CD36基因在小鼠急性肝损伤中的作用及其机制研究

发布时间：2018-01-02 09:33

本文关键词：CD36基因在小鼠急性肝损伤中的作用及其机制研究　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：肝脏是人体内最大的实体脏器,具有多重生物学功能,在维持稳态和保持新陈代谢平衡的过程中起到了非常关键的作用。肝脏是人类重要的免疫器官,当免疫平衡一旦被破坏,就会引发多种免疫相关的肝脏疾病,包括HBV、HCV引起的持续性慢性肝炎、原发性肝癌以及自身免疫性肝炎(AIH)等。AIH自身免疫反应介导的慢性进行性肝脏损伤过程,机体产生针对肝脏自身抗原的免疫反应,从而破坏肝细胞导致肝脏炎症坏死,其发病率在世界范围内逐年增加,严重病例可快速进展为肝硬化和肝衰竭。主要治疗手段为免疫抑制剂和肝移植,但效果欠佳,潜在的病理生理机制仍不清楚,因此尚无明确有效的治疗方案。肝脏对来自体内和体外的许多非营养性物质如各种药物、毒物以及体内某些代谢产物,具有生物转化作用,通过新陈代谢将它们彻底分解或以原形排出体外。而各种药物在使用过程中,因药物本身或/及其代谢产物或由于特殊体质对药物的超敏感性或耐受性降低会导致药物性肝损伤(DILI)。目前DILI已经上升至全球死亡原因的第五位,成为全球不可忽视的公共卫生问题。其致病机制涉及代谢蛋白加合物形成、线粒体功能障碍、氧化应激、过氧亚硝酸盐形成及核DNA断裂等关键过程,目前临床均使用N-乙酰半胱氨酸解毒,但存在很多局限性而导致病程迁延不愈,发展为肝纤维化肝硬化等。分化抗原36(CD36)分子是一种广泛存在于细胞表面的单链糖蛋白,属于B族清道夫受体,可以在多种细胞如巨噬细胞、微血管内皮细胞、血小板、脂肪细胞及上皮细胞等表达,作为模式识别受体,CD36参与一系列生理和病理过程,包括免疫、脂肪酸代谢、血管生成、动脉粥样硬化和吞噬作用等。CD36还与Toll样受体4和6结合以促进无菌炎症。值得注意的是,CD36被证实与α6整合素及CD133共表达于肿瘤干细胞以促进胶质母细胞瘤的进展而抑制CD36受体则会降低人类中黑素瘤、口腔癌及乳腺癌的肿瘤转移。本研究就CD36在急性肝损伤中发挥的作用及机制进行了初步探讨。第一部分:CD36基因敲除在保护小鼠Con A诱导的免疫性肝损伤中的作用及其机制研究刀豆蛋白A(ConA)诱导的免疫性肝损伤模型能较好地模拟人类肝脏自身免疫性疾病和病毒性肝炎的发病机制。通过给小鼠静脉注射Con A建立肝脏损伤的实验动物模型,此模型表现为急性肝炎的临床和病理组织学表现,包括肝脏中白细胞和淋巴细胞浸润、肝细胞坏死、血清转氨酶升高及炎性细胞因子分泌等。现如今此模型已被广泛应用于研究T细胞介导的肝炎致病机制,从而形成了我们目前关于肝损伤过程的认知。目的:虽然目前已有大量关于自身免疫性肝损伤过程的研究,但其确切的细胞和分子机制尚不清楚。越来越多的数据表明T细胞的活化在慢性肝脏损伤过程中具有相当重要的作用。本研究旨在探讨CD36基因对T细胞介导的自身免疫性肝炎的调控作用,为当前临床肝炎的防治提供新的方向。方法:为了探讨CD36基因在急性免疫性肝损伤中是否发挥作用及其作用机制,我们建立了刀豆蛋白A(ConA)诱导的野生型(WT)小鼠及CD36基因缺陷(CD36--)小鼠急性肝损伤模型。(1)内源性CD36对ConA诱导小鼠肝损伤的影响WT小鼠和CD36-/-转基因小鼠经尾静脉按12mg/kg注射Con A诱导肝脏损伤。在8h后收集血清并检测血清中谷丙转氨酶(ALT)的活性;分离小鼠肝组织,利用HE染色方法观察肝组织病变;并利用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定实验(TUNEL)分析肝细胞凋亡情况。