药物诱导肝脏损伤的免疫学机制及人源化小鼠中B细胞特性研究

发布时间：2018-04-05 11:55

本文选题：对乙酰氨基酚　切入点：高迁移率族蛋白-1γδT　出处：《中国科学技术大学》2012年博士论文

【摘要】：对乙酰氨基酚(acetaminophen, APAP)是解热镇痛常用的一种非处方药物,但是该药物过量使用会导致急性的肝脏衰竭,严重的会造成人的死亡。在美国,APAP的过量使用已经给公共健康系统造成沉重负担。在中国,由于HBV的感染,患肝脏疾病的人更多,此药的过量使用对这些人的危害更大。 APAP在肝实质细胞内被细胞色素P450同工酶CYP2E1(cytochrome p450CYP2E1)代谢转化成有毒性的自由基代谢产物N-乙酰-对-苯醌亚胺(N-acetyl-p-benzoquinoneimine, NAPQI),胞内的谷胱甘肽(glutathione,GSH)可以与之结合将其解毒,但是过量的NAPQI会耗尽还原型谷胱甘肽,从而和其他蛋白的半胱氨酸残基结合,接着就会导致氧化应激反应、线粒体异常和DNA损伤,最终造成肝实质细胞坏死。但是药物直接介导的肝实质细胞坏死只是占整个肝脏损伤的一小部分,因为肝实质细胞坏死后活化了肝脏内的天然免疫应答,它们掀起了对肝脏的第二波冲击,而且由后者造成的肝脏损伤远甚过药物直接导致的损伤。在天然免疫介导的损伤中,有许多免疫细胞参与,包括自然杀伤细胞(natural killer cells,NK)、巨噬细胞(macrophages)、树突状细胞(dendrtic cells,DC)、中性粒细胞(neutrophils)。其中中性粒细胞备受关注,因为APAP处理后有大量的中性粒细胞浸润到肝脏中。但是,这些中性粒细胞是如何浸润到肝脏以及触发了这一反应的起因和过程到目前还没有研究清楚。在本文的研究中,我们用过量的APAP处理小鼠,诱导出肝脏的急性损伤,通过病理学染色和谷丙转氨酶(alanine aminotransferase,ALT)水平检测确定肝脏的损伤水平,通过流式细胞术和细胞计数的方法确定肝脏中各种免疫细胞的变化,通过酶联免疫吸附法确定细胞因子的变化,通过细胞清除和细胞因子中和的方法确定它们在体内的功能,同时也通过体外的细胞培养刺激确定细胞的特性变化。通过以上的的研究方法,我们取得的主要结果如下： 1、过量的APAP处理会造成肝脏的急性损伤和中性粒细胞在肝脏的聚集通过检测ALT水平,我们发现过量的APAP注射后会使得肝脏迅速发生损伤,24小时(hours, hr)达到最高,病理染色也显示肝脏组织中有大片的坏死区域。同时肝脏的单个核细胞显著增加,而增加的部分主要是中性粒细胞。 2、中性粒细胞的招募依赖于IL-17A 通过检测血清中和肝脏组织中的细胞因子水平,我们发现IL-17A显著增加。用中和抗体阻断了IL-17A的功能后,小鼠肝脏中的中性粒细胞明显减少,同时肝脏损伤和小鼠的死亡率都降低。 3、γδ T细胞是IL-17A的主要来源通过流式检测,我们发现,肝脏中IL-17A+γδ T细胞的比例明显增加,清除γδT细胞后,血清中IL-17A明显减少。而清除CD4+T细胞和NK1.1+细胞,IL-17A的水平却没有减少。清除γδT细胞也显著降低肝脏的损伤和小鼠的死亡率。另外,肝脏中的中性粒细胞明显减少。我们也发现,在APAP处理后,γδ TCR-/-小鼠肝脏中的中性粒细胞比正常的C57BL/6肝脏中的中性粒细胞显著减少。 4、IL-23刺激γδ T细胞产生IL-17A 那么γδT细胞是在什么条件下分泌IL-17A的?研究发现,APAP处理后血清中和肝脏组织中IL-23显著增加。当用中和抗体阻断IL-23的作用时,血清中IL-17A的水平明显降低,同样的结果也出现在IL-23p40-/-小鼠中。而且IL-23缺失情况下肝脏中性粒细胞的数量明显减少。通过体外实验,我们进一步发现,IL-23单独刺激就可以诱导γδ T细胞产生IL-17A。 5、HMGB1通过TLR4途径刺激巨噬细胞产生IL-23 那么IL-23又是从何处而来?研究发现,抑制巨噬细胞的活性后,IL-23和IL-17A的水平都明显下降。而APAP处理后,HMGB1显著增加。当用特异性抑制剂抑制HMGB1的作用后,IL-23和IL-17A则明显减少,而且中性粒细胞在肝脏中的数量也减少。在体外用HMGB1刺激TLR4+/+巨噬细胞可以使其产生IL-23,但是TLR4-/-的巨噬细胞产生的IL-23却低很多。同时,APAP处理TLR4-/-小鼠后,其血清中的IL-23和IL-17A的含量显著低于C57BL/6小鼠中的含量。结论：巨噬细胞和γδ T细胞之间的相互作用在APAP诱导的组织损伤而造成的急性肝炎中发挥了重要的作用。APAP引起肝实质细胞坏死,释放的HMGB1刺激巨噬细胞分泌IL-23,IL-23诱导γδT细胞产生IL-17A,IL-17A招募中性粒细胞到肝脏中来,HMGB1-TLR4-IL-23-IL17A级联反应对中性粒细胞的浸润起到了关键性作用。
[Abstract]:Acetaminophen (acetaminophen, APAP) is a kind of non prescription drugs commonly used antipyretic analgesic, but the drug overdose can lead to acute liver failure, serious cause of adult death. In the United States, the excessive use of APAP have caused a heavy burden to the public health system. In Chinese, due to HBV infection. More people suffering from liver disease, excessive use of the drug harm to these people more.
APAP cytochrome P450 isoenzyme CYP2E1 in liver parenchyma cells (cytochrome p450CYP2E1) metabolism into metabolites of free radical toxicity of N- acetyl - - quinone imine (N-acetyl-p-benzoquinoneimine, NAPQI), intracellular glutathione (glutathione, GSH) can be combined with the detoxification, but excessive NAPQI will run out of the prototype glutathione and cysteine residues and other binding proteins, then will lead to oxidative stress, mitochondrial dysfunction and DNA damage, resulting in liver cell necrosis. But hepatocyte necrosis drug directly mediated accounts for only a small part of the whole liver, because the liver parenchymal cell necrosis after activation of the innate immune response of liver inside, they set off the second wave of the impact on the liver, and the liver injury caused by the latter is far too directly lead to drug in natural damage. Immune mediated damage in many immune cells, including natural killer cells (natural killer cells, NK), macrophages, dendritic cells (macrophages) (dendrtic cells, DC), neutrophil (neutrophils). The neutrophil concern, because after APAP treatment, a large number of neutrophils infiltration into the liver. However, this is how the neutrophil infiltration into the liver and triggered the cause and process of this reaction is still not clear.
