本文關(guān)鍵詞：CD36基因在小鼠急性肝損傷中的作用及其機(jī)制研究　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： CD36 刀豆蛋白A 肝損傷 CXCL10 染料木素 CD36 對(duì)乙酰氨基酚 肝損傷 PP1

【摘要】：肝臟是人體內(nèi)最大的實(shí)體臟器,具有多重生物學(xué)功能,在維持穩(wěn)態(tài)和保持新陳代謝平衡的過程中起到了非常關(guān)鍵的作用。肝臟是人類重要的免疫器官,當(dāng)免疫平衡一旦被破壞,就會(huì)引發(fā)多種免疫相關(guān)的肝臟疾病,包括HBV、HCV引起的持續(xù)性慢性肝炎、原發(fā)性肝癌以及自身免疫性肝炎(AIH)等。AIH自身免疫反應(yīng)介導(dǎo)的慢性進(jìn)行性肝臟損傷過程,機(jī)體產(chǎn)生針對(duì)肝臟自身抗原的免疫反應(yīng),從而破壞肝細(xì)胞導(dǎo)致肝臟炎癥壞死,其發(fā)病率在世界范圍內(nèi)逐年增加,嚴(yán)重病例可快速進(jìn)展為肝硬化和肝衰竭。主要治療手段為免疫抑制劑和肝移植,但效果欠佳,潛在的病理生理機(jī)制仍不清楚,因此尚無明確有效的治療方案。肝臟對(duì)來自體內(nèi)和體外的許多非營養(yǎng)性物質(zhì)如各種藥物、毒物以及體內(nèi)某些代謝產(chǎn)物,具有生物轉(zhuǎn)化作用,通過新陳代謝將它們徹底分解或以原形排出體外。而各種藥物在使用過程中,因藥物本身或/及其代謝產(chǎn)物或由于特殊體質(zhì)對(duì)藥物的超敏感性或耐受性降低會(huì)導(dǎo)致藥物性肝損傷(DILI)。目前DILI已經(jīng)上升至全球死亡原因的第五位,成為全球不可忽視的公共衛(wèi)生問題。其致病機(jī)制涉及代謝蛋白加合物形成、線粒體功能障礙、氧化應(yīng)激、過氧亞硝酸鹽形成及核DNA斷裂等關(guān)鍵過程,目前臨床均使用N-乙酰半胱氨酸解毒,但存在很多局限性而導(dǎo)致病程遷延不愈,發(fā)展為肝纖維化肝硬化等。分化抗原36(CD36)分子是一種廣泛存在于細(xì)胞表面的單鏈糖蛋白,屬于B族清道夫受體,可以在多種細(xì)胞如巨噬細(xì)胞、微血管內(nèi)皮細(xì)胞、血小板、脂肪細(xì)胞及上皮細(xì)胞等表達(dá),作為模式識(shí)別受體,CD36參與一系列生理和病理過程,包括免疫、脂肪酸代謝、血管生成、動(dòng)脈粥樣硬化和吞噬作用等。CD36還與Toll樣受體4和6結(jié)合以促進(jìn)無菌炎癥。值得注意的是,CD36被證實(shí)與α6整合素及CD133共表達(dá)于腫瘤干細(xì)胞以促進(jìn)膠質(zhì)母細(xì)胞瘤的進(jìn)展而抑制CD36受體則會(huì)降低人類中黑素瘤、口腔癌及乳腺癌的腫瘤轉(zhuǎn)移。本研究就CD36在急性肝損傷中發(fā)揮的作用及機(jī)制進(jìn)行了初步探討。第一部分:CD36基因敲除在保護(hù)小鼠Con A誘導(dǎo)的免疫性肝損傷中的作用及其機(jī)制研究刀豆蛋白A(ConA)誘導(dǎo)的免疫性肝損傷模型能較好地模擬人類肝臟自身免疫性疾病和病毒性肝炎的發(fā)病機(jī)制。通過給小鼠靜脈注射Con A建立肝臟損傷的實(shí)驗(yàn)動(dòng)物模型,此模型表現(xiàn)為急性肝炎的臨床和病理組織學(xué)表現(xiàn),包括肝臟中白細(xì)胞和淋巴細(xì)胞浸潤、肝細(xì)胞壞死、血清轉(zhuǎn)氨酶升高及炎性細(xì)胞因子分泌等�，F(xiàn)如今此模型已被廣泛應(yīng)用于研究T細(xì)胞介導(dǎo)的肝炎致病機(jī)制,從而形成了我們目前關(guān)于肝損傷過程的認(rèn)知。目的:雖然目前已有大量關(guān)于自身免疫性肝損傷過程的研究,但其確切的細(xì)胞和分子機(jī)制尚不清楚。越來越多的數(shù)據(jù)表明T細(xì)胞的活化在慢性肝臟損傷過程中具有相當(dāng)重要的作用。本研究旨在探討CD36基因?qū)細(xì)胞介導(dǎo)的自身免疫性肝炎的調(diào)控作用,為當(dāng)前臨床肝炎的防治提供新的方向。方法:為了探討CD36基因在急性免疫性肝損傷中是否發(fā)揮作用及其作用機(jī)制,我們建立了刀豆蛋白A(ConA)誘導(dǎo)的野生型(WT)小鼠及CD36基因缺陷(CD36--)小鼠急性肝損傷模型。(1)內(nèi)源性CD36對(duì)ConA誘導(dǎo)小鼠肝損傷的影響WT小鼠和CD36-/-轉(zhuǎn)基因小鼠經(jīng)尾靜脈按12mg/kg注射Con A誘導(dǎo)肝臟損傷。在8h后收集血清并檢測血清中谷丙轉(zhuǎn)氨酶(ALT)的活性;分離小鼠肝組織,利用HE染色方法觀察肝組織病變;并利用末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記測定實(shí)驗(yàn)(TUNEL)分析肝細(xì)胞凋亡情況。(2)內(nèi)源性CD36對(duì)ConA誘導(dǎo)小鼠肝內(nèi)T細(xì)胞活化的免疫調(diào)控作用WT小鼠和CD36-/-小鼠經(jīng)尾靜脈注射ConA后,分離小鼠肝內(nèi)單個(gè)核細(xì)胞(mononuclear cells,MNCs),流式細(xì)胞術(shù)檢測T細(xì)胞、NK細(xì)胞、浸潤性巨噬細(xì)胞及中性粒細(xì)胞數(shù),分析細(xì)胞的活化水平;實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(Real-time Reverse transcription polymerase chain reaction,Real-time RT-PCR)分析肝組織中促炎細(xì)胞因子(如TNF-α,CXCL10,IL-1α,MCP-1和IL-6等)的表達(dá);酶聯(lián)免疫吸附測定方法(ELISA)檢測血清中細(xì)胞因子的表達(dá)水平;免疫印跡方法(Western Blot)檢測肝組織中JNK、IKKα/β、ERK及STAT3的磷酸化水平,以及Caspase-3的表達(dá)水平。