(2)内源性CD36对ConA诱导小鼠肝内T细胞活化的免疫调控作用WT小鼠和CD36-/-小鼠经尾静脉注射ConA后,分离小鼠肝内单个核细胞(mononuclear cells,MNCs),流式细胞术检测T细胞、NK细胞、浸润性巨噬细胞及中性粒细胞数,分析细胞的活化水平;实时荧光定量聚合酶链式反应(Real-time Reverse transcription polymerase chain reaction,Real-time RT-PCR)分析肝组织中促炎细胞因子(如TNF-α,CXCL10,IL-1α,MCP-1和IL-6等)的表达;酶联免疫吸附测定方法(ELISA)检测血清中细胞因子的表达水平;免疫印迹方法(Western Blot)检测肝组织中JNK、IKKα/β、ERK及STAT3的磷酸化水平,以及Caspase-3的表达水平。(3)拮抗剂实验验证内源性CD36在ConA诱导小鼠肝损伤中的作用WT小鼠经尾静脉注射Con A后,腹腔注射酪氨酸酶抑制剂染料木素(Genistein),封闭WT小鼠CD36-TLR4-TLR6复合物形成,通过检测ALT水平、HE染色组织学分析以及分离WT小鼠肝内MNCs分析T细胞、NK细胞、浸润性巨噬细胞及中性粒细胞浸润情况验证内源性CD36在Con A诱导的肝损伤中发挥的作用。(4)体外实验分析CD36对CXCL10诱导肝细胞凋亡的调节作用分离WT小鼠和CD36-/-小鼠原代肝细胞,在伴有或者不伴有5 μM genistein 刺激的同时,给予 100ng/mlConA 分别刺激 5min、20min、1h、4h 及8h后收集细胞,检测8h细胞上清谷草转氨酶(AST)表达水平;免疫印迹检测不同时间点刺激后Akt、JNK、IKKα/β及Caspase-3表达水平。结果:Con A刺激后,WT小鼠较CD36-/-小鼠表现出更为严重的肝损伤,ALT水平较高,坏死或凋亡的细胞更多;WT小鼠表达更高水平的炎性细胞因子 TNF-α,CXCL10,IL-1α,MCP-1 及 IL-6;同时,WT 小鼠肝组织中总 MNCs、CD4+、CD8+T细胞、NK细胞、浸润的巨噬细胞及中性粒细胞数均高于CD36-/-小鼠;WT小鼠肝组织中JNK及IKKα/β的磷酸化水平,以及Caspase-3的表达均高于CD36-/-小鼠。此外,实验表明CD36可以通过Akt、JNK及IKKα/β的磷酸化,以及Caspase-3的激活从而调节CXCL10诱导的肝细胞凋亡过程。最后,WT小鼠尾静脉注射Con A的同时腹腔注射genistein可以明显降低ALT水平、肝脏组织坏死以及MNCs的浸润,并且CXCL10刺激肝原代细胞时,孵育genistein后Akt及JNK的磷酸化水平明显降低。结论:实验结果表明CD36在Con A诱导的肝损伤中通过促进肝脏炎性反应及调控CXCL10促细胞凋亡发挥促进炎症应答的作用,这为临床免疫性肝炎的防治提供了新的防治思路。第二部分:CD36基因敲除在保护小鼠APAP诱导的药物性肝损伤中的作用及其机制研究对乙酰氨基酚(APAP)是解热镇痛药的主要成分,其常见的副作用是引起药物性肝损伤,甚至急性肝衰竭。APAP引起的药物性肝损伤在欧美国家尤其严重,近年来我国的发病率也逐渐增加,越来越被重视。APAP所致的肝损伤包括APAP代谢蛋白加合物形成、线粒体功能障碍、氧化应激、过氧亚硝酸盐形成及核DNA断裂等关键过程。最近的研究表明无菌性炎症和先天免疫细胞可能在APAP诱导的肝损伤和修复中起重要作用。