可溶性MULT1通过活化肝脏固有淋巴细胞调控血吸虫病肝纤维化的机制研究

Intervention of liver fibrosis can significantly improve the life quality of patients with advanced schistosomiasis. The activation and the trans-differentiation of hepatic stellate cells into myofibroblasts is the critical event during the formation of liver fibrosis due to the deposition of schistosome eggs. Recent studies have shown that liver NK cells can impact the activated hepatic stellate cells in various ways. NKG2D is a major activating receptor of NK cells, it was reported that both NKG2D and its ligend (NKG2DLs) were markedly modulated after schistosome infection. Our preliminary data indicated an increased level of sMICA, a soluble NKG2DL in human, in sera from schistosomiasis patients, administration of the plasmid loaded with the recombinant sMULT1 alleviated the hepatic fibrosis progress in mice infected with Schistosoma japonicum, and an elevated expression of NKG2D on the NK cells was also detected, which was consistent with the elevated IFN-γ level in serum and enhanced IFN-γ secretion by NK cells in spleen. Our proposal is designed with genetically modified mice with sMULT1 over expression in liver to explore the role of hepatic ILCs, including the NK cells, in regulation of schistosomiasis liver fibrosis. The phenotype and the function of liver ILC1 subgroups would also be measured. The goal of these surveys is to form the cells and cytokines network involved in the regulatory effect of sMULT1 on hepatic fibrosis caused by schistosomiasis. Our research will clarify a novel mechanism of the liver ILCs in schistosomiasis liver fibrosis process and provide theoretical basis for the development of the new therapeutic target on liver fibrosis.

干预肝纤维化进程可显著改善晚期血吸虫病人的生存质量。血吸虫产卵后引起的HSC活化并转分化为成肌纤维细胞是肝纤维化发生的中心环节，肝脏NK细胞能多途径调控HSC。NKG2D 是肝脏NK细胞上的主要活化受体，血吸虫感染能影响HSC的NKG2DLs水平和NK细胞NKG2D水平。前期研究发现，血吸虫病人血清中可溶性NKG2DLs的水平升高；过表达sMULT1可改善血吸虫病小鼠的肝纤维化，并伴有脾NK细胞上NKG2D的表达上调及肝、血清IFN-γ的升高。本项目拟研究肝脏NK细胞在血吸虫病肝纤维化中的作用，利用诱导性肝脏过表达sMULT1基因修饰小鼠，明确感染血吸虫后sMULT1对NK细胞和其它ILC亚群的影响以及sMULT1活化NK细胞后调控HSC的具体机制，为阐明肝脏ILCs对血吸虫病肝纤维化进程和转归的影响提供理论依据，并为探寻纤维化治疗的新靶点提供思路和实验依据。

肝纤维化是血吸虫病患者的主要病理损伤之一，干预血吸虫病肝纤维化，是血吸虫病防治的重点。NK细胞、IFN-γ是血吸虫病肝纤维化的重要调控因素， NKG2D在NK细胞参与肝纤维化调控中有重要作用，sMULT1及sMICA可通过与NKG2D作用增强NK细胞的活性。过表达sMULT1对血吸虫病小鼠肝纤维化的改善作用有待确定，其机制还有待阐明；血吸虫感染后cNK，LrNK和其他ILCs在肝脏内的组成比例、各自表型和功能的变化也有待研究。.本项目构建了sMULT1条件性敲入小鼠，通过注射表达Cre的质粒或他莫昔芬诱导其与肝脏特异性Cre-ERT小鼠杂交双阳性后代都可实现sMULT1的过表达。血清中分别为约每毫升数百纳克和数百微克。体内NKG2D配体在应激环境下上调表达，我们建立sMULT1的基因修饰工具小鼠是诱导性表达的，更接近生物体体内的相应的生理和疾病场景，具有潜在的医药科研应用价值。.进一步研究显示，该基因修饰小鼠在生理条件下过表达sMULT1对小鼠健康和机体的免疫状态几乎没有影响，但是在受到血吸虫感染后过表达sMULT1可以增强NK细胞核NKT细胞表面NKG2D的表达，减小虫卵肉芽肿面积和胶原沉积面积，抑制纤维化进程。但是，机体多种免疫细胞组成、表型无显著性差异；血清中炎性细胞因子IFN-γ、IL-6、IL-10、IL-12P70、MCP1和TNF的水平也无明显变化。结合前期研究数据表明：尽管基因修饰小鼠过表达sMULT1对血吸虫病肝纤维化的抑制作用的机制与预期有差别，但是其较好的干预效果和NKG2DL-NKG2D轴的重要作用是确定的，可溶性NKG2D配体具有作为血吸虫病纤维化的干预工具的应用前景。.我们新建立的新的检测MULT1蛋白方法不仅可用于重组MULT1的检测，理论上也可以用于天然MULT1的检测，包括膜型的和分泌型的，该方法灵敏度比现有报道的ELISA方法提高了一个数量级，有利于将来MULT1相关研究中MULT1蛋白的定量。此外，项目还对血吸虫感染后不同阶段ILC1和B细胞组成和表型、功能变化做了探索性研究。这些都为后续深入研究血吸虫病相关机理及NKG2D配体在干预血吸虫病中的作用机理奠定了良好的基础。