【作者】 杜权；

【导师】 王正国；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 创伤后常发生免疫抑制,易继发感染、脓毒症,甚至多器官功能障碍综合征(multiple organ dysfunction syndrome,MODS),最终影响结局。针对损伤、感染天然免疫很快启动非特异的细胞、体液反应,成为机体的第一道防线,其在创伤后炎症反应的发生、发展动态过程中具有重要作用。巨噬细胞是参与天然免疫的重要细胞,Toll样受体(Toll-like receptor,TLR)家族中的两个重要受体TLR2、TLR4主要表达于巨噬细胞。近来发现TLR4不仅是LPS的模式识别受体(pattern recognition receptors, PRRs),还能识别众多内源性损伤相关分子模式(damage-associated molecular patterns,DAMPs),作为感知组织损伤的“哨兵”而介导无菌性炎症反应。TLR2能识别较TLR4更多的病原体,也参与了组织损伤后炎症反应。TLR2/4与各自的配体结合后,通过相似信号转导最终导致NF-κB活化,产生炎症细胞因子、趋化因子、共刺激分子等,并被认为在机体防御损伤、感染的天然免疫中具有开关作用。在发生天然免疫改变的同时机体发生了神经内分泌反应,表现为交感神经系统兴奋并伴以不同程度的下丘脑-垂体-肾上腺轴活化,产生肾上腺素(epinephrine,E)、去甲肾上腺素(norepinephrine,NE)、糖皮质激素等,除了能调节机体一般功能外,由于神经-内分泌-免疫网络的存在,它们常影响免疫功能,以维持稳态。以往研究显示,创伤后腹腔、脾巨噬细胞对LPS的反应性降低,但其机理不清。TLR2/4是TLR家族两种主要的受体,其表达水平与对配体的敏感性密切相关。尽管对其信号转导进行了大量研究,但是对其表达的调节还不清楚。研究TLR2/4在创伤后表达变化及神经内分泌激素的调节,对于揭示创伤后免疫功能失常的机制并预防、治疗相关并发症以提高临床救治率均具有重要意义。基于以上认识,本研究采用大鼠创伤模型,以腹腔巨噬细胞为研究对象,进行了以下研究:采用RT-PCR动态观察了创伤后巨噬细胞TLR2/4 mRNA表达变化;以不同浓度Pam3CSK4、LPS刺激创伤后24小时的巨噬细胞,测定产生TNF-α水平以反映受体反应性;采用HPLC测定了创伤后24小时不同时间点血清E和NE水平的变化,同时,采用放射免疫法测定了血清ACTH、皮质醇、T3、T4、TSH、β-EP、PRL,观察了神经内分泌激素变化规律;进一步进行离体实验,以不同浓度皮质酮(corticosterone,CORT)、E、NE刺激巨噬细胞不同时间后,以实时定量PCR和半定量RT-PCR方法研究了TLR2/4 mRNA表达;最后,于创伤前行颈交感神经干离断术(TCST)以观察其对创伤后神经内分泌激素的可能调节作用。通过研究,得出以下结果:1.大鼠创伤后巨噬细胞TLR2/4 mRNA表达在伤后2小时后即降低,直至创伤后24小时。创伤后巨噬细胞对Pam3CSK4、LPS刺激后的反应性降低。2.大鼠创伤后2小时血E、NE水平即明显升高,ACTH、GC也明显升高,直至创伤后24小时。垂体-甲状腺轴变化表现为创伤后T3、T4均降低,但是TSH变化不显著。非垂体-肾上腺轴激素β-EP也于创伤后2小时即升高,PRL于创伤后24小时升高。3.CORT刺激巨噬细胞后能下调TLR2 mRNA表达,其作用具有时间、剂量依赖性,但是对TLR4 mRNA表达的影响不明显。E刺激巨噬细胞后能下调TLR2 mRNA、TLR4 mRNA表达,其作用也具有时间、剂量依赖性。不同浓度NE(0～10000ng/ml)刺激巨噬细胞不同时间(0～48小时)后不能明显下调TLR2/4 mRNA的表达。4.TCST不能减轻创伤后2小时明显神经内分泌激素变化,能降低24小时的血清NE、皮质醇水平。总之,本研究得出以下结论:1.大鼠创伤后巨噬细胞TLR2/4 mRNA表达降低,这对于防止创伤后机体过度炎症反应可能具有一定意义,但与此同时,也意味着机体对病原体(如G+、G-菌)处理能力降低。创伤后巨噬细胞反应性降低,可能与TLR2/4表达降低有关。2.大鼠创伤后发生明显神经内分泌反应,交感神经系统、HPA轴活化,并伴以垂体-甲状腺轴、非垂体-肾上腺轴激素明显变化,共同调节机体以维持稳态。3.较高水平CORT、E刺激巨噬细胞较长时间后能下调TLR2 mRNA或TLR4 mRNA表达。4.TCST能一定程度减轻创伤后神经内分泌激素变化,可能有一定意义。通过研究,为创伤后TLR2/4表达变化及其调节提供了一定的实验及理论依据。更多还原

【Abstract】 Immunosuppression is a common complication after trauma, and it may lead to the development of infection, sepsis, and multiple organ dysfunction syndrome (MODS),which eventually influence outcome. Innate immunity is the first line to defend body against infection and injury through rapid initiation of non-specific cellular and fluid response. Innate immunity plays an important role in the occurrence and development of inflammation after trauma. Macrophages are important resident cells to mediate innate immunity. Toll-like receptor(TLR)2 and TLR4 are two major members of TLRs, mainly expressed on monocytes and macrophages. TLR4 is the pattern recognition receptors(PRRs) of LPS, and recently it was found that it can bind to many damage-associated molecular patterns(DAMPs). As a sentinel to detect tissue injury TLR4 mediate sterile inflammation. TLR2 can recognize a wider variety of pathogens, and it also can participate in infla- mmation after injury. After TLR2/4 bind to their respective ligands, they can cause a similar casade of signal transduction, resulting in the NF-κB activation and finally the production of inflammatory cytokines, chemokines, and costimulatory molecules, which are considered to be the switch to innate immune response to pathogenic microorganisms and endogenous damage-associated molecules. With the changes of innate immunity trauma often sets off neuroendocrine changes, which is charactrized by excited sympathetic nervous system and activated hypothalamic-pituitary-adrenal(HPA) axis to some extent. Norepinephrine(NE), epinephrine(E), and glucocorticoids are produced in large quantity. Neuroendocrine hormones may influence immune functions to keep homeostasis through the neuro-endocrine-immune network. Previous research has shown that following trauma peritoneal or spleen macrophages often could display impaired responsiveness