【作者】 刘小伟；

【导师】 张广森； 卢放根；

【作者基本信息】 中南大学 ， 内科学， 2003， 博士

【摘要】 脂多糖（Lipopolysaccharide，LPS）是革兰氏阴性细菌内毒素的主要成分，也是革兰氏阴性细菌激发宿主免疫应答，导致机体损伤的重要因素。人体集聚革兰氏阴性杆菌最丰富的部位是肠道，其所产生的LPS经吸收首先到达肝脏，作为机体主要解毒器官的肝脏，无疑是清除LPS的最活跃场所，但也是免疫损伤的最常见靶位。LPS不但可加重肝硬化、酒精性肝病肝损伤，而且明显增强CCl₄和乙醇等肝毒性物质的肝毒性，单独LPS亦可导致肝损伤。但LPS引起肝损伤过程中涉及的具体信号转导分子及效应分子尚不清楚。 本研究运用常规分子生物学和免疫组织化学方法，动态分析LPS刺激不同时段肝脏LPS／TLR₄途径信号分子及功能分子TNF-α表达与肝脏病理变化的关系，同时比较LPS肝损伤发展不同时相蛋白质组变化，从蛋白质整体水平动态捕捉LPS肝损伤信号转导途径中的特异蛋白质“指纹”，搜查参与到该信号通道的信号、效应分子，探索LPS致肝损伤的发生机理。 首先，Balb／c鼠腹腔注射LPS建立急性内毒素血症动物模型，分别在3小时、6小时、12小时、24小时、30小时处死动物取肝脏组织，HE染色观察病理改变，免疫组织化学方法检测肝细胞凋亡禾月组织TNF－a的表达，逆转录PCR（RT－PCR）法检狈肝组织信号分子 1”LR。（Tolllike Receptor 4，TLR．）、MyD88（MyeloidDifferential Factor 88，MvD88）mRNA 7k平的表达。结果表明，腹腔注射 LPS后 0、 小时小鼠一般情况无明显改变，肝组织学变化不明显，24小时小鼠肝脏出现明显炎性浸润、坏死；而3～6 ’J’时肝细胞凋亡和肝组织 TNF－口的表达达到高峰，12小时即明显减少，24小时凋亡、坏死并存，以坏死为主；肝组织 TLR4mRNA的表达在注射LPS后3小时开始下调，6、12小时明显抑制，24小时则基本恢复：肝组织W088mRNA的表达在不同时间点变化不明显。 然后，本研究为探讨LPS诱发肝损伤相关蛋白质群的变化规律，动态分析了LPS刺激不同时段肝脏的蛋白质谱型。在上述动物实验基础上，匀浆抽提肝脏总蛋白进行双向凝胶电泳，从蛋白质表达丰度角度，寻找差异表达蛋白质；对差异表达蛋白质斑点胰酶酶解后用MALDITOF攸S作肽指纹图，结合蛋白质数据库搜索进行蛋白质鉴定，并分析各差异蛋白质在内毒素肝损伤中的作用。结果发现腹腔注射LPS后6 ’J’时(早期人 蛋白质谱显示TNF－a相关蛋白TNFAIPI（TNF－a inducible endothelial protein，TNFAIPI）表达明显上调，TRA IL受体2蛋白表达下调，线粒体相关蛋白MetaxinZ、TIMMSA（Mitochondrlal import Inner membranetranslocase subunit TIMS A，TIMMSA）开始表达上调，部分蛋白酶体相关蛋白质如磷脂酶AZ、PA28（proteasome activator 28，PA28）、MDPP（Maggesiumdependent protein phosphatase，MDPP） V等的表达被抑制；24小时（后期，包括30小时3只小鼠死亡）的蛋白质谱显示，TRAIL受体2表达抑制，线粒体相关蛋白MetaxinZ、TIMMSA达到表达高峰，蛋白酶体相关蛋白磷脂酶AZ、PA28、MDPP恢复表达，并开始表达增殖相关蛋白P34d＊激酶。 本研究结果提示，LPS致肝损伤呈时间依从性：早期，TNF－a相关蛋白出现表达高峰，线粒体凋亡途径相关蛋白质开始表达上调，出现以TNF－口为中心的肝细胞凋亡，肝组织TLR．mRNA的表达表现为抑制状态；中后期，线粒体凋亡途径相关蛋白质达到表达高峰，肝组织出现明显病理变化，凋亡和坏死并存，以坏死为主，同时机体开始启动损伤修复程序，肝组织TLR．mRNA的表达开始恢复，与肝脏的病理变化在时间上呈一致性，可能与LPS耐受性产生有关，提示LPS信号传导分子TLR．在介导肝损伤中起重要作用，而MmD88mRNA的表达在LPS刺激不同时限无明显变化，提示其为胞内保守信号分子。更多还原

