【作者】 林燕；

【导师】 王红阳；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2011， 硕士

【摘要】 【研究背景及目的】肝炎是全世界范围内发病率较高的肝脏疾病之一,可由多种原因引起,如病毒感染、自身免疫功能紊乱、酗酒、药物毒性、营养不良和胆汁淤积等,我国是肝炎高发国家,大部分肝炎是病毒感染引起,尤其是以HBV感染为主。病毒性肝炎的主要发病机制是病毒抗原进入机体后引起的免疫反应造成肝细胞损伤,以细胞免疫反应为主,主要的效应细胞是特异性细胞毒性T淋巴细胞。病人免疫状况不同导致肝炎的临床表现各异。急性肝损伤(Acute liver failure)是由急性重型病毒性肝炎引起的致死性很高的肝脏疾病,病死率高达40-80%。大多数慢性病毒性肝炎病人在接受长期药物治疗后最终仍会发展为肝硬化、肝癌,肝脏的慢性炎症是导致肝癌的重要原因,因此对病毒性肝炎的研究具有重要的临床意义。自身免疫性肝炎(AIH)是一种病因不明的肝脏慢性炎症疾病,在西方国家发病率较高,目前认为细胞免疫反应和体液免疫反应均参与了其发病机制,细胞毒性T细胞是主要的致病因素,多数患者最终发展为肝硬化甚至肝癌。刀豆蛋白A(ConA)是一种植物蛋白成分提取物,T淋巴细胞表面有其受体,为TCR、CD3等在内的糖蛋白的糖残基,肝脏是ConA作用的靶器官之一。由于肝内存在Kupffer细胞、T细胞等大量免疫细胞,因此尾静脉注射后ConA能够经血液循环特异性地与肝窦内T细胞表面的受体结合并引起T细胞活化,从而导致肝脏炎症及损伤。ConA诱导的小鼠肝炎主要表现为转氨酶的升高以及严重的肝脏病理损伤,可以很好地模拟病毒性肝炎、自身免疫性肝炎等各种主要由免疫因素介导的肝脏疾病。由于解剖学上的联系,肝脏长期暴露于较低浓度的肠道细菌及细菌产物的刺激下,并且肝脏是最主要的肠道菌群过滤器官。肝脏内有大量的免疫细胞如巨噬细胞、树突状细胞、T淋巴细胞以及B淋巴细胞,因此正常肝脏能够迅速有效地清除肠道来源的细菌及细菌产物。然而,当肝脏的功能受损时,肠源性细菌产物可通过门静脉或其侧支循环进入血液形成内毒素血症。肠源性内毒素是强有力的致炎因子,能够引起肝内大量免疫细胞的活化,从而引起肝内大量炎症因子的分泌,这些炎症因子加重了肝脏已有炎症的发生、发展。内毒素(LPS)通过其受体Toll样受体4(TLR4)发挥作用。TLR4与LPS结合后,通过MyD88通路和TRIF通路活化下游信号,前者可引起多种炎症介质的释放,后者则可引起I型和II型干扰素的释放。在肝脏中,TLR4也表达于Kupffer细胞、星状细胞、胆管上皮细胞、内皮细胞和树突状细胞,在多种肝脏疾病的发生发展中可能发挥重要作用。本课题拟应用ConA诱导的小鼠肝炎模型研究肠源性内毒素在免疫因素介导的肝炎中的作用,同时建立TLR4敲除的小鼠ConA肝炎模型,探讨肠源性内毒素LPS在本模型中的作用机制,为病毒性肝炎、自身免疫性肝炎等免疫因素介导肝炎的防治提供有潜在临床价值的实验数据。【实验方法】1.建立ConA诱导的小鼠肝炎模型,检测注射ConA后小鼠体内LPS水平;2.利用抗生素清除肠源性内毒素,比较抗生素处理组和对照组小鼠ConA引起的肝损伤程度和LPS水平的变化;3.用抗生素清除小鼠肠源性内毒素,比较抗生素处理组和对照组小鼠注射ConA后肝脏内T细胞的浸润情况以及CD4~+ T细胞的活化情况;4.用ELISA方法检测抗生素处理组和对照组小鼠注射ConA后血清中Th1和Th2相关细胞因子的表达情况;5.用Western Blot方法检测抗生素处理组和对照组小鼠注射ConA后肝脏组织中相关促进和抑制凋亡分子的表达情况;6.比较LPS受体Toll样受体4(TLR4)敲除小鼠和对照小鼠的ConA引起的肝损伤程度;7.用流式细胞仪检测TLR4敲除小鼠和对照小鼠肝脏内参与引起ConA肝炎的T细胞的浸润情况以及CD4~+ T细胞的活化情况;8.分离TLR4敲除小鼠和对照小鼠脾细胞以及分选纯化CD4~+ T细胞,体外实验验证ConA对CD4~+ T细胞活化的影响;9.用ELISA方法检测TLR4敲除小鼠和对照小鼠注射ConA后血清中Th1和Th2相关细胞因子的表达情况;用Real-time PCR方法检测Th1细胞分化调节分子T-bet的表达情况;10.用Western Blot方法检测TLR4敲除小鼠和对照小鼠注射ConA后肝脏组织中相关促进和抑制凋亡分子的表达情况;用Real-time PCR方法检测穿孔素Perforin和Granzyme B的表达情况。【实验结果】1.在ConA诱导的小鼠肝炎模型中存在血清LPS水平的升高;2.抗生素处理组小鼠在ConA注射后血清中LPS的升高明显低于对照组,并且利用抗生素清除肠源性LPS可明显减轻ConA引起的肝脏损伤及肝细胞凋亡;3.清除肠源性LPS能够明显降低ConA引起的肝内T细胞浸润以及CD4~+ T细胞的活化;4.清除肠源性LPS能够抑制ConA引起的Th1相关细胞因子的产生;5.清除肠源性LPS能够明显抑制ConA引起的肝细胞凋亡;6. TLR4缺失明显减轻ConA诱导的肝脏损伤和肝细胞的凋亡;7. TLR4缺失抑制T细胞的肝内浸润并能有效抑制CD4~+ T细胞的活化;8. TLR4缺失能够抑制ConA引起的Th1相关细胞因子的产生,而促进Th2相关细胞因子IL-10的产生;9. TLR4缺失能够明显抑制ConA引起的肝细胞凋亡;10.穿孔素/颗粒酶B途径可能参与了TLR4缺失对肝细胞凋亡的保护作用。【结论】本研究证明肠源性LPS在ConA诱导的免疫因素介导肝炎的发生发展中发挥了重要作用。肠源性LPS通过与其受体TLR4结合,促进ConA引起的肝内T细胞浸润以及肝脏中的CD4~+ T细胞的活化,继而促进一系列Th1相关细胞因子的生成,从而加重ConA引起的小鼠肝损伤;清除肠源性LPS和TLR4缺失小鼠在ConA处理后肝脏损伤较轻,并且肝内T细胞浸润明显减轻,CD4~+ T细胞的活化和体内Th1相关细胞因子水平明显低于对照组,而对Th1相关细胞因子有拮抗作用的Th2相关细胞因子IL-10有明显升高。清除肠源性LPS和TLR4缺失能够上调Mcl-1的表达而下调Bax的表达,并能够增强Erk1/2的磷酸化,从而减轻ConA引起的肝细胞凋亡,穿孔素/颗粒酶B途径可能参与了TLR4缺失对肝细胞凋亡的保护作用。我们的实验结果提示抑制肠道细菌的异常转位以及清除肠源性LPS可能会有效改善肝炎病人的肝功能,减轻肝脏炎症的发生发展。更多还原

