本文選題：對乙酰氨基酚　切入點：高遷移率族蛋白-1γδT　出處：《中國科學(xué)技術(shù)大學(xué)》2012年博士論文

【摘要】：對乙酰氨基酚(acetaminophen, APAP)是解熱鎮(zhèn)痛常用的一種非處方藥物,但是該藥物過量使用會導(dǎo)致急性的肝臟衰竭,嚴(yán)重的會造成人的死亡。在美國,APAP的過量使用已經(jīng)給公共健康系統(tǒng)造成沉重負(fù)擔(dān)。在中國,由于HBV的感染,患肝臟疾病的人更多,此藥的過量使用對這些人的危害更大。 APAP在肝實質(zhì)細(xì)胞內(nèi)被細(xì)胞色素P450同工酶CYP2E1(cytochrome p450CYP2E1)代謝轉(zhuǎn)化成有毒性的自由基代謝產(chǎn)物N-乙酰-對-苯醌亞胺(N-acetyl-p-benzoquinoneimine, NAPQI),胞內(nèi)的谷胱甘肽(glutathione,GSH)可以與之結(jié)合將其解毒,但是過量的NAPQI會耗盡還原型谷胱甘肽,從而和其他蛋白的半胱氨酸殘基結(jié)合,接著就會導(dǎo)致氧化應(yīng)激反應(yīng)、線粒體異常和DNA損傷,最終造成肝實質(zhì)細(xì)胞壞死。但是藥物直接介導(dǎo)的肝實質(zhì)細(xì)胞壞死只是占整個肝臟損傷的一小部分,因為肝實質(zhì)細(xì)胞壞死后活化了肝臟內(nèi)的天然免疫應(yīng)答,它們掀起了對肝臟的第二波沖擊,而且由后者造成的肝臟損傷遠(yuǎn)甚過藥物直接導(dǎo)致的損傷。在天然免疫介導(dǎo)的損傷中,有許多免疫細(xì)胞參與,包括自然殺傷細(xì)胞(natural killer cells,NK)、巨噬細(xì)胞(macrophages)、樹突狀細(xì)胞(dendrtic cells,DC)、中性粒細(xì)胞(neutrophils)。其中中性粒細(xì)胞備受關(guān)注,因為APAP處理后有大量的中性粒細(xì)胞浸潤到肝臟中。但是,這些中性粒細(xì)胞是如何浸潤到肝臟以及觸發(fā)了這一反應(yīng)的起因和過程到目前還沒有研究清楚。 在本文的研究中,我們用過量的APAP處理小鼠,誘導(dǎo)出肝臟的急性損傷,通過病理學(xué)染色和谷丙轉(zhuǎn)氨酶(alanine aminotransferase,ALT)水平檢測確定肝臟的損傷水平,通過流式細(xì)胞術(shù)和細(xì)胞計數(shù)的方法確定肝臟中各種免疫細(xì)胞的變化,通過酶聯(lián)免疫吸附法確定細(xì)胞因子的變化,通過細(xì)胞清除和細(xì)胞因子中和的方法確定它們在體內(nèi)的功能,同時也通過體外的細(xì)胞培養(yǎng)刺激確定細(xì)胞的特性變化。通過以上的的研究方法,我們?nèi)〉玫闹饕Y(jié)果如下： 1、過量的APAP處理會造成肝臟的急性損傷和中性粒細(xì)胞在肝臟的聚集 通過檢測ALT水平,我們發(fā)現(xiàn)過量的APAP注射后會使得肝臟迅速發(fā)生損傷,24小時(hours, hr)達到最高,病理染色也顯示肝臟組織中有大片的壞死區(qū)域。同時肝臟的單個核細(xì)胞顯著增加,而增加的部分主要是中性粒細(xì)胞。 2、中性粒細(xì)胞的招募依賴于IL-17A 通過檢測血清中和肝臟組織中的細(xì)胞因子水平,我們發(fā)現(xiàn)IL-17A顯著增加。用中和抗體阻斷了IL-17A的功能后,小鼠肝臟中的中性粒細(xì)胞明顯減少,同時肝臟損傷和小鼠的死亡率都降低。 3、γδ T細(xì)胞是IL-17A的主要來源 通過流式檢測,我們發(fā)現(xiàn),肝臟中IL-17A+γδ T細(xì)胞的比例明顯增加,清除γδT細(xì)胞后,血清中IL-17A明顯減少。而清除CD4+T細(xì)胞和NK1.1+細(xì)胞,IL-17A的水平卻沒有減少。清除γδT細(xì)胞也顯著降低肝臟的損傷和小鼠的死亡率。另外,肝臟中的中性粒細(xì)胞明顯減少。我們也發(fā)現(xiàn),在APAP處理后,γδ TCR-/-小鼠肝臟中的中性粒細(xì)胞比正常的C57BL/6肝臟中的中性粒細(xì)胞顯著減少。 4、IL-23刺激γδ T細(xì)胞產(chǎn)生IL-17A 那么γδT細(xì)胞是在什么條件下分泌IL-17A的?研究發(fā)現(xiàn),APAP處理后血清中和肝臟組織中IL-23顯著增加。當(dāng)用中和抗體阻斷IL-23的作用時,血清中IL-17A的水平明顯降低,同樣的結(jié)果也出現(xiàn)在IL-23p40-/-小鼠中。而且IL-23缺失情況下肝臟中性粒細(xì)胞的數(shù)量明顯減少。通過體外實驗,我們進一步發(fā)現(xiàn),IL-23單獨刺激就可以誘導(dǎo)γδ T細(xì)胞產(chǎn)生IL-17A。 5、HMGB1通過TLR4途徑刺激巨噬細(xì)胞產(chǎn)生IL-23 那么IL-23又是從何處而來?研究發(fā)現(xiàn),抑制巨噬細(xì)胞的活性后,IL-23和IL-17A的水平都明顯下降。而APAP處理后,HMGB1顯著增加。當(dāng)用特異性抑制劑抑制HMGB1的作用后,IL-23和IL-17A則明顯減少,而且中性粒細(xì)胞在肝臟中的數(shù)量也減少。在體外用HMGB1刺激TLR4+/+巨噬細(xì)胞可以使其產(chǎn)生IL-23,但是TLR4-/-的巨噬細(xì)胞產(chǎn)生的IL-23卻低很多。同時,APAP處理TLR4-/-小鼠后,其血清中的IL-23和IL-17A的含量顯著低于C57BL/6小鼠中的含量。 結(jié)論：巨噬細(xì)胞和γδ T細(xì)胞之間的相互作用在APAP誘導(dǎo)的組織損傷而造成的急性肝炎中發(fā)揮了重要的作用。APAP引起肝實質(zhì)細(xì)胞壞死,釋放的HMGB1刺激巨噬細(xì)胞分泌IL-23,IL-23誘導(dǎo)γδT細(xì)胞產(chǎn)生IL-17A,IL-17A招募中性粒細(xì)胞到肝臟中來,HMGB1-TLR4-IL-23-IL17A級聯(lián)反應(yīng)對中性粒細(xì)胞的浸潤起到了關(guān)鍵性作用。
[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.

【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392

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