【作者】 马洪第；

【导师】 廉哲雄；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2013， 博士

【摘要】 原发性胆汁性肝硬化是一种由于免疫系统对自身肝脏进行攻击而引起的慢性胆汁淤积性肝脏自身免疫性疾病,高发于中年女性。患者发病后会导致胆管阻塞和胆汁淤积,造成特异性的胆管上皮细胞损伤,从而引发肝脏的纤维化,最后演变为肝硬化和肝功能衰竭,严重威胁患者的生命。由于获取早期发病的临床肝脏样本十分困难,因此对原发性胆汁性肝硬化发病机制的探索及治疗靶点的开发均主要以该疾病的动物模型-dnTGF-β RⅡ小鼠作为研究工具。该模型很好地模拟了人类原发性胆汁性肝硬化的主要表型特征：自发产生抗线粒体的自身抗体；血清中炎性细胞因子水平升高；病理组织学检查可发现伴随肝内胆管的破坏,肝脏汇管区有大量的CD4T细胞、CD8T细胞等淋巴细胞的浸润；此外dnTGF-β小鼠还有自发的结肠炎,也可以作为研究肝脏炎症和肠道微环境相互作用的模型。虽然以往的工作利用dnTGF-β RⅡ小鼠这一疾病模型对于该自身免疫性肝病发病早期的一些免疫学致病机制进行了初步探究,包括CD8T细胞的致病性,细胞因子和调节性B细胞对疾病进程的影响等；但是对该疾病的致病机制仍未有全面的了解,还有很多未知的领域,包括天然免疫在该疾病中所发挥的作用以及趋化因子及其配体在介导该疾病炎性浸润中的机制等均未有明确的报道。因此,本研究以dnTGF-β RⅡ小鼠以及Toll样受体缺失或者趋化因子受体缺失的dnTGF-β RⅡ小鼠为研究原发性胆汁性肝硬化的动物模型,利用细胞免疫学、分子生物学以及医学病理学的实验方法方法对该疾病致病过程中天然免疫受体Toll样受体以及趋化因子受体CXCR3发挥作用的机制进行深入探讨,为阐明原发性胆汁性肝硬化的致病机制以及开发该疾病有效的治疗靶点提供证据支持。本研究得到的实验结果如下：1.环境因素的改变(肠道菌群清除)影响了原发性胆汁性肝硬化小鼠模型的发病程度我们用含有甲硝锉,氨苄青霉素,新霉素以及万古霉素这四种抗生素的水从4周龄起饲养SPF级的dnTGF-β RⅡ (TG)小鼠以清除肠道中绝大多数的共生菌。在抗生素喂养12周,即小鼠15周龄时杀鼠,对其肝脏的病理学分析发现与非抗生素水喂养的对照组相比,抗生素喂养加重了TG小鼠的胆管炎,表现为肝脏炎性浸润的增多以及胆管破坏的加重。2. Toll样受体2缺失加重了dnTGF-β RⅡ小鼠自发的胆管炎及结肠炎与野生型(WT)小鼠相比,tlr2在dnTGF-β RⅡ (TG)小鼠中有较高的表达,因此我们将TG小鼠与Toll样受体2(TLR2)敲除小鼠进行杂交,得到TLR2-/-dnTGF-β RⅡ (TGT2)小鼠。首先,对其肝脏的病理学分析发现,与TG小鼠相比,TGT2小鼠肝脏汇管区的炎性浸润及肝内胆管的破坏有明显的加重。细胞学分析的结果显示与TG小鼠相比,TGT2小鼠肝脏中T细胞尤其是CD4T细胞的浸润增加了,并且这些浸润的CD4T和CD8T中呈现效应性记忆性表型(CD44highCD62Llow)的细胞明显增多。其次,肠道病理学的分析发现TLR2敲除加重了TG小鼠的自发结肠炎。与TG小鼠相比TGT2小鼠不仅体重明显下降,结肠重量显著上升,而且结肠中的炎性浸润已经扩展到整个粘膜的厚度,腺体和杯状细胞受到严重破坏,并伴随有隐窝囊肿的存在。再次,对细胞因子的检测结果表明,与TG小鼠相比,TGT2小鼠血清中TNF-α和IFN-γ的浓度明显升高,并且在TGT2小鼠的肝脏中il6的基因表达显著增加。对于肝脏中IL-6相关的信号通路的检测发现与TG小鼠相比,TGT2小鼠肝脏中STAT3磷酸化的水平明显升高。最后,为了从相反的方向证明TLR2在TG小鼠发病过程中的作用,我们计划用TLR2的特异性配体Pam3CSK4来对TG小鼠进行高剂量单次注射和低剂量连续注射的处理,从而更加全面地阐明TLR2在TG小鼠发病中的作用机制。3. Toll样受体4缺失有加重dnTGF-β RⅡ小鼠肝脏炎症及肠道炎症的趋势与WT小鼠相比,tlr4在TG小鼠中的表达升高,因此我们对杂交所得到的TLR4-/-dnTGF-β RⅡ (TGT4)小鼠进行了一系列的细胞免疫学和医学病理学的分析。结果发现：与TG小鼠相比TGT4小鼠肝脏中浸润的T淋巴细胞的比例有所增加；并且CD8T细胞在总的T细胞中所占的比例有明显的上升；其肝脏中浸润的效应性记忆CD8T细胞(CD8Tem)无论在CD8T细胞中所占的比例还是其绝对数都有明显的增加。虽然TLR4敲除对于dnTGF-β RⅡ小鼠结肠炎症的影响没有统计学上的显著性差异,但是无论是通过病理切片的观察还是统计数据的分析,我们都可以发现TGT4小鼠与TG小鼠相比结肠炎有加重的趋势。4. CXCR3在dnTGF-β RⅡ小鼠致病过程中作用的初步探讨首先,我们发现与WT小鼠相比TG小鼠肝脏中趋化因子CXCL9和CXCL10的浓度有明显上升。然后,流式分析的结果显示在TG小鼠肝脏中表达这两种趋化因子的受体CXCR3的T细胞比WT明显增多。接下来,来自TG小鼠或者敲除了CXCR3的TG小鼠中的T细胞的过继转输实验将为证明CXCR3介导T细胞向TG小鼠肝脏迁移提供更加有力的证据。综上所述,本研究证明了TLR2缺失可以加重原发性胆汁性肝硬化小鼠模型中的胆管炎以及自发性结肠炎。并且发现CXCR3可能介导了该小鼠模型中T细胞向肝脏炎性部位的迁移。我们的研究首次证明了在该疾病模型中天然免疫受体TLR2的敲除促进了肝脏和肠道的炎症。并且首次在该疾病模型中探讨肠道微环境与肝脏炎症之间的关系,这为阐明原发性胆汁性肝硬化的致病机制以及寻找该疾病有效的治疗靶点提供了理论依据。更多还原

【Abstract】 Primary biliary cirrhosis (PBC) is a chronic cholestatic and autoimmune liver disease, which caused by abnormal immune responses to the patients’ own livers. This disease occurs at high frequency among middle-aged women. After onset, the following symptoms show up:biliary obstruction and cholestasis, biliary epithelial cells injury, liver fibrosis, and ultimately, cirrhosis and liver failure, which seriously threaten patients’ lives. As it is very difficult to acquire clinical liver samples of patients in the early onset, the pathogenesis studies and therapeutic developments of primary biliary cirrhosis are mainly based on an animal model-dnTGF-βRⅡ mice. The mice spontaneously develop an autoimmune biliary disease closely resembling