【作者】 曾议；

【导师】 王曙光；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 硕士

【摘要】 一、背景在临床中,由于严重外伤,肝脏大手术、药物中毒、细菌、病毒感染等原因导致的急性肝损伤容易导致急性肝衰竭。目前对于急性肝损伤所致的肝功能不全或急性肝功能衰竭,临床治疗十分棘手,药物保肝难以收到理想的疗效。在此病理过程中,由于外伤、细菌、病毒等直接造成的肝细胞损害是有限的,而机体自身对一些轻微的损害或病原体的不适当的炎症或免疫反应可能造成严重的后果。过强的炎症反应使大量炎性介质进入血循环可造成全身炎症反应综合征(systemic inflammatory response syndrome, SIRS)、引起休克和多系统脏器功能衰竭(multi-system/organ failure, MSOF),急性肝损伤是属于全身炎症反应综合征的一个脏器表现。炎症的发生与发展是一个复杂的过程,受到很多因素的影响和调节。参与机体炎症反应的调控因素可大致分为体液和神经两大类,其中单核巨噬细胞在炎症中发挥极其关键的作用。当前有关炎症反应与肝损伤的研究主要集中在体液调节方面,对于神经调控方面的研究由于缺乏良好的动物模型,研究资料较少显示。肝脏交感神经调控系统由腹腔神经节、脊髓胸腰段、下丘脑交感中枢三个不同层次的中枢构成。肝脏中kupffer细胞是单核巨噬细胞系统中的主要成员,是居留在肝脏中的巨噬细胞,占全身巨噬细胞总数的80%以上,占肝脏非实质细胞数量的35%,起重要的防御功能。Kupffer细胞的激活与肝脏急、慢性炎症关系密切。综合近年来国内外的研究结果,我们推断在急性肝损伤的发生、发展中,随着交感神经活性的改变,交感神经系统各级中枢完全有可能通过对神经递质释放的调节直接调控Kupffer细胞表达炎症相关因子,从而参与启动和触发了过度炎症反应。二、目的本研究拟从肝脏交感神经调控系统的不同节段去交感处理,探讨交感神经系统各个节段在急性肝损伤中的调控效能,并通过体外实验初步验证交感神经活化在急性肝损伤发生、发展中的调节效应。以从新的角度重新认识全身炎症反应及相应脏器损伤的调节机制,为过度炎症反应(SIRS)以及MODS的治疗提供了新的思路。三、方法与结果1、不同节段去交感神经对CCL4所致大鼠急性肝损伤的影响:从颈交感神经干、内脏大神经以及肝内神经末稍不同调控节段阻断交感神经的传导通路,在此基础上应用CCL4制作急性肝损伤模型。用记录电极测定交感神经冲动的发放频率和波幅变化,肝功能、血清TNF-a浓度以及肝组织病理学、高效液相色谱法测定肝组织内NE等有关指标。①正常大鼠颈交感神经干放电波幅较低、频率均一、电压趋于平稳,经CCL4腹腔内注射行肝损伤刺激以后,交感神经干放电立刻增加、波伏增大,呈脉冲式增强,持续记录约40秒钟后,交感神经电活动回复正常时波形。②颈交感干离段、肝内去交感组分别和shame组比较:白蛋白和前白蛋白增高,AST、ALT,总胆红素等各项肝功能指标显著降低,差异具有显著性(P<0.05, P<0.01)。内脏大神经离断和shame组比较各项肝功能指标差异无显著性(P>0.05)。③颈交感干离断组和肝内去交感分别与shame组比较,肝组织内去甲肾上腺素浓度明显降低,差异非常显著(P<0.01)。内脏大神经离断和shame组比较无明显变化,结果统计无差异(P>0.05)。④颈交感干离断、肝内去交感组血清TNF-a水平较shame明显降低(P<0.05),内脏大神经离断组较shame组升高。2、交感神经递质NE对内毒素诱导大鼠kupffer细胞TNF-a/ IL-1β表达的影响采用原位灌注、IV型胶原酶消化、percoll密度梯度离心,选择性贴壁等方法分离纯化得到的kupffer细胞分为三组培养。A组(空白对照组);B组(LPS);C组(LPS+NE )分别采用Real time PCR和ELISA检测KC的TNF-α、IL-1βmRNA和蛋白水平表达。①KC细胞正常情况下几乎不表达TNF-a和IL-1β,经外源性内毒素LPS(终浓度为10μg/ml)的刺激后,在12小时TNF-a、IL-1βmRNA表达显著增加;而在加入去甲肾上腺素终浓度为1μmol/L及10μmol/L培养液的处理组中,TNF-a mRNA水平与LPS组比较分别增加50.9% (P<0.05)和59.1% (P<0.05) ,IL-1βmRNA水平较LPS组增加53.7%(P<0.05)和57.8%(P<0.05),其表达量与去甲肾上腺素浓度呈正相关;②TNF-a、IL-1β在蛋白水平和mRNA水平结果一致。正常情况下几乎不表达或微量表达,LPS(10μg/ml)刺激后表达显著增高。(LPS+NE)共刺激后,TNF-a、IL-1β浓度在去甲肾上腺素浓度为1μmol/L及10μmol/L时较LPS组明显增加(P<0.05),而在低浓度(0.1μmol/L)情况下,结果无显著差异。结论:1、交感神经调控系统不同节段去交感神经,可显著减轻CCL4所致急性肝损伤时肝功能损害,肝组织内去甲肾上腺素水平随之降低。交感神经系统各个节段具有相对独立的调控功能以调控维持机体机能稳定,另外也可能是交感传入通路破坏后迷走神经中枢功能反射性增强后拮抗所致。2、交感神经调控不同节段激活在急性肝损伤早期病程中有促进炎症效应。交感神经活化可能通过直接作用kupffer细胞并促进炎症相关细胞因子的表达,在一定浓度范围内,随着去甲肾上腺素浓度增加,促炎相关细胞因子表达增加。更多还原

