热习服上调Treg细胞减轻小鼠热应激损伤的研究

发布时间：2018-03-27 23:28

本文选题：热应激损伤　切入点：热习服　出处：《第三军医大学》2016年硕士论文

【摘要】：研究背景与目的热应激损伤是指机体在持续的热应激作用下,发生的一系列应激性生理变化和病理性的损伤,俗称中暑,现代医学中的热痉挛、热衰竭、热射病等,均属于热应激损伤,多见于持续的热环境暴露或高强度运动,不仅影响在热环境下的作业能力,更对机体的健康造成严重危害。其主要的发生机制是由于机体从外界热环境获得的热量和自身产热之和,超出了机体的最大散热效率,热量逐渐蓄积无法散失,导致下丘脑体温调节中枢调控失能,机体核心体温被动升高,内脏血流大量向皮肤转移,各脏器功能紊乱,细胞发生缺血缺氧损伤,释放多种物质激活炎症相关信号通路,诱导急性系统性炎症的发生,而过度激烈的炎症反应的发生,又对脏器造成了更严重的损伤,以至于可进一步发展为多器官衰竭综合征,甚至导致死亡。热习服(heat acclimation,HA)是机体在长期的热环境暴露后,身体组织在各个水平达到一个新的稳态,产生一系列有利于适应热环境,提高耐热能力的变化;机体在形成热习服后,能够防止热应激损伤的发生,或降低热应激损伤的严重程度。热习服后热耐受能力提高的机制复杂,尚不完全明确,已有大量的相关研究发现,热习服提高热耐受、减轻热应激损伤的原理包括热习服后基础代谢率降低、血容量增加、排汗功能改善、心血管系统功能增强、对汗液中钠离子的重吸收增强等;而在后期的全身炎症反应发生期间,热习服是否也存在相应的机制减轻炎症的发生,从而降低损伤程度,目前尚不清楚。在本研究中,我们对热习服小鼠的外周免疫器官中免疫细胞亚群进行了分析,最后以调节性T细胞(regulatory T cell,Treg)对炎症的负调控作用为切入点,探究了热习服对热应激损伤的保护作用与Treg细胞的关系,并对其机制进行了初步的研究探讨,为探明热习服提高机体热耐受、减轻热应激损伤的免疫机制研究提供思路。方法:1.建立热习服小鼠模型,初步明确热习服提高小鼠热耐受与免疫细胞亚群的改变。2.分选未热习服小鼠脾脏、淋巴结的Treg细胞,尾静脉输入同样未热习服的小鼠,观察Treg细胞的输入对热应激环境下小鼠生存率的影响,进行组织病理切片对比脏器损伤情况,探讨Treg细胞的上升是否对热应激损伤具有保护作用。3.引入treg细胞缺陷的microrna-155(mir-155)基因敲除小鼠,与普通小鼠在同样热应激下的死亡情况进行对比,明确treg细胞缺乏是否降低小鼠热应激耐受。并对热应激小鼠的脾脏中性粒细胞和treg进行流式细胞检测细胞频率,进一步明确treg细胞与热应激损伤程度的相关性。4.通过流式细胞术检测热习服和未热习服小鼠在热应激下血液中性粒细胞频率变化情况,以及脾脏中性粒细胞与treg细胞频率,分析脾脏treg细胞频率与中性粒细胞频率的相关性。5.通过免疫组化对脾脏叉头状转录因子3(forkheadboxprotein-3,foxp3)和程序性死亡受体-配体1(programmedcelldeath-ligand1,pd-l1)分子表达进行检测,细胞共培养实验验证treg与中性粒细胞之间的相互作用,对热习服提高机体热耐受的机制进行初步研究。主要结果:1.对热习服小鼠和未热习服小鼠的主要免疫细胞分群进行比较,发现热习服小鼠treg细胞在脾脏、淋巴结中频率明显上调。2.通过对比同样热应激下热习服小鼠与未热习服小鼠存活情况,发现未热习服组小鼠死亡率更高、脏器受损程度更为明显,推测热习服提高小鼠的热耐受与treg细胞上调有关。3.采用流式分选获取未热习服小鼠脾脏、淋巴结的treg细胞,尾静脉输入同样未热习服的小鼠,发现小鼠在热应激下的死亡率显著降低,脏器损伤也有所减轻,证明treg细胞输入能够达到类似于热习服的效果,减轻热应激损伤的严重程度。4.treg细胞缺陷的mir-155基因敲除小鼠和普通小鼠进行热应激实验,发现mir-155基因敲除小鼠在同样强度热应激下的死亡率更高,进一步证实了treg细胞与热应激损伤的相关性。5.对高热应激后脾脏中性粒细胞频率与treg细胞频率进行相关性分析,发现呈良好的负相关,推测treg细胞能够抑制急性炎症反应中的中性粒细胞募集,从而减轻热应激下脏器损伤程度。6.免疫组化分析结果发现,与未热习服小鼠相比,热习服小鼠脾脏pd-l1和foxp3分子显著上调,提示热习服能够上调pd-l1和foxp3的表达,从而诱导treg细胞的增加,发挥更强的免疫负调控作用。7.细胞共培养实验发现treg细胞与中性粒细胞共培养体系中,热刺激诱导的髓过氧化物酶释放减少,提示treg细胞能够抑制中性粒细胞的活化;通过阻断不同的信号通路,对其机制进行初步探讨,发现Treg主要通过PD-1/PD-L1信号通路抑制中性粒细胞的活化。结论:我们的研究检测了小鼠在形成热习服后,外周免疫器官的各种免疫细胞的频率变化,发现Treg细胞频率有所上调,热习服提高机体热耐受的机制可能与Treg细胞对急性炎症的负调控作用相关。热习服能够诱导小鼠脾脏Treg细胞频率上调,可能与热习服后PD-L1分子、Foxp3分子的上调有关,Treg上调可减轻热应激损伤中脏器的中性粒细胞募集和浸润,通过PD1/PD-L1发挥接触性抑制,在一定程度上抑制中性粒细胞的活化,减轻炎症强度和脏器的损伤程度。
[Abstract]:Background and objective heat stress damage refers to the body in the heat stress under the effect of continuous, the occurrence of a series of stress-induced physiological changes and pathological damage, commonly known as heat stroke, heat cramps, heat exhaustion in modern medicine, heatstroke etc., are more common in heat stress injury, exposure or high intensity exercise heat the environment continued, not only affect on the thermal environment of the working ability, more of the body causing serious harm to health. The main pathogenesis is because the body is obtained from the thermal environment outside the heat and heat generation and exceeds the maximum cooling efficiency of the body, the heat accumulation can gradually dissipated, resulting in hypothalamic thermoregulation central regulation of disability, the core temperature of the body passive increase splanchnic blood flow to the skin, a large number of transfer, the viscera function disorder, cell ischemia and hypoxia