In this study, we used APAP treated mice excess, acute injury induced by liver, pathology and GPT (alanine aminotransferase, ALT) level of liver damage level determined, determine the changes of immune cells in the liver by flow cytometry and cell counting method, determine the change cytokine by ELISA method determined by cell clearance and cytokines and their functions in vivo, and in vitro cell culture stimulation to determine changes in cell properties. Through the above method, our main results are as follows:
1, excessive APAP treatment can cause acute liver damage and the aggregation of neutrophils in the liver
By testing the levels of ALT, we found that an excess of APAP after injection can cause liver damage occurred rapidly, 24 hours (hours, HR) reached the highest, pathological staining also showed large areas of necrosis in the liver. The liver mononuclear cells also increased significantly, and the increase of mainly neutrophils.
2, the recruitment of neutrophils depends on IL-17A
By detecting the levels of cytokines in serum and liver tissue, we found that IL-17A increased significantly. Neutralizing antibodies blocked the function of IL-17A, and neutrophils in mice liver decreased significantly, while liver damage and mortality in mice decreased.
3, gamma delta T cells are the main source of IL-17A
By flow cytometry, we found that IL-17A+ in liver of gamma delta T cells increased obviously, removal of gamma delta T cells, serum IL-17A decreased significantly. While the removal of CD4+T and NK1.1+ cells, the IL-17A level is not reduced. Removal of gamma delta T cells also significantly reduced liver injury and mortality in mice. In addition, neutrophils in the liver decreased. We also found that, after APAP treatment, neutrophil gammadelta TCR-/- in liver were significantly decreased than normal PMN C57BL/6 in the liver.
4, IL-23 stimulates the production of IL-17A by gamma delta T cells
Then gamma delta T cells secrete IL-17A under what conditions? The study found that serum IL-23 after treatment with APAP and liver tissue was significantly increased. When the blockade of IL-23 neutralizing antibody function, serum IL-17A levels were significantly lower, similar results were seen in IL-23p40-/- mice. In the absence of IL-23 and the number of the liver of neutrophils was significantly reduced. In vitro, we further found that IL-23 alone can be induced by stimulation of gamma delta T cells to produce IL-17A.
5, HMGB1 stimulates macrophages to produce IL-23 through TLR4 pathway
So IL-23 is where you came from? The study found that inhibiting the activity of macrophages, IL-23 and IL-17A levels were significantly decreased. After APAP treatment, HMGB1 was significantly increased. When the inhibition of HMGB1 by specific inhibitors, IL-23 and IL-17A were significantly reduced, and the number of neutrophils in the liver. Also reduced. TLR4+/+ macrophages stimulated with HMGB1 in vitro can produce IL-23, but the TLR4-/- IL-23 produced by macrophages was much lower. At the same time, APAP TLR4-/- mice after treatment, the serum content of IL-23 and IL-17A were significantly lower in C57BL/6 mice.
Conclusion: acute hepatitis interaction between macrophages and T cells in APAP induced tissue injury caused by play the important role of.APAP induced hepatic parenchymal cell necrosis, the release of HMGB1 stimulated macrophages to secrete IL-23 and IL-17A gamma delta T cells induced by IL-23 IL-17A, the recruitment of neutrophils to the liver, HMGB1-TLR4-IL-23-IL17 a chain reaction of neutrophil infiltration plays a key role.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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