【Abstract】 Background:In the clinic, acute liver injury due to serious trauma, major surgery, poisoning, bacteria, virus infection may easily lead to acute liver failure. At present ,the drug treatment is hard to get an ideal effect. In this pathological process, due to trauma, bacteria, viruses, etc as a direct result of liver cell damage is limited. In fact, the body,s inappropriate inflammatory or immune response to pathogens may result in serious consequences. Excessive inflammatory response caused by a large number of inflammatory mediators in the blood circulation may end in systemic inflammatory response syndrome (SIRS) even MOSF, acute liver injury is one part of it.The occurrence and development of Inflammation is a complex process which affected by many factors regulation. Inflammatory regulation can be broadly divided into two major categories: body fluids regulation and neural regulation. At present, the research of inflammatory response and liver injury focused on fluid conditioning, the data of neural regulation show less for the lack of a good animal model. The hepatic sympathetic system was composed of three different levels: the abdominal sympathetic ganglia, thoracolumbar spinal cord, hypothalamus. Kupffer cells are the main member of mononuclear macrophage system which resident in the liver. accounting for more than 80% of the total body macrophages, 35% of liver non-parenchymal cells. The activation of Kupffer cells play an important role in acute and chronic liver inflammation.Objective :By review recent research results, our research based on the hypothesis : With the changes in sympathetic activity. sympathetic nervous system directly regulate Kupffer cell,s secretion by means of neurotransmitter release, as well as involved in start-up and trigger the excessive inflammatory response and lead to the occurrence of acute liver injury. The aims of the present study are elucidate the mechanism of inflammatory response and to propose a new perspective on the systemic inflammatory response and MOSF .methods and results:1. To observe the effects of denervation of different segment of sympathetic nerve system on liver function in the carbon tetrachloride (CCl4)induced rats and the process of synchronization associated with the inflammatory changes. From the cervical sympathetic trunk, splanchnic nerves and nerve endings in liver block the sympathetic regulation pathway, all animal model was prepared as literature reported . After 24 hours of acute liver injury model was established by using CCl4. Liver function as well as histopathological examination and other relevant indicators, serum TNF-a , neuro-electrophysiology and the norepinephrine concentrations of liver tissue were determined in each group.①Normal discharge of cervical sympathetic trunk are low amplitude, frequency uniform, voltage stabilized .After intraperitoneal injection of CCL4 ,the discharge of cervical sympathetic trunk wave V was immediate increased, pulse was enhanced and sustained record About 40 seconds later, the sympathetic nerve electrical activity back to normal .②Transection of cervical sympathetic trunk (TCST)and denervation of sympathetic nerve in liver group resulted in significant changes of parameters under acute liver injury, and has beneficial effects on protein anabolism, metabolism of bilirubin.(P < 0.05 , P < 0.01),but not transection of greater splanchnic nerve groups(P>0.05)③Compared with the shame, the liver tissue norepinephrine of TCST and sympethtic denervation in liver group were lower, the difference was significant(P<0.01)④Compared with the shame, the serum TNF-a levels of TCST and sympethtic denervation in liver group were lower, the difference was significant(P<0.01).2. To evaluate the effect of noradrenalin on the expression of TNF-αand IL-1βin rat Kupffer cells induced by lipopolysaccharide(LPS). The isolated rat kupffer cells cultured in vitro divided into three groups, Given lipopolysaccharide (10μg/ml ) with or without norepinephrine (0.1μmol / L ~ 10μmol / L) respectively for 12 hours. Using quantitative reverse transcription-polymerase chain reaction to detect TNF-a and IL-1βmRNA expression, ELISA detect TNF-a, IL-1βprotein expression of cell culture supernatant.①In the LPS and norepinephrine (1μmol / L and 10μmol / L ) group, TNF-a mRNA expression were higher than that in LPS group 50.9% (P <0 . 05) and 59.1% (P <0. 05) Respectively;IL-1βmRNA expression increased 53.7% and 57.8% than that in LPS group (P <0 . 05).②The cell culture supernatant TNF-a and IL-1βprotein were significantly higher than that in LPS group (P <0. 05) , And in low concentration (0.1μmol / L) case, the results showed no significant difference.Conclusion :1. Sympathetic system has proinflammatory effect under acute liver injury due to CCl42. The high concentration of noradrenalin could enhance the expressions of TNF-αand IL-1βof hepatic Kupffer cells induced by lipopolysaccharide and has pro-inflammatory effects. The activation of sympathetic system may direct act kupffer cells and promote the expression of inflammatory cytokines.In a certain concentration range, with the increase of norepinephrine, pro-inflammatory cytokines expression incresed as well.更多还原