(3)拮抗劑實(shí)驗(yàn)驗(yàn)證內(nèi)源性CD36在ConA誘導(dǎo)小鼠肝損傷中的作用WT小鼠經(jīng)尾靜脈注射Con A后,腹腔注射酪氨酸酶抑制劑染料木素(Genistein),封閉WT小鼠CD36-TLR4-TLR6復(fù)合物形成,通過檢測ALT水平、HE染色組織學(xué)分析以及分離WT小鼠肝內(nèi)MNCs分析T細(xì)胞、NK細(xì)胞、浸潤性巨噬細(xì)胞及中性粒細(xì)胞浸潤情況驗(yàn)證內(nèi)源性CD36在Con A誘導(dǎo)的肝損傷中發(fā)揮的作用。(4)體外實(shí)驗(yàn)分析CD36對(duì)CXCL10誘導(dǎo)肝細(xì)胞凋亡的調(diào)節(jié)作用分離WT小鼠和CD36-/-小鼠原代肝細(xì)胞,在伴有或者不伴有5 μM genistein 刺激的同時(shí),給予 100ng/mlConA 分別刺激 5min、20min、1h、4h 及8h后收集細(xì)胞,檢測8h細(xì)胞上清谷草轉(zhuǎn)氨酶(AST)表達(dá)水平;免疫印跡檢測不同時(shí)間點(diǎn)刺激后Akt、JNK、IKKα/β及Caspase-3表達(dá)水平。結(jié)果:Con A刺激后,WT小鼠較CD36-/-小鼠表現(xiàn)出更為嚴(yán)重的肝損傷,ALT水平較高,壞死或凋亡的細(xì)胞更多;WT小鼠表達(dá)更高水平的炎性細(xì)胞因子 TNF-α,CXCL10,IL-1α,MCP-1 及 IL-6;同時(shí),WT 小鼠肝組織中總 MNCs、CD4+、CD8+T細(xì)胞、NK細(xì)胞、浸潤的巨噬細(xì)胞及中性粒細(xì)胞數(shù)均高于CD36-/-小鼠;WT小鼠肝組織中JNK及IKKα/β的磷酸化水平,以及Caspase-3的表達(dá)均高于CD36-/-小鼠。此外,實(shí)驗(yàn)表明CD36可以通過Akt、JNK及IKKα/β的磷酸化,以及Caspase-3的激活從而調(diào)節(jié)CXCL10誘導(dǎo)的肝細(xì)胞凋亡過程。最后,WT小鼠尾靜脈注射Con A的同時(shí)腹腔注射genistein可以明顯降低ALT水平、肝臟組織壞死以及MNCs的浸潤,并且CXCL10刺激肝原代細(xì)胞時(shí),孵育genistein后Akt及JNK的磷酸化水平明顯降低。結(jié)論:實(shí)驗(yàn)結(jié)果表明CD36在Con A誘導(dǎo)的肝損傷中通過促進(jìn)肝臟炎性反應(yīng)及調(diào)控CXCL10促細(xì)胞凋亡發(fā)揮促進(jìn)炎癥應(yīng)答的作用,這為臨床免疫性肝炎的防治提供了新的防治思路。第二部分:CD36基因敲除在保護(hù)小鼠APAP誘導(dǎo)的藥物性肝損傷中的作用及其機(jī)制研究對(duì)乙酰氨基酚(APAP)是解熱鎮(zhèn)痛藥的主要成分,其常見的副作用是引起藥物性肝損傷,甚至急性肝衰竭。APAP引起的藥物性肝損傷在歐美國家尤其嚴(yán)重,近年來我國的發(fā)病率也逐漸增加,越來越被重視。APAP所致的肝損傷包括APAP代謝蛋白加合物形成、線粒體功能障礙、氧化應(yīng)激、過氧亞硝酸鹽形成及核DNA斷裂等關(guān)鍵過程。最近的研究表明無菌性炎癥和先天免疫細(xì)胞可能在APAP誘導(dǎo)的肝損傷和修復(fù)中起重要作用。目的:藥物性肝損傷已經(jīng)成為一個(gè)不容忽視的嚴(yán)重的公共衛(wèi)生問題,盡管一直一來對(duì)APAP誘導(dǎo)的肝損傷的機(jī)制研究已達(dá)數(shù)十年之久,但是仍然存在很多不足之處,只有對(duì)其機(jī)制更深入研究,才能對(duì)APAP誘導(dǎo)的肝損傷提供更為高效有效的預(yù)防治療措施。本研究旨在探討CD36基因是否能夠調(diào)控影響APAP誘導(dǎo)的肝損傷,為當(dāng)前臨床藥物性肝炎的防治提供新的方向。方法:為了探討CD36基因在藥物性肝損傷中是否發(fā)揮作用及其作用機(jī)制,我們建立了刀對(duì)乙酰氨基酚(APAP)誘導(dǎo)的野生型(WT)小鼠及CD36基因缺陷(CD36./.)小鼠急性肝損傷模型。(1)CD36是否參與了 APAP誘導(dǎo)的肝損傷過程WT小鼠隨機(jī)分為兩組,給予APAP(250mg/kg)或等量PBS處理,處理后1h、3h、6h、12h及24h取小鼠肝臟,實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(qPCR)檢測肝臟中CD36mRNA表達(dá)水平;蛋白印跡法(Western blot)檢測肝臟中CD36蛋白表達(dá)水平。(2)內(nèi)源性CD36對(duì)APAP誘導(dǎo)小鼠肝損傷的影響WT及CD36-/-小鼠停止喂食16h后腹腔注射APAP(250mg/kg),在8h及24h后收集血清并檢測血清中谷丙轉(zhuǎn)氨酶(ALT)的活性;分離小鼠肝組織,利用HE染色方法觀察肝組織病變;并利用末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記測定實(shí)驗(yàn)(TUNEL)分析肝細(xì)胞凋亡情況。(3)內(nèi)源性CD36對(duì)APAP誘導(dǎo)小鼠肝內(nèi)炎癥反應(yīng)的調(diào)控作用WT小鼠和CD36-/-小鼠經(jīng)腹腔注射APAP后,分離小鼠肝內(nèi)單個(gè)核細(xì)胞(mononuclear cells,MNCs),流式細(xì)胞術(shù)檢測浸潤性巨噬細(xì)胞及中性粒細(xì)胞數(shù),分析細(xì)胞的活化水平;實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(Real-time Reverse transcription polymerase chain reaction,Real-time RT-PCR)分析肝組織中促炎細(xì)胞因子(如TNF-α,IL-1β,KC,MCP-1和IL-6等)的表達(dá);酶聯(lián)免疫吸附測定方法(ELISA)檢測血清中細(xì)胞因子的表達(dá)水平;免疫印跡方法(Western Blot)檢測肝組織中JNK、Caspase-3、APAP的代謝產(chǎn)物N-乙�；鶎�(duì)苯醌亞胺(NAPQI)蛋白加合物和細(xì)胞色素P450 2E1(CYP2E1)的蛋白表達(dá)水平。