CRH与Urocortin经RhoGTPases调节巨噬细胞吞噬作用的机制研究

发布时间：2018-02-03 05:38

本文关键词： 神经肽天然免疫神经内分泌 CRH UCN 腹腔巨噬细胞吞噬作用 F-actin 细胞骨架重塑 RhoA Rac1 CRH受体 PKA PKC ERK1/2　出处：《第三军医大学》2009年博士论文　论文类型：学位论文

【摘要】： 神经内分泌与免疫功能的相互渗透作用和关联作用,对机体在不同条件下稳态的维持起有决定性的作用,尤其在创伤与感染的病理生理条件下。神经内分泌与免疫系统之间相互关系的研究已经成为神经免疫内分泌学(Neuroimmun-endocrinology)的一个重要研究热点。研究发现,下丘脑分泌的神经肽可参与外周免疫功能的调节机制,如促肾上腺皮质激素释放激素(Corticotropin-releasing hormone,CRH)及其结构相关肽urocortin(UCN)经过下丘脑-垂体-肾上腺(hypothalamicpituitary-adrenal,HPA)轴发挥间接地免疫/炎症调节作用,但是外周局部的CRH与UCN可以自分泌或旁分泌的方式直接参与免疫/炎症的调节。严重创伤或感染后机体免疫功能出现失调,尤其是巨噬细胞免疫功能改变可导致机体防御感染的免疫功能紊乱,导致全身性炎症反应综合征(systemic inflammatory response syndrome,SIRS)、多器官功能衰竭(multiple organ failure,MOF)的易感性增加。巨噬细胞功能紊乱的机制很复杂,可能与微环境中神经内分泌激素等有关。因此,深入探讨神经肽CRH与UCN对细胞免疫功能影响及机制有利于维持机体内环境的稳态,丰富创伤或感染后免疫功能紊乱的免疫平衡调控策略。CRH与UCN,以及它们的受体在机体中枢神经系统与外周组织与器官分布非常广泛。CRH与UCN通过与其受体结合,激活多种G蛋白,从而可能激活下游多种信号通路,发挥不同的生物学效应。目前关于CRH在调节机体行为、内分泌、自律性以及应激免疫等方面的研究已积累了很多资料。越来越多的证据表明外周CRH与UCN在调节免疫炎症反应过程发挥着重要的作用。吞噬清除作用作为巨噬细胞重要的天然免疫功能,在机体严重创伤后感染后往往受到抑制。尽管对巨噬细胞吞噬机制已有广泛的认识,但是关于CRH与UCN,在巨噬细胞天然吞噬功能中的调节作用,目前知之甚少。本研究通过采用原代培养大鼠腹腔巨噬细胞,观察CRH与UCN对巨噬细胞吞噬荧光微球的吞噬率以及平均荧光强度的影响、及其对巨噬细胞在吞噬过程中细胞骨架蛋白重塑与解聚的动态影响,明确CRH与UCN对巨噬细胞天然吞噬功能的调节作用。接着深入研究CRH与UCN调控巨噬细胞骨架蛋白调节因子Rho GTPases亚家族蛋白RhoA、Rac1磷酸化的量效关系和时效关系,进而利用信号分子阻断策略探讨CRH与UCN调控巨噬细胞RhoA、Rac1磷酸化的受体途径和信号转导通路,旨在揭示CRH与UCN调控巨噬细胞天然吞噬功能的信号转导机制。主要研究内容与结果: 1.通过流式细胞技术结合荧光微球分析了CRH与UCN对巨噬细胞吞噬功能的调节作用。结果显示CRH在10-8M水平可显著增强巨噬细胞吞噬微球功能。UCN在10-10~10-7浓度均能够明显增强巨噬细胞吞噬功能。 2.正常巨噬细胞的骨架蛋白均匀分布在胞核与胞浆,在胞膜周边有轻度的集中。在CRH孵育细胞1h后,F-actin骨架蛋白向细胞周边聚集,膜上出现单层丝状伪足。UCN刺激后F-actin改变与CRH刺激后相似,吞噬30min时骨架蛋白重塑更明显, 60min时骨架蛋白表达量却下降。这种动态变化现象与CRH与UCN刺激后巨噬细胞吞噬改变相一致。 3.CRH以10-8M浓度孵育巨噬细胞30min后Rac1磷酸化水平明显增加,60min后RhoA磷酸化水平达到峰值。UCN在10-10~10-7M浓度孵育细胞时均可显著增强RhoA、Rac1磷酸化水平。其中10-8M的UCN孵育细胞30min后Rac1磷酸化水平增加最明显,但是RhoA磷酸化水平呈现双峰状增加,持续时间更长。 4.Rho-ROCK的特异性抑制剂W56、选择性Rac1抑制剂Y27632预孵育细胞后,可明显抑制CRH、UCN对巨噬细胞的促吞噬作用。Y27632、W56预孵育后细胞周边丝状伪足量少,周边聚集不明显,丝状、板状伪足伸展变短,可阻碍F-actin重塑。 5.CRHR1的特异性拮抗剂Antalarmin、CRHR1/R2非特异性拮抗剂Astressin预处理后,CRH与UCN孵育细胞对荧光微球的吞噬率明显下降。在Antalarmin预孵育后, CRH所致的RhoA磷酸化可被完全抑制,而对由CRH所致的Rac1磷酸化抑制的程度与Astressin预孵育后的效应接近。Antalarmin预孵育后,对由UCN所致的RhoA磷酸化水平没有影响,Rac1磷酸化水平轻微降低,但是在Astressin预孵育后, UCN孵育所致的RhoA、Rac1磷酸化水平均明显降低。 6.CRH与UCN孵育静息状态下的大鼠腹腔巨噬细胞,PKA活性增强非常显著。PKA活性在CRH作用后15min达到高峰,而在UCN作用后的10min至60min呈逐渐增加趋势。PKC活性在CRH与UCN孵育细胞后增加比较迅速,在15min时到达高峰。MDL-12330A预孵育细胞阻断PKA后,CRH、UCN对细胞RhoA磷酸化的影响均显著下降。在应用CR预孵育细胞后CRH对RhoA磷酸化增强,对Rac1磷酸化水平没有明显影响。但是CR预孵育细胞后UCN对RhoA磷酸化影响下降约63%,对Rac1磷酸化影响显著下降。 7.CRH以10-8M的浓度刺激巨噬细胞后ERK1/2磷酸化增强,5min时最显著,持续到15min。UCN孵育巨噬细胞后ERK1/2磷酸化水平5-30min持续高水平。在PD98059预处理后可完全抑制CRH对RhoA的表达升高作用,预处理后UCN对RhoA磷酸化升高完全被逆转,对Rac1的表达影响的抑制率达47.7% 结论 1.CRH与UCN可明显增强巨噬细胞的吞噬功能,UCN的促吞噬作用强于CRH。CRH对吞噬作用的影响具有浓度依赖性,其中在10-8M浓度时对细胞吞噬效应最强;UCN在10-10~10-7浓度时均能够明显增强巨噬细胞吞噬功能。 