肝脏交感神经调控与急性肝损伤关系的实验研究

发布时间：2018-01-04 04:01

本文关键词：肝脏交感神经调控与急性肝损伤关系的实验研究　出处：《第三军医大学》2010年硕士论文　论文类型：学位论文

【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(P0.05, P0.01)。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(P0.05)。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(P0.01)。内脏大神经离断和shame组比较无明显变化,结果统计无差异(P0.05)。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(P0.05),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% (P0.05)和59.1% (P0.05) ,IL-1βmRNA水平较LPS组增加53.7%(P0.05)和57.8%(P0.05),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(P0.05),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。
[Abstract]:First, the background
In the clinic, due to severe liver trauma, surgery, drug poisoning, bacteria, virus infection induced acute liver injury and other reasons can lead to acute liver failure. The liver function of acute hepatic injury induced by incomplete or acute liver failure, clinical treatment is very difficult, it is difficult to get ideal medicine hepatoprotective effect. This pathology in the process, due to trauma, bacteria, viruses and other direct cause of liver damage is limited, and the body of some minor damage or pathogen inappropriate inflammatory or immune response may cause serious consequences. The inflammatory reaction is too strong so that a large number of inflammatory mediators into the blood circulation can cause systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), caused by shock and multiple system organ failure (multi-system/organ, failure, MSOF), acute liver injury is a systemic inflammatory reaction A viscera manifestation of the syndrome.
The occurrence and development of inflammation is a complex process, influenced by many factors and regulation. Regulatory factors are involved in the inflammatory reaction can be roughly divided into two categories of fluids and nerve, which macrophages play a crucial role in inflammation. The inflammation and liver injury research focus adjustment in the aspect of body fluids, in the study of neural regulation due to the lack of good animal models, fewer studies on display. Hepatic sympathetic regulation system by celiac ganglion, thoracolumbar spinal cord, hypothalamus sympathetic center three different levels of the central composition. Kupffer in liver cells is the main member of monocyte macrophage system, is in residence liver macrophages accounted for more than 80% of the total body macrophages, accounting for 35% of the number of liver nonparenchymal cells, activating.Kupffer cells of the defense function It is closely related to acute liver and chronic inflammation.
Research results at home and abroad in recent years, we conclude that in the occurrence of acute liver injury in the development, along with the change of sympathetic activity, sympathetic nervous system central is entirely possible by adjusting the direct regulation of Kupffer cell expression of inflammatory neurotransmitter release related factors, and thus participate in the start and trigger the excessive inflammatory reaction.
Two, the purpose
This study from different segments of liver sympathetic regulation system to sympathetic treatment, the regulation of the sympathetic nervous system performance of each segment in acute liver injury, and in vitro validation occurs in acute liver injury of sympathetic activation, regulating effect on development. From a new point of view to understand the systemic inflammatory response and the corresponding organ damage regulation mechanism for excessive inflammatory response (SIRS) provides a new way of thinking and the treatment of MODS.
Three, methods and results
1, the influence of different segments of sympathetic denervation on acute liver injury induced by CCL4 in rats: from the cervical sympathetic trunk, splanchnic nerve and nerve endings in the liver of different control section blocking the sympathetic nerve, making the acute liver injury model on the basis of the application of CCL4. The distribution frequency and amplitude changes of electrode determination. Sympathetic with the record of liver function, serum TNF-a level and liver histopathology of liver tissue were measured NE the indexes such as high performance liquid chromatography. The normal rat cervical sympathetic trunk on the wave amplitude of low frequency, uniform voltage stabilized, after CCL4 intraperitoneal injection for liver injury stimulation, sympathetic nerve discharge increased immediately, wave V increases, was pulse enhancement, continuous recording after about 40 seconds, sympathetic nerve activity is back to normal. The waveform of the sympathetic and sympathetic group were compared with shame group in the liver: Albumin and prealbumin increased, AST, ALT, liver function indexes of total bilirubin decreased significantly, the difference was significant (P0.05, P0.01). The greater splanchnic nerve transection and shame group of liver function indexes had no significant difference (P0.05). The TCST group and hepatic sympathetic respectively. Compared with shame group, liver tissue norepinephrine concentrations decreased significantly, very significant difference (P0.01). The greater splanchnic nerve transection and shame were not significantly changed, the statistical difference (P0.05). The transection of cervical sympathetic trunk, to the level of serum TNF-a in liver sympathetic group was significantly lower than that in shame (P0.05), splanchnic nerve transection group was higher than that in shame group.
2, the sympathetic neurotransmitter NE on endotoxin induced by in situ perfusion on expression of Kupffer cell TNF-a/ IL-1 beta rats, type IV collagenase digestion, Percoll density gradient centrifugation and selective adherence method of purified Kupffer cells were divided into three groups (blank control group.A group); B group (LPS); group C (LPS+NE) Real time and ELISA PCR were used to detect the expression of KC TNF- alpha, IL-1 beta and mRNA protein level. KC cells were normally almost no expression of TNF-a and IL-1 beta by exogenous endotoxin LPS (final concentration 10 g/ml) after stimulation of TNF-a increased significantly in 12 hours, the expression of IL-1 beta mRNA; while adding norepinephrine concentration were 1 mol/L and 10 mol/L culture liquid in the treatment group, TNF-a mRNA levels compared with LPS group were increased by 50.9% (P0.05) and 59.1% (P0.05), IL-1 beta mRNA levels increased compared with group LPS 53.7% (P0.05) and 57.8% (P0.05) the expression. The amount and concentration of noradrenaline was positively correlated; the TNF-a IL-1 beta on mRNA and protein level were consistent. Under normal conditions, almost no expression or low expression, LPS (10 g/ml) was increased significantly after stimulation. (LPS+NE) Co stimulation, TNF-a was higher than LPS group IL-1 beta concentration to noradrenaline concentration was 1 mol/L and 10 mol/L (P0.05), and in the low concentration (0.1 mol/L) cases showed no significant difference.
Conclusion:
1, different segments of the sympathetic nervous system to regulate sympathetic nerve, can significantly reduce the damage of liver function in acute liver injury induced by CCL4, liver tissue norepinephrine levels decreased. The sympathetic nervous system of each segment is relatively independent of the control function to control the machine can maintain body stability, also may be the sympathetic afferent pathway damage after the central vagal reflex reinforcement function after induced by antagonistic.
2, the regulation of different segments of sympathetic nerve activation in acute liver injury in early stage can promote the inflammatory effect. Sympathetic nerve activation may directly through Kupffer cells expression of inflammation related cytokines and promote, in a certain range of concentration, with the concentration of noradrenaline increased proinflammatory cytokine expression.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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