肝肠轴：肝脏及肠道B淋巴细胞对非酒精性脂肪性肝病作用机制的研究

Hepatic microenviornment variation is the major pathophysiological change during the progression of nonalcoholic fatty liver disease (NAFLD). Although many investigations about the immune factors in NAFLD had done, they were mainly focused on the function of T lymphocytes. Few studies were related with the influence of B lymphocytes in the pathogenesis of NAFLD. However, B lymphocytes play an important role in the pathogenesis of obesity and other diseases often associated with NAFLD. Our previous data showed the level of B lymphocytes was increased in the liver of NAFLD mice model, which indicated the hepatic B cells promoted this disease. Meanwhile, our previous study showed the B lymphocytes of mesenteric lymph node (MLN) could migrate to the liver. And the transwell assay showed the NAFLD liver homogenate attracted the mesenteric B lymphocytes more effectively than the control groups did. These facts provided clues that intestinal B cells were involved in the pathogenesis of NAFLD and could influence the liver of NAFLD as well. Therefore, this study aims to investigate the role of B lymphocytes in both the liver and the gut, especially the CCR9+ and α4β7+ B cells, during the progression of NAFLD by conducting experiments, such as flow cytometry analysis, Real-time PCR and cell reinfusion, both in vivo and in vitro. The former one includes the studies with wild-type NAFLD mice model and B cell-deficient mice model feeding with high fat diet, and the latter consists the coculture of B lymphocytes and T lymphocytes et al. To sum up, in this present study, we will investigate the alterations and the influences of hepatic and intestinal B lymphocytes and the CCR9+ α4β7+ B lymphocytes in the pathogenesis of NAFLD to shed light on the immune mechanisms of the liver-gut axis.

肝脏微环境变化是非酒精性脂肪性肝病(NAFLD)发病中的主要病理生理改变，其中关于免疫因素的研究主要集中在肝脏T细胞方面，而B淋巴细胞的研究鲜见。但研究表明B细胞在肥胖等与NAFLD常伴随的疾病中却发挥着重要作用。我们前期实验发现NAFLD小鼠肝脏内B细胞含量升高，提示肝脏B细胞参与了疾病发生；并且肠系膜淋巴结的B细胞回输后可向肝脏迁移，NAFLD小鼠肝脏匀浆上清对肠系膜淋巴结B细胞趋化能力更强，这些研究提示肠道B细胞也参与了NAFLD发生。因此本研究拟通过野生型NAFLD小鼠和B细胞缺陷型高脂喂养小鼠模型进行体内实验；并通过T细胞和B细胞共培养等体外实验，采用流式细胞术，RT-PCR及细胞回输实验等研究肝脏及肠道B细胞，尤其是表达CCR9、α4β7分子的B细胞在NAFLD发生中的变化情况及功能机制，进一步诠释NAFLD发生时肝肠轴的免疫机制。

多种免疫因素参与了非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)发病过程,但关于B细胞的研究鲜见。但B细胞通过多种途径促进肥胖、胰岛素抵抗、二型糖尿病等代谢综合征相关疾病的发生发展。NAFLD 发病机制的 “多重 打击”(multiple hits)学说强调了肠源性因素的重要作用,肠道菌群失调促进了NAFLD的发展,而肠道相关淋巴组织(gut associated lymphoid tissue, GALT) 包含大量 B 淋巴细胞。课题组前期研究发现NAFLD小鼠肠道来源淋巴细胞具有迁移至肝脏的潜能。因此本研究主要探索了B细胞,尤其是肝脏B细胞和肠道B细胞在NAFLD中变化和作用，并初步研究了是否存在肠道B细胞迁移至肝脏。研究结果如下（1）NAFLD小鼠脾脏、肝脏、肠道多个器官及组织内B细胞增多, 并通过B细胞缺陷小鼠证明B淋巴细胞参与了NAFLD 发生。(2)肝脏B细胞不仅自身分泌促炎性IL-6、TNF-α细胞因子及IgG2a抗体增多,而且能够促进CD4+T细胞活化和向Th1细胞分化而参与NAFLD发生。(3)NAFLD时肠道B细胞不但分泌 sIgA及IL-6增多，而且具有向肝脏迁移的潜能,其向肝脏迁移的机制可能涉及趋化因子CCL5及其受体 CCR3 的相互作用。同时过继回输NAFLD小鼠肠道来源B淋巴细胞能够诱导受体小鼠发生肝损害,提示肠道来源淋巴细胞促进了NAFLD的发生。(4)完成原计划内容前提下，课题组还发现肠系膜脂肪组织与NAFLD发生关系密切，并且炎症早期其B细胞浸润早于巨噬细胞等经典促炎性因素，并可能通过调节巨噬细胞分化等参与促进NAFLD发生。综上，在 NAFLD 的发生发展中,肝脏B细胞及肠道B细胞均发挥了十分重要的作用，其中肠道B细胞不仅能够分泌促炎因子,而且能够迁移至肝脏引起肝损伤,为肠源性因素参与NAFLD发病提供了证据,也是“肠肝对话”的有力体现。