2.CRH与UCN可增强巨噬细胞骨架蛋白重塑,这与CRH与UCN增强吞噬作用相一致。表现为CRH与UCN孵育后的巨噬细胞F-actin骨架蛋白向细胞周边聚集,膜上出现粘附斑以及伪足增加,尤其UCN刺激后细胞出现板状伪足延伸。 3.Rho GTPases在CRH与UCN调节巨噬细胞天然吞噬免疫中起到重要作用。CRH与UCN可通过增强巨噬细胞Rho GTPases中的RhoA、Rac1磷酸化水平,以促进巨噬细胞骨架蛋白的重塑,从而增强巨噬细胞的吞噬功能。 4.CRH与UCN通过不同的信号转导机制,最后由Rho特异性的通路调节大鼠腹腔巨噬细胞骨架蛋白重塑。CRH通过与CRHR1结合,激活cAMP-PKA/ERK1/2信号通路,促进RhoA、Rac1磷酸化;UCN主要与CRHR2结合,通过PKC/ERK1/2信号通路促进RhoA、Rac1磷酸化。 5.CRH与UCN是结构相关肽,对巨噬细胞天然吞噬功能的作用相似,但是信号通路不同。这种差异性反映了它们在调节巨噬细胞吞噬过程中对吞噬活性以及细胞骨架蛋白重塑的影响有所不同。这些肽可能决定了微环境中由外源性刺激引发的细胞吞噬功能改变的程度。
[Abstract]:The mutual infiltration and interaction between neuroendocrine and immune function, maintenance of the body under different conditions of steady state plays a decisive role in pathophysiological conditions in trauma and infection. Study on the relationship between the neuroendocrine and immune system has become neuroimmunoendocrinology (Neuroimmun-endocrinology) is an important research topic. The study found that the hypothalamus neuropeptide can regulate the mechanism involved in the peripheral immune function, such as corticotropin releasing hormone (Corticotropin-releasing hormone, CRH) and urocortin (UCN) peptide structure through the hypothalamic pituitary adrenal axis (hypothalamicpituitary-adrenal, HPA) regulate inflammation / immune indirectly, but peripheral local CRH and UCN can be directly involved in autocrine or paracrine immune / inflammatory regulation of severe trauma. After infection or immune function imbalance, especially the change of macrophage immunity can lead to immune dysfunction of defense against infection, resulting in systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), multiple organ failure (multiple organ failure, MOF). The mechanism of the increased susceptibility of macrophage dysfunction is very complex. May be associated with nerve endocrine hormone in the micro environment. Therefore, to further explore the effect of neuropeptide CRH and UCN on cell immune function and mechanism of steady state to maintain the internal environment, rich in immune dysfunction after trauma or inflammation of the immune balance and control strategy of.CRH and UCN, and their receptors in the central nervous system and peripheral the tissues and organs is widely distributed and UCN.CRH binding with its receptor activation of G protein, which may activate multiple downstream channel Signaling pathway plays different biological effects. At about CRH in the regulation of the body's behavior, endocrine, immune and stress on self-discipline and other aspects have accumulated a lot of information. More and more evidence that peripheral CRH and UCN play an important role in regulating the immune inflammatory reaction process. The phagocytosis of macrophages as a natural immune function importantly, in the body after infection of severe trauma often suppressed. Although there has been broad understanding of the mechanisms of phagocytosis of macrophages, but on the CRH and UCN in macrophages phagocytic function in the natural regulation, is poorly understood.
