本文選題：熱應(yīng)激損傷　切入點(diǎn)：熱習(xí)服　出處：《第三軍醫(yī)大學(xué)》2016年碩士論文

【摘要】：研究背景與目的熱應(yīng)激損傷是指機(jī)體在持續(xù)的熱應(yīng)激作用下,發(fā)生的一系列應(yīng)激性生理變化和病理性的損傷,俗稱中暑,現(xiàn)代醫(yī)學(xué)中的熱痙攣、熱衰竭、熱射病等,均屬于熱應(yīng)激損傷,多見于持續(xù)的熱環(huán)境暴露或高強(qiáng)度運(yùn)動(dòng),不僅影響在熱環(huán)境下的作業(yè)能力,更對(duì)機(jī)體的健康造成嚴(yán)重危害。其主要的發(fā)生機(jī)制是由于機(jī)體從外界熱環(huán)境獲得的熱量和自身產(chǎn)熱之和,超出了機(jī)體的最大散熱效率,熱量逐漸蓄積無法散失,導(dǎo)致下丘腦體溫調(diào)節(jié)中樞調(diào)控失能,機(jī)體核心體溫被動(dòng)升高,內(nèi)臟血流大量向皮膚轉(zhuǎn)移,各臟器功能紊亂,細(xì)胞發(fā)生缺血缺氧損傷,釋放多種物質(zhì)激活炎癥相關(guān)信號(hào)通路,誘導(dǎo)急性系統(tǒng)性炎癥的發(fā)生,而過度激烈的炎癥反應(yīng)的發(fā)生,又對(duì)臟器造成了更嚴(yán)重的損傷,以至于可進(jìn)一步發(fā)展為多器官衰竭綜合征,甚至導(dǎo)致死亡。熱習(xí)服(heat acclimation,HA)是機(jī)體在長期的熱環(huán)境暴露后,身體組織在各個(gè)水平達(dá)到一個(gè)新的穩(wěn)態(tài),產(chǎn)生一系列有利于適應(yīng)熱環(huán)境,提高耐熱能力的變化;機(jī)體在形成熱習(xí)服后,能夠防止熱應(yīng)激損傷的發(fā)生,或降低熱應(yīng)激損傷的嚴(yán)重程度。熱習(xí)服后熱耐受能力提高的機(jī)制復(fù)雜,尚不完全明確,已有大量的相關(guān)研究發(fā)現(xiàn),熱習(xí)服提高熱耐受、減輕熱應(yīng)激損傷的原理包括熱習(xí)服后基礎(chǔ)代謝率降低、血容量增加、排汗功能改善、心血管系統(tǒng)功能增強(qiáng)、對(duì)汗液中鈉離子的重吸收增強(qiáng)等;而在后期的全身炎癥反應(yīng)發(fā)生期間,熱習(xí)服是否也存在相應(yīng)的機(jī)制減輕炎癥的發(fā)生,從而降低損傷程度,目前尚不清楚。在本研究中,我們對(duì)熱習(xí)服小鼠的外周免疫器官中免疫細(xì)胞亞群進(jìn)行了分析,最后以調(diào)節(jié)性T細(xì)胞(regulatory T cell,Treg)對(duì)炎癥的負(fù)調(diào)控作用為切入點(diǎn),探究了熱習(xí)服對(duì)熱應(yīng)激損傷的保護(hù)作用與Treg細(xì)胞的關(guān)系,并對(duì)其機(jī)制進(jìn)行了初步的研究探討,為探明熱習(xí)服提高機(jī)體熱耐受、減輕熱應(yīng)激損傷的免疫機(jī)制研究提供思路。方法:1.建立熱習(xí)服小鼠模型,初步明確熱習(xí)服提高小鼠熱耐受與免疫細(xì)胞亞群的改變。2.分選未熱習(xí)服小鼠脾臟、淋巴結(jié)的Treg細(xì)胞,尾靜脈輸入同樣未熱習(xí)服的小鼠,觀察Treg細(xì)胞的輸入對(duì)熱應(yīng)激環(huán)境下小鼠生存率的影響,進(jìn)行組織病理切片對(duì)比臟器損傷情況,探討Treg細(xì)胞的上升是否對(duì)熱應(yīng)激損傷具有保護(hù)作用。3.引入treg細(xì)胞缺陷的microrna-155(mir-155)基因敲除小鼠,與普通小鼠在同樣熱應(yīng)激下的死亡情況進(jìn)行對(duì)比,明確treg細(xì)胞缺乏是否降低小鼠熱應(yīng)激耐受。并對(duì)熱應(yīng)激小鼠的脾臟中性粒細(xì)胞和treg進(jìn)行流式細(xì)胞檢測(cè)細(xì)胞頻率,進(jìn)一步明確treg細(xì)胞與熱應(yīng)激損傷程度的相關(guān)性。4.通過流式細(xì)胞術(shù)檢測(cè)熱習(xí)服和未熱習(xí)服小鼠在熱應(yīng)激下血液中性粒細(xì)胞頻率變化情況,以及脾臟中性粒細(xì)胞與treg細(xì)胞頻率,分析脾臟treg細(xì)胞頻率與中性粒細(xì)胞頻率的相關(guān)性。