human PBC. High level of anti-mitochondrial antibodies (AMAs) and increased serum cytokines have been detected in the mice.Histologically, dnTGF-βRⅡ mice manifest extensive CD4and CD8lymphocytic infiltration in portal tracts, associated with biliary destruction. Moreover, dnTGF-β R Ⅱ mice, developing colitis spontaneously as well, could be established as a model to investigate the interactions between hepatic inflammation and intestinal microenvironment.Primary studies based on dnTGF-βRⅡ mouse model about the pathogenesis of autoimmune liver disease in early onset have been reported, including the pathogenic role of CD8T, the regulatory role of Bregs, and the functions of different cytokines. However, the pathogenic mechanisms of this disease have not been fully understood. Many unknown areas still exist in this disease, such as the role of innate immune responses, and the complexity of chemokines and their receptors, which induce inflammatory infiltration.Therefore, in this study, dnTGF-βRⅡ mice and dnTGF-βRⅡ mice lacking either Toll-like receptors or chemokine receptor were established as mouse models to investigate the role of innate immune receptors-Toll-like receptor2and4, as well as chemokine receptors, CXCR3, in the progress of this autoimmune liver disease. Methods used in this study were supported by cellular immunology, molecular biology and medical pathology. The aim of this study is to elucidate the pathogenesis of primary biliary cirrhosis and to search for therapeutic targets. The results obtained in this study are as follows: 1. Changed environmental factors (intestinal flora depletion) affected the severity of primary biliary cirrhosis-like disease in mouse modelWe added antibiotics including metronidazole, ampicillin, neomycin, and vancomycin into drinking water of dnTGF-βR Ⅱ (TG) mice (at SPF level) since the fourth week after birth to deplete the vast majority of the gut symbionts.12weeks later, when15-week-old, the mice were sacrificed and the liver pathology analysis demonstrated that drinking antibiotic water could aggravate cholangitis in TG mice compared with control group without antibiotic water. TG mice in antibiotic water drinking group had increased hepatic inflammatory infiltrations and worse bile duct injury.2. Toll-like receptor2depletion aggravated cholangitis and colitis in dnTGF-βR Ⅱ miceCompared with wild-type (WT) mice, dnTGF-βR Ⅱ (TG) mice have higher expression of tlr2gene in liver. Thus, we crossed TG mice with Toll-like receptor2(TLR2) knockout mice and got TLR2-/-dnTGF-βR Ⅱ (TGT2) mice. First, liver pathology analysis showed that, compared with TG mice, TGT2mice had more severe portal inflammation and bile duct damage in liver. Cytological analysis revealed that, compared with TG mice, infiltrating T cells in TGT2mice liver, especially CD4T cells, were increased. There were also more infiltrating CD4T and CD8T cells presenting effector memory phenotype (CD44highCD62Llow) in TGT2mice than in TG mice. Secondly, intestinal pathology analysis showed aggravated colitis in TGT2mice. Compared with TG mice, the body weight of TGT2mice decreased significantly, but