(4)拮抗劑實(shí)驗(yàn)驗(yàn)證內(nèi)源性CD36在APAP誘導(dǎo)小鼠肝損傷中的作用WT小鼠經(jīng)腹腔注射APAP后,腹腔注射酪氨酸酶抑制劑PP1,通過檢測ALT水平;以及原代肝細(xì)胞培養(yǎng)后檢測上清液AST水平等實(shí)驗(yàn)驗(yàn)證內(nèi)源性CD36在Con A誘導(dǎo)的肝損傷中發(fā)揮的作用。結(jié)果:APAP刺激后,WT小鼠較CD36-/-小鼠表現(xiàn)出更為嚴(yán)重的肝損傷,ALT水平較高,壞死或凋亡的細(xì)胞更多;WT小鼠表達(dá)更高水平的炎性細(xì)胞因子KC,IL-1β,MCP-1及IL-6;同時(shí),WT小鼠肝組織中浸潤的巨噬細(xì)胞及中性粒細(xì)胞數(shù)均高于CD36-/-小鼠;此外,WT小鼠腹腔注射APAP的同時(shí)腹腔注射PP1可以明顯降低ALT水平,以及WT來源原代肝細(xì)胞在PP1孵育后上清液AST水平遠(yuǎn)低于APAP處理組。結(jié)論:APAP處理的CD36--小鼠與WT小鼠相比,表現(xiàn)出較輕的肝損傷。證實(shí)了 CD36在APAP誘導(dǎo)的肝損傷中通過促進(jìn)肝臟炎性反應(yīng)及促進(jìn)細(xì)胞凋亡發(fā)揮促進(jìn)炎癥應(yīng)答的作用,這為臨床藥物性肝炎的防治提供了新的防治思路。
[Abstract]:The liver is the largest organ in the human body, has multiple biological functions in maintaining homeostasis and keep the balance in the process of The new supersedes the old. plays a key role. The immune organ of the human liver is important, when the immune balance once destroyed, it will lead to a variety of immune related liver diseases, including HBV, persistent chronic hepatitis B HCV the primary liver cancer and autoimmune hepatitis (AIH) and.AIH autoimmune reaction mediated by chronic liver injury, immune responses to self antigens in liver, and liver cell damage lead to liver inflammation and necrosis, its incidence increased year by year in the world, serious cases may progress rapidly to cirrhosis and liver failure. The main treatment method for immunosuppression and liver transplantation, but the effect is poor, the potential pathophysiological mechanism is still not clear, so there is no clear and effective The treatment of liver from in vivo and in vitro of many non nutritive substances such as drugs, poisons and some metabolites in vivo, with biotransformation, they will be completely decomposed by The new supersedes the old. or discharged to the prototype. And a variety of drugs in use process, because of the drug itself or / and its metabolites or due to the special constitution to reduce drug sensitivity or tolerance will lead to drug-induced liver injury (DILI). At present, DILI has risen to the fifth cause of death in the world, become a global public health problem can not be ignored. The pathogenic mechanisms involved in metabolism protein adduct formation, mitochondrial dysfunction, oxidative stress, and other key nuclear DNA fracture during the formation of peroxynitrite, the clinical use of N- acetylcysteine detoxification, but there are many limitations due to delayed healing of disease, for the development of hepatic fibrosis Sclerosis. Differentiation antigen 36 (CD36) is a single chain glycoprotein molecule exists widely in cell surface, which belongs to the class B scavenger receptor family, can