目的:药物性肝损伤已经成为一个不容忽视的严重的公共卫生问题,尽管一直一来对APAP诱导的肝损伤的机制研究已达数十年之久,但是仍然存在很多不足之处,只有对其机制更深入研究,才能对APAP诱导的肝损伤提供更为高效有效的预防治疗措施。本研究旨在探讨CD36基因是否能够调控影响APAP诱导的肝损伤,为当前临床药物性肝炎的防治提供新的方向。方法:为了探讨CD36基因在药物性肝损伤中是否发挥作用及其作用机制,我们建立了刀对乙酰氨基酚(APAP)诱导的野生型(WT)小鼠及CD36基因缺陷(CD36./.)小鼠急性肝损伤模型。(1)CD36是否参与了 APAP诱导的肝损伤过程WT小鼠随机分为两组,给予APAP(250mg/kg)或等量PBS处理,处理后1h、3h、6h、12h及24h取小鼠肝脏,实时荧光定量聚合酶链式反应(qPCR)检测肝脏中CD36mRNA表达水平;蛋白印迹法(Western blot)检测肝脏中CD36蛋白表达水平。(2)内源性CD36对APAP诱导小鼠肝损伤的影响WT及CD36-/-小鼠停止喂食16h后腹腔注射APAP(250mg/kg),在8h及24h后收集血清并检测血清中谷丙转氨酶(ALT)的活性;分离小鼠肝组织,利用HE染色方法观察肝组织病变;并利用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定实验(TUNEL)分析肝细胞凋亡情况。(3)内源性CD36对APAP诱导小鼠肝内炎症反应的调控作用WT小鼠和CD36-/-小鼠经腹腔注射APAP后,分离小鼠肝内单个核细胞(mononuclear cells,MNCs),流式细胞术检测浸润性巨噬细胞及中性粒细胞数,分析细胞的活化水平;实时荧光定量聚合酶链式反应(Real-time Reverse transcription polymerase chain reaction,Real-time RT-PCR)分析肝组织中促炎细胞因子(如TNF-α,IL-1β,KC,MCP-1和IL-6等)的表达;酶联免疫吸附测定方法(ELISA)检测血清中细胞因子的表达水平;免疫印迹方法(Western Blot)检测肝组织中JNK、Caspase-3、APAP的代谢产物N-乙酰基对苯醌亚胺(NAPQI)蛋白加合物和细胞色素P450 2E1(CYP2E1)的蛋白表达水平。(4)拮抗剂实验验证内源性CD36在APAP诱导小鼠肝损伤中的作用WT小鼠经腹腔注射APAP后,腹腔注射酪氨酸酶抑制剂PP1,通过检测ALT水平;以及原代肝细胞培养后检测上清液AST水平等实验验证内源性CD36在Con A诱导的肝损伤中发挥的作用。结果:APAP刺激后,WT小鼠较CD36-/-小鼠表现出更为严重的肝损伤,ALT水平较高,坏死或凋亡的细胞更多;WT小鼠表达更高水平的炎性细胞因子KC,IL-1β,MCP-1及IL-6;同时,WT小鼠肝组织中浸润的巨噬细胞及中性粒细胞数均高于CD36-/-小鼠;此外,WT小鼠腹腔注射APAP的同时腹腔注射PP1可以明显降低ALT水平,以及WT来源原代肝细胞在PP1孵育后上清液AST水平远低于APAP处理组。结论:APAP处理的CD36--小鼠与WT小鼠相比,表现出较轻的肝损伤。证实了 CD36在APAP诱导的肝损伤中通过促进肝脏炎性反应及促进细胞凋亡发挥促进炎症应答的作用,这为临床药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.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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