This study by using primary cultured rat peritoneal macrophages, phagocytosis rate effect observation of CRH and UCN on macrophage phagocytosis of fluorescent microspheres and the average fluorescence intensity, and dynamic effect on macrophage cells in the phagocytic process of cytoskeletal remodeling and depolymerization, clear CRH and UCN natural function of macrophage phagocytosis regulation. Then we study CRH and UCN regulate macrophage cytoskeleton regulator Rho GTPases subfamily protein RhoA, dose and time effect relationship of Rac1 phosphorylation, and then use the signal blocking strategy of CRH and UCN molecules regulate macrophage RhoA receptor pathway and signal transduction pathway of Rac1 phosphorylation, signal transduction mechanism of CRH and UCN to reveal the natural control of macrophage phagocytosis function.
The main research contents and results are as follows:
1., the regulation effect of CRH and UCN on macrophage phagocytosis was analyzed by flow cytometry combined with fluorescent microspheres. Results showed that CRH could significantly enhance macrophage phagocytosis function at 10-8M level, and.UCN could significantly enhance macrophage phagocytosis in 10-10~10-7 concentration.
2. normal cytoskeletal protein macrophages homogeneously distributed in the nucleus and cytoplasm, with slight concentration in the membrane surrounding. Incubated in CRH cells after 1h, F-actin to the cell skeleton protein gathered around, single filopodia after.UCN stimulation of F-actin change and CRH stimulate the emergence of the membrane after similar, phagocytic 30min cytoskeleton remodeling obviously, 60min skeleton protein expression was decreased. The dynamic changes of CRH and UCN after stimulation with macrophage consistent change.
The phosphorylation level of Rac1 3.CRH in 10-8M 30min concentration increased significantly after incubation of macrophages, 60min phosphorylation of RhoA and.UCN reached the peak in the 10-10~10-7M concentration of incubating the cells significantly enhanced RhoA phosphorylation of Rac1. The 10-8M UCN 30min after Rac1 cells were incubated in the phosphorylation level increased obviously, but the phosphorylation of RhoA the level of present Shuangfeng increases lasted longer.
