Cbl-b/C-Cbl双敲除DC在小鼠肝硬化发生发展中的作用及机制研究

Cirrhosis is a chronic disease results from different mechanisms of liver injury that lead to necroinflammation and fibrogenesis. The mortality and mobility are increased in the worldwide. Recently, it was reported that immune cells play import roles in the formation of liver cirrhosis. In the present study, the mice display sever liver fibrosis and cirrhosis under HE staining when Cbl-b and C-Cbl both were ablated in dendritic cells. The subsets of DC either in spleen or in liver were changed. Also Cbl-b/C-Cbl deficient DC produce higher IL-6, IL-12, IFNr and IL-10, compared to the control DC. Finally, the dKO mice died from liver cirrhosis in 4-6 month years old. FACS analysis suggests that CD4/CD8 T cells were activated in liver and spleen. The percentage of MDSC was increased. Based on above preliminary data, we intent to further study the role of Cbl-b/C-Cbl on the DC biological function by modern cellular and molecular techniques. Through Knock out and chimera mice model to extensively point out it is DC itself or induce liver cirrhosis by other immune cells, and explore the detail mechanisms of Cbl-b/C-Cbl in DC.

肝硬化是由多种机制诱导的肝细胞损伤，从而引起肝脏的坏死性炎症和纤维化形成所导致的一类慢性肝病。其发病率和致死率在全球呈现不断上升的趋势。近年来报道认为免疫细胞在肝硬化发生，发展过程中同样发挥很重要的作用。本研究中我们的预实验结果显示当在DC中特异性同时敲除Cbl-b/C-Cbl后，HE染色显示小鼠有严重的肝硬化症状。肝脏和脾脏DC亚群比例发生变化，且DC分泌的IFNr、IL-12、IL-6、IL10等细胞因子升高。小鼠在4-6月左右死于肝硬化。肝脏和脾脏中CD4、CD8T细胞呈现活化状态，MDSC比例升高。在此预实验基础上，我们拟通过现代细胞生物学和分子生物学手段进一步明确Cbl-b/C-Cbl对DC生物学功能的影响，通过基因敲除小鼠和嵌合小鼠模型，明确是DC细胞本身还是DC通过其它免疫细胞导致小鼠发生肝硬化；并进一步探讨Cbl-b/C-Cbl影响DC生物学功能的具体分子机制。

为探讨Cbl-b、C-Cbl在DC中的作用，我们将Cbl-b-/-和C-Cblf/f小鼠和CD11C Cre小鼠杂交得到在DC中特异性敲除Cbl-b、C-Cbl的小鼠， 使我们吃惊的是在小鼠发育到3-4个月时，双敲除Cbl-b，C-Cbl的小鼠发生100%的死亡，解剖发现小鼠肝脏有显著的硬化，病理切片检查发现2月龄小鼠肝脏呈现肝纤维化，而4个月左右得小鼠呈现肝硬化的症状。进一步分析CD8+、CD103+、CD11b+ DC细胞发现主要以CD8+以及CD103+DC增多为主。但DC表面的CD80、CD86等DC活化指标并未发生显著变化。肝脏中T细胞的活化指标CD44、CD62L等指标均提示T细胞被活化。实时定量PCR检测发现DC的细胞因子表达谱发生变化，表现为：IL-6、IL-12、IFNr等表达升高，TGFb表达不变。而将CD11CCre+Cbl-b-/-C-Cblf/f小鼠和IL-6-/-、IL-12-/-、IL-17-/-及IL-1R-/-小鼠杂交得到CD11CCre+Cbl-b-/-C-Cblf/f IL-6-/-、CD11CCre+Cbl-b-/-C-Cblf/f IL-12-/-、CD11CCre+Cbl-b-/-C-Cblf/f IL-17-/-、CD11CCre+Cbl-b-/-C-Cblf/f IL-1R-/-小鼠，观察发现IL-6、IL-1R敲除后小鼠的死亡时间显著延长，而IL-12、IL-17敲除后小鼠死亡时间没显著变化。说明IL-6、IL-1对小鼠肝硬化的发展起一定作用。通过转染293T细胞体外研究Cbl-b和C-Cbl和各种DC分化相关的转录因子发现Cbl-b和C-Cbl可能通过调控IRF4、IRF8、Notch1、Batf3、ID2、PU.1等在DC中其重要调控作用的转录因子相互作用进而影响DC的分化发育。仔细研究发现dKO CD103+DC细胞的凋亡减少，而Brdu染色发现其Brdu+细胞增多，Western-blot检测caspase3/7/8/9发现caspase7和caspase9的cleavge片段水平显著降低，提示Cbl-b可能由于Cbl-b/C-Cbl双敲除后导致DC细胞凋亡，进而影响了DC细胞对自身抗原的递呈，从而引起自身免疫性肝硬化。预试验结果初步证明可能通过Erk的磷酸化而影响BIM的降解，从而参与了线粒体凋亡。