【Abstract】 Acute liver failure is a devastating liver disease associated with signi?cant mortality （40%-80%）, characterized by the sudden onset in a patient of severe acute hepatitis. It can be caused by many kinds of reasons, such as virus infection, autoimmunity dysfunction, alcohol consumption, hepatotoxins intake and so on, however, the underlying pathophysiological mechanisms are not well understood. Development of new drugs depends primarily on the availability of suitable animal models.Concanavalin A has high affinity toward the hepatic sinus, leading to T-cell activation in the liver. ConA-induced hepatitis is a well-known experimental murine model that can mimic many kinds of human T cell-mediated hepatitis, with significantly elevated levels of transaminase as well as severe liver injury. Previous studies have identified that in Concanavalin A-mediated hepatitis model activated T cells and NKT cells release a series of cytokins that play great roles in the liver injury. Different cytokins can be categorized as Th1 and Th2 associated cytokines by their biological function. Studies have illustrated that Th1 associated cytokines such as IL-12 and IFN-γare dominant factors inducing the development of Th1 cells. Conversely, IL-4 production directs the development of a Th2 response. Th1 cells mediate delayed type hypersensitivity responses and provide protection against intracellular pathogens and viruses, Th2 cells provide help to B cells and eradicate helminthes and other extra-cellular parasites. Th1/Th2 differentiation of T cells is largely dependent on the balance of GATA-3 and T-bet. The balance of Th1 and Th2 cells exerts an crucial role in keeping normal immunoregulation, however, in pathologic conditions such as allergic and autoimmune diseases, polarized Th1 and Th2 responses are found and believed to be critical to the outcome of these conditions.Emerging evidence suggests a strong interaction between the gut microbiota and human disease. Receiving～70% of its blood supply from the intestine through the portal circulation, the liver is the ?rst line of defense against gut-derived antigens. In order to cope with these latent systemic pathogens the liver contains a large number of resident immune cells such as macrophages, natural killer cells, dendritic cells, T and B lymphocytes. The resident macrophages of the liver, Kupffer cells, are able to take up ef?ciently endotoxin and phagocytose bacteria carried through portal vein blood, hepatocytes are also a major cell type involved in LPS uptake. Despite the constant exposure to low levels of gut-derived bacteria and bacterial products, there are no obvious inflammation in the healthy liver. However, when normal liver physiology is disrupted and inflammatory cells are activated, gut-derived