【Abstract】 Bacterial infection is still a major threat to humans. Invasion of Gram-negative bacteria elicits immune responses, a major mediator of which is lipopolysaccharide(LPS), a component of the cell wall of Gram-negative bacteria. Liver is the first target organ to be attacked by the gut-derived lipopolysaccharide. LPS appears to provoke liver inflammation with focal hepatocyte injury and increases the sensitivity to CCU, ethanol liver injury. Recent study has shown that LPS can cause liver injury in normal subjects and exaggarate the liver injury in alcoholic liver disease. The prevailing hypothesis for LPS-induced signaling activation is that LPS/Toll-like receptor 4 (TLR4) signaling pathway is activated, proinflammatory factors are released and results in secondary tissue injury. However, the precise mechanism for LPS-induced liver injury remains unclear. It is conceivable that specific signaling and effector molecules are involved in the initiation and development of the processes.In this study, signaling molecules involved in LPS/TLR4 signaling pathway and some effector molecules were detected bymolecular biological and immunohistochemistry technology at indicated timepoints in LPS-induced liver injury model. Simultaneously, the functional molecules involved in the pathogenesis of LPS-induced liver injury were detected by use of proteomics methods.First, LPS-induced endotoxemic mouse were sacrificed at 3 hours, 6 hours, 12 hours, 24 hours and 30 hours respectively. The liver tissues were stained with hematoxylin and eosin for histopathologic analyses. Hepatocyte apoptosis and hepatic TNF-a was detected by immunohistochemical method. Simultaneously, the expression of TLR4mRNA and MyD88mRNA was detected by use of semi-quantitative RT-PCR. A large number of inflammatory cells infiltrated portal areas with additional focal hepatocellular necrosis 24 hours after i.p.LPS. Hepatocyte apoptosis and hepatic TNF- a expression increased significantly 3-6 hours after i.p LPS, while recruited to normal level at 12 hours. Hepatic TLR4mRNA expression was significantly depressed 6 through 12 hours after i.p.LPS, but recruited at 24 hours. Hepatic MyD88 mRNA expression did not change among different timepoints.Second, we identified several possible target proteins in order to provide some information regarding the mechanism of LPS-induced liver injury. The total liver protein was separated by two-dimensional gel electrophoresis at indicated different timepoints. After in-gel trypticdigestion, the peptide mass footprints were drawn up and the protein database was searched to identify the proteins. Then, analyzed how the indicated proteins participated in the pathogenesis of endotoxin-induced liver injury. After i.p. LPS: at earlier stage (6 hours), peak expression of TNFAIP1 was accompanied. TRAIL receptor 2 was downregulated. Mitochondrial related proteins (Metaxin2, TIMM8A) were upregulated. The proteasome related proteins were inhibited; Later stage (24 hours, even 3 mouse died at the 30 hour), significant histopathological changes took place with recruitment of proteasome related proteins and peak expression of metaxin2, TIMM8A . At the same time, p34 cdc2 kinase was also upregulated.It was concluded that LPS-induced liver injury was time dependent. At earlier stage: TNF- a related proteins reached peak expression and mitochondrial apoptotic pathway related proteins began upregulating. TNF-a-dependent hepatocyte apoptosis played predominant role at this stage. The expression of liver TLR4mRNA was depressed significantly. At latter stage: Mitochondrial apoptotic pathway related proteins reached peak expression and the repair program was initiated at the appropriate timepoint. Hepatocyte necrosis made a major role at this stage. The expression of liver TLR4mRNA was recruited from the initial depressed states. It indicated that the signal molecule TLR4mRNA took an important role in LPS-inducedliver injury and may participated in LPS tolerance. MyD88mRNA expression di更多还原