肝脏微环境中IGF-2在移植细胞再殖肝脏的作用及其机制研究

At present cell transplantation, instead of orthotopic liver transplantation, is a promising option for cellular therapy of end-stage liver diseases. However, the inability to efficiently expand transplanted cells in recipient liver is one of the main bottlenecks of therapeutic liver repopulation. And the underlying molecular mechanisms of cell repopulation need to be better defined in order to translate this technique into clinical use. Creation of a suitable hepatic microenvironment that can stimulate proliferation in transplanted cells or senescent hepatocytes still remain elusive and are not well defined. Our early work has been concluded that proliferation of senescent hepatocytes is reversed after transplanted into young microenvironment. And we also found that insulin-like growth factor 2 (IGF-2) was up-regulated in young hepatic microenvironment of FAH-/- mice and secreted by the host hepatocytes. When down-regulation the expression of IGF-2, the repopulated rate of transplanted hepatocytes was decreased. In addition, we identified IGF-2 declined in aged environment when compared with young mice, followed with weak repopulation. All of these results indicated a hypothesis that IGF-2 could promote liver repopulation of cell transplantation and reverse proliferated ability of senescent hepatocytes. In this project, based on Fumarylacetoacetate hydrolase knockout (Fah-/-) mouse that is an excellent repopulation model, we will carry out hepatocytes transplantation to confirm the valid effect and the mechanism of IGF-2 on increased proliferation of transplanted hepatocytes or senescent hepatocytes. Then we try to create the optimal dosage, route, and interval of administration of IGF-2 by intraperitoneal (i.p.) injection, which are more suitable for clinical use. Our aim is to further characterize IGF-2 is one of the possible driving forces for the growth of transplanted cells and understanding the mechanisms regulating repopulation can improve the effect of transplantation, which is very important in clinic in the future, and offer a possible technological strategy to achieve therapeutic repopulation.

作为原位肝移植的替代方案，细胞移植在终末期肝病治疗中具有重要的应用前景。而移植细胞在宿主肝脏内增殖缓慢以及动物模型中实现肝脏再殖的调控机制还缺乏认识，极大地限制了其临床应用。现有的研究还未提出具有临床转化的促增殖方案以及如何解决老年患者衰老肝细胞增殖抑制问题。前期研究中，申请人证明了年轻肝脏微环境可以使得衰老肝细胞恢复增殖能力，并发现宿主微环境中存在着潜在促进细胞增殖的胰岛素样生长因子IGF-2，也证明了IGF-2与移植细胞的增殖速率和再殖效率成正相关。因此申请人提出“年轻微环境中IGF-2促进并逆转衰老供体细胞增殖能力，实现衰老逆转”的设想。本课题拟采用可实现肝脏再殖的FAH-/-小鼠模型，探讨IGF-2促进移植细胞再殖肝脏并逆转细胞衰老的作用及调控机制。通过建立基于IGF-2的干预手段，力争找到可以破解限制肝脏疾病细胞治疗向临床过度的瓶颈因素并为基于衰老的增殖相关疾病治疗提供理论基础。

细胞移植作为原位肝移植的替代方案在终末期肝病治疗中具有重要的应用前景。而移植细胞在宿主肝脏内增殖缓慢以及动物模型中实现肝脏再殖的调控机制还缺乏认识，极大地限制了其临床应用。现有的研究还未提出具有临床转化的促增殖方案以及如何解决老年患者衰老肝细胞增殖抑制问题。本研究主要基于可实现肝脏再殖的FAH-/-小鼠动物模型，探讨移植细胞能够实现肝脏再殖的作用机制并试途建立临床上的治疗方案。首先，我们发现随着宿主肝脏的损伤，肝细胞逐渐表达衰老相关标志物如衰老相关β半乳糖苷酶、细胞周期抑制因子和DNA损伤标志物等。通过对衰老相关分泌因子的检测发现衰老的肝细胞明显表达胰岛素样生长因子IGF2。随着移植细胞的增殖与再殖，宿主衰老的肝细胞逐渐被替换而减少，与此同时IGF2的表达也随之减少。通过对再殖肝脏中移植细胞集落和宿主肝脏的比较发现衰老相关指标只表达在宿主肝细胞中，而增殖相关的标志物则主要表达于移植集落中。体外培养的原代肝细胞加入IGF2后其增殖速率明显上升，而且IGF2主要是通过激活PI3K和MAPK信号通路促进细胞增殖的。体内抑制宿主肝细胞中IGF2的表达后移植细胞的再殖能力明显降低，相应的细胞增殖相关蛋白如CyclinA2和CyclinD1的表达也随之降低。其次，我们在倒千里光碱加三分之二肝切以及倒千里光碱加四氯化碳损伤模型中同样证实了IGF2可以促进移植肝细胞的增殖。进一步为了探讨宿主微环境对移植细胞再殖的作用，我们对肝实质中细胞外基质以及细胞间连接相关蛋白的检测，发现肝脏损伤后细胞间连接减弱，细胞外基质成分降低，可能为移植细胞的再殖提供了空间。而在人肝病组织中同样发现损伤肝脏中广泛存在衰老的肝细胞，且表达IGF2。通过这些研究结果，我们得出结论是宿主肝脏损伤会促进肝细胞发生衰老并分泌衰老相关因子IGF2，促进移植细胞的增殖。同样宿主肝脏的结构也会发生改变，为移植细胞的再殖提供空间环境。本研究为临床上破解限制细胞移植治疗肝衰竭等终末期肝病提供新的策略。