。本课题拟在前期工作基础上用基质细胞培养液模拟体内微环境；通过SELDI-TOF MS、Tricine-SDS-PAGE、LC-MS/MS和蛋白质芯片（PNT227）等技术靶标老、中、青年肝脏微环境中的差异蛋白；pSOS酵母双杂交技术明确蛋白质相互作用；病毒感染和siRNA干扰整体调节微环境蛋白因子，找到逆转早衰的因子组合。进而在大鼠肝脏移植模型中离体干预老年供肝，通过检测移植后缺血再灌注损伤、肝功能、干细胞衰老及Wnt通路等指标，明确干预老年微环境对防止干细胞早衰的作用，为提高临床老年供肝的利用率提供理论和实验基础。

肝脏移植供体匮乏的解决方案是启用边缘供肝。在老龄化严重的社会背景下老年供肝备受关注。干细胞微环境（stem cell niche）是干细胞生活的特定环境，维持并调节干细胞的生物学特性。本课题组在外源干细胞改善老年供肝功能的研究中发现外源干细胞在老年受体中的早衰现象，因此怀疑老年器官的微环境中存在导致外源干细胞早衰的因素。深入研究肝脏干细胞微环境的结构和功能有助有充分激发肝脏干细胞在肝脏再生中的修复潜力。肝星状细胞作为肝脏干细胞niche中的重要支持细胞在肝脏干细胞的增殖和分化中的作用及机制尚未及时阐明。.本研究首先研究了大鼠和人静息态肝星状细胞（HSCs）的分离和无血清原代培养技术，观察不同活化阶段HSCs的生物学特性。探索并掌握了肝脏类器官的培养技术。同时通过HSCs与细胞肝干细胞共培养发现，与老年小鼠肝脏提取的HSCs相比，年轻小鼠肝脏提取的HSCs能够提高肝干细胞的增殖能力且衰老相关指标低表达。通过对老年小鼠和青年小鼠HSCs培养基超滤和浓缩提取了HSCs中分泌蛋白。通过差异蛋白质组学技术，对不同龄来源的HSCs分泌蛋白进行差异蛋白分析，寻找具有明显差异的蛋白谱。提取了老年小鼠和青年小鼠HSCs分泌的外泌体，并进行全转录组测序，寻找了具有明显差异的非编码RNA。本课题进行过程中，通过对HSCs分离技术的探索，找到了一种更加简便，高效率的HSCs分离方法，并成功实现了HSCs的无血清培养。本研究中发现与不同年龄来源小鼠HSCs共培养后，肝脏干细胞的增殖和衰老指标有显著区别。通过对不同年龄来源小鼠HSCs外泌体测序后发现了显著的差异非编码RNA。.本研究解决了小鼠和人部分肝脏组织静息态肝星状细胞分离的难题，探索了不同年龄来源HSCs对肝干细胞增殖和衰老的影响，寻找了其中的差异蛋白和差异非编码RNA。