be in a variety of cells such as macrophages, endothelial cells, platelets, the expression of fat cells and epithelial cells, as a pattern recognition receptor, CD36 is involved in a series of physiological and pathological processes, including immune, fatty acid metabolism, angiogenesis, atherosclerosis and phagocytosis of.CD36 and Toll like receptor 4 and 6 in order to promote the combination of sterile inflammation. It is worth noting that CD36 expression was confirmed with alpha 6 integrin and CD133 in tumor stem cells to promote the progression of glioblastoma and inhibition of CD36 receptors will reduce the human melanoma, oral cancer and breast cancer metastasis. The study on CD36 play in acute liver injury and its action mechanism were discussed. The first part: CD36 gene knockout in Paul Immunological liver injury mice induced by A in Con supporting the role and mechanism of concanavalin A (ConA) the pathogenesis of immunological liver injury model induced by can simulate human autoimmune liver diseases and viral hepatitis. Through the establishment of experimental animal models of liver injury mice by intravenous injection of Con A, the the model for the clinical and histopathological manifestations of acute hepatitis, including white blood cells and lymphocytes infiltration in the liver, liver cell necrosis, serum transaminase and secretion of inflammatory cytokines. The pathogenesis of hepatitis now this model has been widely applied in the study of T cell mediated, thus forming our present knowledge about the course of liver damage. Objective: Although there are a large number of autoimmune liver injury in the process of research, but its exact cellular and molecular mechanism is still unclear. More and more data show that The activation of T cells play an important role during chronic liver injury. This study aimed to investigate the regulatory effect of CD36 gene on T cell mediated autoimmune hepatitis, provide a new direction for the prevention of clinical hepatitis. Methods: in order to investigate whether CD36 gene plays a role and mechanism in acute immunity liver injury, we established the concanavalin A (ConA) induced by wild type (WT) mice and CD36 deficient (CD36--) mice model of acute liver injury. (1) endogenous CD36 on ConA induced liver injury in mice WT mice and CD36-/- transgenic mice by tail vein injection of Con 12mg/kg in A induced liver injury. Serum was collected and serum alanine aminotransferase in 8h (ALT) activity; liver tissue of mice were separated by HE to observe the pathological changes of hepatic tissue staining method; and using terminal deoxynucleotidyl transferase mediated dUTP. Experimental determination of export end labeling (TUNEL) analysis of liver cell