to LPS, but the mechanism remains unclear. As two important receptors, the levels of TLR2/4 expression are closely related with the sensitivity to their ligands. Although extensive research is focused on TLRs and their signaling, how to regulate TLR2/4 expression remains unknown. To study the expression of TLR2/4 and its regulation by neuroendocrine hormones after trauma is of importance, it will provide valuable knowledge to explore the mechanism behind immune dysfunction and prevent and treat its complications to improve survival rate.Based on the above knowledge, the present research established a rat trauma model, peritoneal macrophages were collected from rats and the following studies were done: the level of TLR2/4 mRNA expression on rat macrophages was evaluated dynamically after trauma by RT-PCR; tumor necrosis factor-α(TNF-α) secretion of macrophages stimulated by different concentrations of Pam3CSK4 and LPS respectively was observed 24h after trauma to analyze receptors reactivity; changes of serum E and NE were determined by HPLC after trauma for 24 hours; at the mean time, the levels of serum adrenocorticotrophic hormone (ACTH), cortisol,T3,T4,TSH,β-EP,and PRL were determined by radioimmunity assay; following study in vitro is done to observe the effects of corticosterone (CORT), E, and NE on TLR2/4 mRNA expression on macrophages by real-time quantitative PCR and semi-quantitative RT-PCR in different concentrations and for different durations; finally, transection of the cervical sympathetic trunk(TCST) was performed to observe its effect on the changes of neuroendocrine hormone after trauma.Following results were made from the research:1. The expression of TLR2/4 mRNA could be downregulated by trauma, which continued from 2h to 24h following trauma. Trauma resulted in impaired responsiveness to both LPS and Pam3CSK4 on macrophages.2. Following trauma, the elevation of serum levels of E, NE, ACTH, and cortisol continued from 2h to 24h. Although the levels of T3 and T4 were decreased, those of TSH remained instant following trauma. The serum levels ofβ-EP and PRL not from pituitary-adrenal axis were also increased.3. After macrophages exposure to CORT, its down regulation of TLR2 mRNA expression was time-dosedependent, but no significant influence on TLR4 mRNA expression. The down regulation of TLR2 mRNA and TLR4 mRNA expression by E was also time-dosedependent in macrophages. After macrophages exposure to NE, it(0-10000ng/ ml) could not downregulate the expression of TLR2/4 mRNA significantly, and neither did NE(1000ng/ml) 48h after exposure.4. Although TCST can not attenuate remarkable changes of neuroendocrine hormones 2h following trauma, the levels of serum NE and cortisol were decreased significantly 24h following trauma.In conclusion, TLR2/4 expression is downregulated, which may help to prevent excessive inflammatory response following trauma and means that the organism has less ability to cope with pathogens then. Trauma results in impaired macrophages responsiveness, which may be related with the down regulation of TLR2/4 expression. Following trauma remarkable neuroendocrine changes occur, which is mainly characterized with excited sympathetic nervous system and activated hypothalamic-pituitary-adrenal axis. The pituitary-thyroid axis is changed, and the serum hormones levels not from pituitary-adrenal axis are increased, which cooperate to regulate body functions to keep homeostasis. After macrophages exposure to higher concentrations of CORT and E for longer durations, they may downregulate the levels of TLR2 mRNA or TLR4 mRNA expression . TCST can attenuate neuroendocrine hormones changes following trauma, which may be a useful measure.The present study provided valuable laboratorial and theoretical evidence for the research on the expression and regulation of TLR2/4 following trauma.更多还原