injury, the release of a variety of substances related to activation of inflammatory signal pathway, induced by acute Systemic inflammation and excessive inflammatory reaction fierce occurred, and caused more serious damage to the organ, that can be further developed to multiple organ failure syndrome, and even lead to death. Heat acclimatization (heat acclimation HA) is the body in the thermal environment of long-term exposure to body tissues a new steady state in each level, produced a series of favorable to improve the thermal environment, changes in heat capacity; the body in the form of heat acclimatization, can prevent heat stress injury, or reduce the severity of heat stress injury. Heat acclimatization mechanism after heat tolerance improved complex, is not completely clearly, a lot of research has found that heat acclimatization improve heat tolerance, reduce heat stress damage including principle of heat acclimatization after basal metabolic rate, increased blood volume, perspiration function improved, cardiovascular system function enhancement, to sweat Sodium reabsorption enhancement; during systemic inflammatory response occurred in the late heat acclimatization whether there is a corresponding mechanism to reduce inflammation, thereby reducing the degree of injury, is unclear. In this study, the peripheral immune organ of our heat acclimatization in mice of immune cell subsets the analysis, finally to regulatory T cells (regulatory T cell, Treg) the negative regulatory role of inflammation as a starting point, to explore the heat acclimatization to heat stress injury and the relationship between the protective effect of Treg cells, and the mechanism for a preliminary study, to explore the heat acclimatization improve body heat tolerance, provide ideas reduce the immune mechanism of heat stress injury. Methods: 1. to establish a mouse model of preliminary heat acclimatization, clear heat acclimatization improve heat tolerance in mice with immune cell subsets changes without.2. sorting heat acclimatization mice spleen, lymph node of Treg cells Mouse tail vein input, also not heat acclimatization, the input of Treg cells was observed on mice survival rate under heat stress, tissue biopsy contrast organ injury, to explore the rise of Treg cells is introduced into Treg cell defects in the protective effect of.3. microRNA-155 on heat stress injury (miR-155) gene knockout mice, compared with the ordinary mice died in the same under heat stress, lack of clear Treg cells could reduce heat stress in mice. And the heat stress of mice spleen neutrophils and Treg flow cytometry to detect the cell frequency, to further clarify the correlation between.4. Treg cells and heat stress damage by flow cytometry for detection of heat acclimatization and heat acclimatization in mice under heat stress blood neutrophil frequency changes, and splenic neutrophils and Treg cells of spleen Treg frequency. The correlation between.5. cell frequency and neutrophil frequency by immunohistochemistry in spleen of forkhead transcription factor 3 (forkheadboxprotein-3, Foxp3) and programmed death ligand receptor 1 (programmedcelldeath-ligand1, PD-L1) was used to detect the expression of molecular interactions between Treg, experimental