apoptosis. (2) endogenous CD36 on ConA induced activation of T cells in the liver of mice and the immune regulation of WT mice and CD36-/- mice by tail vein injection of ConA, mononuclear cells isolated from mouse liver (mononuclear cells MNCs), T cells were detected by flow cytometry. Cells were NK cells, infiltrating macrophages and neutrophil count, the level of cell activation analysis; real-time fluorescence quantitative polymerase chain reaction (Real-time Reverse transcription polymerase chain reaction, Real-time RT-PCR) analysis of proinflammatory cytokines in the liver tissue (such as TNF- alpha, CXCL10, IL-1 alpha, MCP-1 and IL-6) expression; enzyme ELISA method (ELISA) to detect the expression of cytokines in serum; Western blot method (Western Blot) in the detection of liver tissue JNK, IKK alpha / beta, ERK and STAT3 phosphorylation, and Caspase-3 The level of expression. (3) antagonist experiments of endogenous CD36 in ConA induced liver damage in mice WT mice by intravenous injection of Con A after intraperitoneal injection of tyrosinase inhibitor genistein (Genistein), closed WT mice CD36-TLR4-TLR6 complex formation, by testing the levels of ALT, analysis and separation of WT in the liver of mice MNCs analysis T cells, HE staining of NK cells, play the infiltration of liver injury induced by CD36 in Con A to verify the endogenous infiltrating macrophages and neutrophils in vitro. (4) the in vitro analysis of CD36 on CXCL10 induced liver cell apoptosis regulating effect of separation of WT and CD36-/- mice primary hepatocytes with or in with 5 M genistein stimulation and 100ng/mlConA stimulation of 5min, 20min, 1H, 4H and 8h were collected after detection of 8h cell supernatant aspartate aminotransferase (AST) expression; Western blot Detected at different time points after stimulation with Akt, JNK, IKK levels of alpha / beta and Caspase-3 expression. Results: Con after A stimulation, WT mice compared with CD36-/- mice showed more serious liver damage, higher levels of ALT, necrosis or apoptosis of cells; expression of higher levels of inflammatory cytokines TNF-, WT mouse CXCL10, IL-1 alpha, MCP-1 and IL-6; at the same time, total MNCs, WT in liver tissue of mice in CD4+, CD8+T cells, NK cells, macrophages and neutrophil count was higher than that of CD36-/- mice in JNK and IKK; alpha / beta phosphorylation of WT in liver tissue of mice, and the expression of Caspase-3 was higher than that of CD36-/- mice. In addition, experiments show that CD36 can Akt, JNK and IKK alpha / beta phosphorylation and activation of Caspase-3, thereby regulating the liver cell apoptosis induced by CXCL10. Finally, WT mice tail vein injection of Con A and intraperitoneal injection of Genistein can significantly reduce