factors likely augment or exacerbate certain liver diseases leading to enhanced tissue damage and propagation of inflammation via activation of TLR-signaling pathways.The role of gut-derived endotoxin in liver disease has excited much interest in investigators for a long time, and the critical role of gut-derived endotoxin as a cofactor in acute and chronic liver disease, both experimental and clinical, was already established more than 30-40 years ago. Our previous study also demonstrated that the endotoxin/TLR4 regulates the survival and proliferation of hepatocytes and their preneoplastic derivatives during chemically induced hepatocarcinogenesis.Toll like receptors （TLRs） recognize pathogen-associated molecular patterns and activate signaling pathways that lead to the induction of innate immune and inflammatory genes. In addition, there is accumulating evidence that TLRs contribute signi?cantly to activation of adaptive immune responses such as dendritic cell maturation and T- and B-cell responses,specially, the TH1-dependent immune responses. TLR4 is an important member of TLRs, which could sense endotoxin and activate transcription factors that initiate innate immunity. Previous study displayed that TLR4 was also expressed on T lymphocyte, playing a vital role in adaptive immunity. Recent studies have demonstrated the contribution of TLR4 signaling to the trapping of CD8⁺ T cells within the murine liver. Moreover, other very recent studies indicate that direct interactions of gut-derived antigens signaling through Toll like receptor 4 on CD4⁺ T cells contributes to their regulatory development and function. We hypothesize that gut-derived LPS and TLR4 may have some roles in ConA-induced hepatitis, which predominantly mediated by T lymphocytes in liver.In the current study, we report that there is a sustained LPS accumulation in ConA-induced hepatitis model. Deletion of gut microflora using antibiotics and TLR4 deficiency can decrease the accumulation of endotoxin efficiently and markedly suppress the liver injury induced by ConA. This attenuated hepatitis seen in both antibiotics-treatment mice and TLR4 knockout (TLR4^-/-) mice primarily results from the reduced infiltration of T lymphocytes and suppressed activation of CD4⁺ T cells as well as decreased levels of Th1 cytokine production in response to ConA stimulation. Furthermore, deletion of gut microflora and TLR4 deficiency lighten liver injury induced by ConA through promoting the activation of Erk1/2, in addition, perforin/granzyme B mechanism may involve in the protection of TLR4 ablation. These results suggest that preventing plasma endotoxin accumulation could have a beneficial impact on liver function for patients with T cell-mediated hepatisis.更多还原