verification and neutrophil cells were cultured on heat acclimatization improve the mechanism of heat tolerance were studied. The main results: 1. of heat acclimatization in mice and without heat acclimatization in mice of immune cells clustering comparison, found that heat acclimatization of mouse Treg cells in the spleen and lymph nodes in the frequency of.2. is upregulated by comparing the same under heat stress heat acclimatization and heat acclimatization in mice take the mice survived, found no heat acclimatization group has a higher mortality rate, organ damage degree is more obvious, that heat acclimatization improve the heat tolerance of mice with Treg cells increased about.3. by flow Separating acquire non thermal acclimation in mice spleen, lymph node of Treg cells, mouse tail vein input also not heat acclimatization, found that mortality in heat stress mice significantly reduced, organ damage has been reduced, Treg cell input can achieve similar to the heat acclimatization effect, relieve the severity of miR-155 gene.4.treg cell deficiency heat stress injury of the knockout mice and normal mice were subjected to heat stress experiment, found that miR-155 knockout mice at the same intensity under heat stress has a higher mortality rate, further confirmed that Treg cells and heat stress injury correlation.5. correlation analysis of spleen neutrophil Treg cell frequency and frequency after heat stress and found a good negative correlation, suggesting that Treg cells can inhibit neutrophil recruitment in acute inflammation, thereby reducing the heat stress of organ damage degree of.6. immune group The results showed that compared with non heat acclimatization mice, heat acclimatization of mouse spleen PD-L1 and Foxp3 molecules was significantly increased, suggesting heat acclimatization could upregulate the expression of PD-L1 and Foxp3, and thus induce Treg cells, play a role in the negative regulation of immune.7. cells more experiments showed that Treg cells and neutrophils in co culture system in the co culture, thermal stimulation induced myeloperoxidase release decreased, suggesting that the activation of Treg cells to inhibit neutrophil; by blocking different signaling pathways, to preliminarily explore its mechanism, found that activation of Treg through PD-1/PD-L1 signaling pathway inhibition of neutrophil. Conclusion: our study examined mice in the form of heat acclimatization after the change of frequency of various immune cells in the peripheral immune organs, found that the frequency of Treg cells increased, heat acclimatization improve body heat tolerance and the possible mechanism of Treg cells Related to the negative regulation on acute inflammation. Heat acclimatization can induce mouse spleen Treg cells frequency increases, and heat acclimatization after PD-L1 molecule, Foxp3 molecule upregulation of neutrophil Treg upregulation can alleviate the heat stress injury in organ recruitment and infiltration, through the PD1/ PD-L1 contact inhibition play, inhibiting the activation of neutral granulocyte in a certain extent, reduce the degree of inflammation and organ damage strength.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R594.1

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