The specific inhibitor of W56 4.Rho-ROCK, a selective inhibitor of Rac1 preincubation of Y27632 cells, can inhibit CRH and UCN on macrophage phagocytosis promoting.Y27632, pretreatment of W56 cells after peripheral filopodia less, gathered around is not obvious, filiform, lamellipodia extension becomes shorter, can hinder F-actin remodeling.
Specific antagonist Antalarmin 5.CRHR1, CRHR1/R2 specific antagonist after pretreatment with Astressin, CRH and UCN cells were incubated on the fluorescent microspheres phagocytosis rate decreased significantly. At Antalarmin after preincubation, phosphorylation of RhoA induced by CRH can be inhibited completely, and close to the effect degree of inhibition by phosphorylation of Rac1 induced by CRH the preincubation with Astressin after pretreatment of.Antalarmin, by the influence on the phosphorylation of RhoA induced by UCN, slightly reduced the phosphorylation level of Rac1, but in Astressin after preincubation, incubated in UCN induced RhoA and Rac1 phosphorylation were significantly reduced.
6.CRH and UCN were incubated with rat peritoneal macrophages in resting state, PKA activity was significantly increased the activity of.PKA in 15min after CRH reached the peak, increasing the activity of.PKC in CRH and UCN increased quickly after the cells were incubated in UCN after 10min to 60min, reached a peak of.MDL-12330A pre incubation cells PKA in 15min, CRH, the effect of UCN on the phosphorylation of RhoA significantly decreased. In CRH on RhoA phosphorylation enhanced by CR preincubation of cells, has no obvious effect on the phosphorylation of Rac1. But CR pre incubation decreased by about 63% the effect of UCN on the phosphorylation of RhoA cells and the effect on Rac1 phosphorylation decreased significantly.
Enhanced ERK1/2 phosphorylation of 7.CRH at a concentration of 10-8M stimulated macrophages, 5min was most significant, until the sustained phosphorylation of ERK1/2 5-30min 15min.UCN were incubated with macrophages after high level. Can completely inhibit the expression of CRH of RhoA increased in PD98059 after pretreatment, pretreatment of UCN increased the phosphorylation of RhoA was completely reversed, inhibited the expression of Rac1 on the rate of 47.7%
conclusion
1.CRH and UCN can significantly enhance the phagocytic function of macrophage, promoting phagocytosis of UCN is stronger than the effect of CRH.CRH on phagocytosis in a dose-dependent manner, which had the strongest effect on phagocytic cells in concentration of 10-8M; UCN can significantly enhance the phagocytic function of macrophages in 10-10~10-7 concentration.
2.CRH and UCN can induce cytoskeletal remodeling in macrophages, and the CRH and UCN enhanced phagocytosis. Consistent performance for CRH and UCN after incubation of macrophages to F-actin cytoskeleton surrounding cells aggregation, membrane appeared focal adhesion and pseudopodia increased, especially after the stimulation of UCN cells showed lamellipodia extension.
3.Rho GTPases plays an important role in regulating the natural phagocytosis of macrophages in CRH and UCN..CRH and UCN can enhance the phosphorylation level of macrophages in Rho GTPases by promoting the remodeling of macrophage skeleton protein, thereby enhancing the phagocytosis of macrophages.
4.CRH and UCN through different signal transduction pathways, and finally by Rho specific regulation of rat peritoneal macrophage protein skeleton remodeling in.CRH combined with CRHR1, cAMP-PKA/ERK1/2 signal pathway to promote RhoA phosphorylation of Rac1; UCN and CRHR2 combining RhoA PKC/, through the promotion of ERK1/2 signaling pathway, phosphorylation of Rac1.
5.CRH and UCN are related to the structure of natural peptide on the immune function of macrophage is similar, but different signaling pathway. This difference reflects in the regulation of macrophage phagocytosis of phagocytic activity and the cytoskeletal remodeling are different. These peptides may determine caused by exogenous stimuli in the microenvironment of phagocytic cells the function of the degree of change.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392.1

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