colon weight increased. In the colon of TGT2mice, ulceration with transmural inflammatory cell infiltrates was observed. Moreover, crypts were destroyed and goblet cells were inconspicuous in the colon of TGT2mice, which due to the dense inflammatory cell infiltrates, and accompanied by crypt abscesses and mucin depletion. Again, analysis of cytokines exhibited that compared with TG mice, serum TNF-α and IFN-γ levels were significantly increased and il6gene expression in liver was distinctly up-regulated in TGT2mice. By detecting IL-6related signaling pathway, we found up-regulated phosphorylation of STAT3in the liver of TGT2mice compared with TG mice. Finally, in order to define the role of TLR2in the pathogenesis of primary biliary cirrhosis-like disease in TG mice in another way, we plan to treat TG mice with TLR2specific ligand Pam3CSK4by administrating a single injection at high-dose or a series of injections weekly at low-does. The results will provide us more evidence to elucidate the pathogenic role of TLR2in TG mice.3. Increasing trend of aggravated liver inflammation and colitis in TLR4-/-dnTGF-βRⅡCompared with wide type (WT) mice, tlr4gene expression was up-regulated in TG mice in liver. We obtained TLR4-/-dnTGF-βR Ⅱ (TGT4) mice by crossing TLR4-/-micewith dnTGF-βR Ⅱ mice. Cellular immunology and medical pathology analysis showed that compared with TG mice, TGT4mice had increased proportion of T lymphocytes in liver mononuclear cells. Among these infiltrating T cells, the percentage of CD8T cells was higher than in TG mice. Furthermore, the frequency and absolute number of effector memory CD8T cells in liver of TGT4mice were increased compared with TG mice. Although there was no significant difference in colitis between TG and TGT4mice, increasing trend of aggravated liver inflammation and colitis in TLR4-/-dnTGF-βR Ⅱ mice was observed in both pathological analysis and statistical analysis.4. The exploration of pathogenic role of CXCR3in dnTGF-βR Ⅱ miceFirst, the concentration of chemokine CXCL9and CXCL10was up-regulated in the liver of TG mice compared with wide type mice. Then, the flow cytometry analysis revealed increased CXCR3(receptor of CXCL9and CXCL10) expressing T cells in the liver of TG mice compared with wide type mice. Next, we plan to adoptively transfer T cells from TG mice or CXCR3-/-TG mice, to elucidate the role of CXCR3in T cell migration to liver in TG mice.In summary, this study demonstrated that the absence of TLR2led to aggravated spontaneous cholangitis and colitis in the mouse model of primary biliary cirrhosis. And we also found that CXCR3may participate in guiding T cell migration to inflammatory sites of liver in this mouse model. Our study demonstrated for the first time that depletion of innate immune receptor TLR2aggravated inflammation in liver and colon in TG mice. And we explored the relationship between intestinal micro-environment and liver inflammation in TG mice firstly. All the evidence we provided in this study could shed light on the pathogenic mechanism of primary biliary cirrhosis and make a contribution to the therapeutic developments.更多还原