the level of ALT, liver Tissue necrosis and MNCs infiltration, and CXCL10 stimulation of primary hepatocytes when incubated with Akt and JNK phosphorylation level was significantly decreased after genistein. Conclusion: the experimental results showed that the liver injury induced by A in CD36 Con by promoting inflammatory response and regulation of liver CXCL10 in promoting cell apoptosis play promote inflammatory responses, this provides a new idea for clinical prevention of autoimmune hepatitis prevention. The second part: CD36 gene knockout of drug-induced liver injury in mice induced by APAP protection mechanisms and the effects of acetaminophen (APAP) is the main component of antipyretic analgesics, the common side effects were caused by drug induced liver injury even.APAP, acute liver failure caused by drug induced liver injury is particularly serious in western countries, the incidence rate in our country has gradually increased in recent years, more and more attention to liver injury induced by.APAP including the metabolism of APAP eggs Bai Jiahe The formation, mitochondrial dysfunction, oxidative stress, and nuclear DNA fracture and other key during the formation of peroxynitrite. Recent studies have shown that aseptic inflammation and innate immune cells may play an important role in liver damage and repair induced by APAP. Objective: drug induced liver injury has become an unavoidable serious public health problem. Although the result of research has been mechanism of liver injury induced by APAP has been for decades, but there are still many deficiencies, only the mechanism more in-depth research, in order to liver injury induced by APAP to provide a more efficient and effective prevention and treatment measures. The purpose of this study was to investigate whether CD36 gene can regulate the effects of liver injury APAP induction, provide a new direction for the prevention and treatment of the clinical drug hepatitis. Methods: in order to investigate whether the CD36 gene function and its role in drug-induced liver injury The mechanism, we established a knife of acetaminophen (APAP) induced by wild type (WT) mice and CD36 deficient (CD36./.) mice model of acute liver injury (1). CD36 is involved in the process of liver injury induced by APAP WT mice were randomly divided into two groups, treated with APAP (250mg/kg) or equivalent PBS after treatment, 1H, 3h, 6h, 12h and 24h in mice liver, real-time fluorescence quantitative polymerase chain reaction (qPCR) level of CD36mRNA expression in the liver; Western blot (Western blot) the expression level of CD36 protein was detected in the liver of mice. (2) effects of WT and CD36-/- endogenous CD36 on APAP induced liver injury in mice stop feeding after