5.通過免疫組化對(duì)脾臟叉頭狀轉(zhuǎn)錄因子3(forkheadboxprotein-3,foxp3)和程序性死亡受體-配體1(programmedcelldeath-ligand1,pd-l1)分子表達(dá)進(jìn)行檢測(cè),細(xì)胞共培養(yǎng)實(shí)驗(yàn)驗(yàn)證treg與中性粒細(xì)胞之間的相互作用,對(duì)熱習(xí)服提高機(jī)體熱耐受的機(jī)制進(jìn)行初步研究。主要結(jié)果:1.對(duì)熱習(xí)服小鼠和未熱習(xí)服小鼠的主要免疫細(xì)胞分群進(jìn)行比較,發(fā)現(xiàn)熱習(xí)服小鼠treg細(xì)胞在脾臟、淋巴結(jié)中頻率明顯上調(diào)。2.通過對(duì)比同樣熱應(yīng)激下熱習(xí)服小鼠與未熱習(xí)服小鼠存活情況,發(fā)現(xiàn)未熱習(xí)服組小鼠死亡率更高、臟器受損程度更為明顯,推測(cè)熱習(xí)服提高小鼠的熱耐受與treg細(xì)胞上調(diào)有關(guān)。3.采用流式分選獲取未熱習(xí)服小鼠脾臟、淋巴結(jié)的treg細(xì)胞,尾靜脈輸入同樣未熱習(xí)服的小鼠,發(fā)現(xiàn)小鼠在熱應(yīng)激下的死亡率顯著降低,臟器損傷也有所減輕,證明treg細(xì)胞輸入能夠達(dá)到類似于熱習(xí)服的效果,減輕熱應(yīng)激損傷的嚴(yán)重程度。4.treg細(xì)胞缺陷的mir-155基因敲除小鼠和普通小鼠進(jìn)行熱應(yīng)激實(shí)驗(yàn),發(fā)現(xiàn)mir-155基因敲除小鼠在同樣強(qiáng)度熱應(yīng)激下的死亡率更高,進(jìn)一步證實(shí)了treg細(xì)胞與熱應(yīng)激損傷的相關(guān)性。5.對(duì)高熱應(yīng)激后脾臟中性粒細(xì)胞頻率與treg細(xì)胞頻率進(jìn)行相關(guān)性分析,發(fā)現(xiàn)呈良好的負(fù)相關(guān),推測(cè)treg細(xì)胞能夠抑制急性炎癥反應(yīng)中的中性粒細(xì)胞募集,從而減輕熱應(yīng)激下臟器損傷程度。6.免疫組化分析結(jié)果發(fā)現(xiàn),與未熱習(xí)服小鼠相比,熱習(xí)服小鼠脾臟pd-l1和foxp3分子顯著上調(diào),提示熱習(xí)服能夠上調(diào)pd-l1和foxp3的表達(dá),從而誘導(dǎo)treg細(xì)胞的增加,發(fā)揮更強(qiáng)的免疫負(fù)調(diào)控作用。7.細(xì)胞共培養(yǎng)實(shí)驗(yàn)發(fā)現(xiàn)treg細(xì)胞與中性粒細(xì)胞共培養(yǎng)體系中,熱刺激誘導(dǎo)的髓過氧化物酶釋放減少,提示treg細(xì)胞能夠抑制中性粒細(xì)胞的活化;通過阻斷不同的信號(hào)通路,對(duì)其機(jī)制進(jìn)行初步探討,發(fā)現(xiàn)Treg主要通過PD-1/PD-L1信號(hào)通路抑制中性粒細(xì)胞的活化。結(jié)論:我們的研究檢測(cè)了小鼠在形成熱習(xí)服后,外周免疫器官的各種免疫細(xì)胞的頻率變化,發(fā)現(xiàn)Treg細(xì)胞頻率有所上調(diào),熱習(xí)服提高機(jī)體熱耐受的機(jī)制可能與Treg細(xì)胞對(duì)急性炎癥的負(fù)調(diào)控作用相關(guān)。熱習(xí)服能夠誘導(dǎo)小鼠脾臟Treg細(xì)胞頻率上調(diào),可能與熱習(xí)服后PD-L1分子、Foxp3分子的上調(diào)有關(guān),Treg上調(diào)可減輕熱應(yīng)激損傷中臟器的中性粒細(xì)胞募集和浸潤,通過PD1/PD-L1發(fā)揮接觸性抑制,在一定程度上抑制中性粒細(xì)胞的活化,減輕炎癥強(qiáng)度和臟器的損傷程度。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R594.1

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