intraperitoneal injection of APAP 16h (250mg/kg), serum was collected and serum alanine aminotransferase in 8h and 24h (ALT) activity; liver tissue of mice were separated by HE to observe the pathological changes of hepatic tissue staining method; and using terminal deoxynucleotidyl transferase mediated dUTP Experimental determination of nick end labeling (TUNEL) analysis of liver cell apoptosis. (3) endogenous CD36 on APAP induced inflammatory reaction in the liver of mice in the regulation of WT and CD36-/- mice by intraperitoneal injection of APAP, mononuclear cells isolated from mouse liver (mononuclear cells, MNCs), detection of infiltrating macrophages and neutrophils flow cytometry analysis of activation level of cell; real-time fluorescence quantitative polymerase chain reaction (Real-time Reverse transcription polymerase chain reaction, Real-time RT-PCR) analysis of liver tissue proinflammatory cytokines (such as TNF- alpha, IL-1 beta, KC, MCP-1 and IL-6) expression; enzyme linked immunosorbent assay (ELISA) to detect the expression of cytokines in serum; Western blot method (Western Blot) in the detection of liver tissue JNK, Caspase-3, metabolite of N- acetyl APAP to quinone imine (NAPQI) protein adducts and cells Cytochrome P450 2E1 (CYP2E1) level of protein expression. (4) antagonist experiments of endogenous CD36 in APAP induced liver damage in mice WT mice by intraperitoneal injection of APAP after intraperitoneal injection of tyrosinase inhibitor PP1, by detecting the level of ALT; and the primary cultured hepatocytes after detection of liver injury play supernatant AST level test validation of endogenous CD36 in Con induced A. Results: after APAP stimulation, WT mice compared with CD36-/- mice showed more serious liver damage, higher levels of ALT, necrosis or apoptosis of cells; inflammatory cytokines KC, higher levels of expression of WT in mouse IL-1 beta, MCP-1 and IL-6; at the same time WT, liver tissue in mice macrophages and neutrophil count was higher than that of CD36-/- mice; in addition, WT mice were injected intraperitoneally with APAP and intraperitoneal injection of PP1 can significantly reduce the level of ALT, WT and the source of hepatocytes in PP1 group The supernatant AST level after incubation is much lower than that of APAP group. Conclusion: compared with CD36-- mice and WT mice treated with APAP, showed mild liver injury. CD36 was confirmed in APAP induced liver injury by promoting liver inflammatory reaction and promote cell apoptosis promoting inflammatory responses, provide new ideas for prevention and treatment this is a clinical drug hepatitis prevention.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575

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