本文選題：小膠質(zhì)細胞 + M2型極化��； 參考：《第二軍醫(yī)大學(xué)》2016年博士論文

【摘要】：研究目的:多發(fā)性硬化癥(Multiple Sclerosis,MS)是一種主要累及中樞神經(jīng)系統(tǒng)白質(zhì)的炎性脫髓鞘性疾病,臨床表現(xiàn)為全身多系統(tǒng)的神經(jīng)功能障礙。MS在發(fā)病初期尚可在治療后緩解,但后期轉(zhuǎn)變?yōu)槔^發(fā)進展型,進行性惡化不再緩解。目前,進展型MS尚缺乏確切有效的治療手段,尋找合適的治療策略和新靶點至關(guān)重要。在目前的研究和治療中,中樞神經(jīng)系統(tǒng)的炎癥調(diào)控一直是控制MS發(fā)生、發(fā)展的關(guān)鍵。最近的研究表明,MS不僅是一種中樞神經(jīng)系統(tǒng)自身免疫性疾病,也是神經(jīng)退行性疾病,退行性改變與炎癥性反應(yīng)相互促進加重疾病的進展。小膠質(zhì)細胞作為中樞神經(jīng)系統(tǒng)的固有免疫細胞以及聯(lián)結(jié)固有免疫和獲得性免疫的橋梁,不僅影響了MS的炎癥進程,而且在MS的神經(jīng)退行性改變中也發(fā)揮重要作用。小膠質(zhì)細胞在外界不同因子刺激下激活后可表現(xiàn)為兩種不同的功能狀態(tài),一種狀態(tài)下大量分泌炎性因子和毒性物質(zhì),發(fā)揮促炎和清除異物作用;另一種功能狀態(tài)下則大量分泌抗炎因子和神經(jīng)保護性物質(zhì),發(fā)揮抗炎和促修復(fù)作用。這兩種功能狀態(tài)分別稱為M1型/經(jīng)典型和M2型/非經(jīng)典型極化。闡明小膠質(zhì)細胞M1型與M2型的極化機制是調(diào)控其功能的重要著力點,而目前對M2型極化的調(diào)控機制了解更少,亟待進一步研究。長鏈非編碼RNA(long non-coding RNAs,lnc RNAs)是一類轉(zhuǎn)錄本長度大于200bp,沒有蛋白編碼功能的RNA分子。近年來隨著芯片和測序技術(shù)的進步,人們發(fā)現(xiàn)lnc RNAs不僅被廣泛轉(zhuǎn)錄,而且在幾乎所有生物學(xué)過程中都存在相應(yīng)的表達變化,而大量的相關(guān)研究也使得越來越多有功能的lnc RNAs被發(fā)現(xiàn)。隨著功能研究的深入,lnc RNAs對環(huán)境變化高度敏感、廣泛存在、作用方式多樣等特性得到公認,被認為是表觀遺傳調(diào)控機制的重要承擔者。在中樞神經(jīng)系統(tǒng)中,lnc RNAs不僅表達于各種類型的腦細胞中,而且腦中表達譜占所有已發(fā)現(xiàn)的lnc RNAs的比例可能是所有器官中最高的,可能具有較其他系統(tǒng)更為重要的作用,而對lnc RNAs在小膠質(zhì)細胞功能和MS的疾病進展中發(fā)揮何種作用,目前還不清楚。研究方法:本研究擬采用lnc RNAs芯片比較分析靜息型與M2型小膠質(zhì)細胞的lnc RNAs表達譜,通過差異表達分析和共表達分析等生物信息學(xué)手段尋找可能在小膠質(zhì)細胞M2型極化調(diào)控中具有作用的lnc RNAs。針對特定篩選出的分子,將綜合運用分子生物學(xué)、細胞生物學(xué)、形態(tài)學(xué)和行為學(xué)等研究手段進一步闡明其作用形式與分子機制,以期深入認識lnc RNAs在小膠質(zhì)細胞極化過程中的作用。研究結(jié)果:我們通過芯片篩選發(fā)現(xiàn),總共有120條lnc RNAs在小膠質(zhì)細胞向M2型極化過程中存在表達變化,其中l(wèi)nc RNA growth arrest-specific 5(GAS5)在IL-4刺激后的M2型小膠質(zhì)細胞中表達明顯下調(diào)。在體外原代培養(yǎng)的小膠質(zhì)細胞中,通過過表達與干擾實驗我們發(fā)現(xiàn)GAS5可以抑制Ym-1、Fizz-1、CD206、IGF-1等M2型小膠質(zhì)細胞的標志物表達,同時增加TNF-α、IL-1β等M1型炎性因子的分泌。GAS5基因修飾的小膠質(zhì)細胞條件上清顯著影響共培養(yǎng)的少突膠質(zhì)細胞前體細胞(oligodendrocyte precursor cells,OPCs)的凋亡、分化和神經(jīng)元的突起生長。在體內(nèi),將GAS5基因修飾的小膠質(zhì)細胞移植入MS的動物模型EAE小鼠可影響疾病進展。干擾GAS5的小膠質(zhì)細胞移植入溶血軟磷脂(lysophosphatidylcholine,LPC)誘導(dǎo)的局部脫髓鞘模型小鼠可促進髓鞘再生。通過RNA-IP、RNA-pull down和Ch IP等實驗進一步研究發(fā)現(xiàn),GAS5通過與PRC2結(jié)合,調(diào)節(jié)控制極化的關(guān)鍵轉(zhuǎn)錄因子IRF4的轉(zhuǎn)錄。此外,我們發(fā)現(xiàn)GAS5在老年小鼠小膠質(zhì)細胞中的表達較青年小鼠明顯升高。在MS病人腦片中,我們發(fā)現(xiàn)GAS5在傾向于M1型的阿米巴樣的小膠質(zhì)細胞中表達更高。我們的結(jié)果表明lnc RNAs參與了對小膠質(zhì)細胞極化的調(diào)控,GAS5在小膠質(zhì)細胞極化調(diào)控中發(fā)揮重要作用,其作用是通過調(diào)節(jié)控制極化的關(guān)鍵轉(zhuǎn)錄因子IRF4的轉(zhuǎn)錄實現(xiàn)的。此外,我們的研究也部分揭示了老年小鼠中樞神經(jīng)系統(tǒng)小膠質(zhì)細胞M2型極化障礙的原因。結(jié)論:通過本研究,我們可得到如下結(jié)論:1、Lnc RNAs參與了小膠質(zhì)細胞極化調(diào)控過程,其中l(wèi)nc RNA GAS5抑制了小膠質(zhì)細胞向M2型極化并促進其向M1型極化;2、GAS5過表達或干擾的小膠質(zhì)細胞顯著影響MS的動物模型EAE小鼠的疾病進展和LPC模型小鼠髓鞘再生過程,顯示GAS5在體內(nèi)小膠質(zhì)細胞功能發(fā)揮中的重要作用;3、GAS5主要通過結(jié)合體內(nèi)主要抑制性復(fù)合物PRC2抑制IRF4的轉(zhuǎn)錄,從而抑制小膠質(zhì)細胞向M2型極化。就我們所知,本工作首次揭示了一個lnc RNA分子在小膠質(zhì)細胞中的功能,也是第一次證明了一個lnc RNA通過影響免疫反應(yīng)參與EAE和MS的發(fā)病過程。
[Abstract]:Objective: Multiple Sclerosis (MS) is an inflammatory demyelinating disease mainly involved in the white matter of the central nervous system. The clinical manifestation of the systemic multisystem neurologic dysfunction (.MS) can be relieved at the beginning of the onset of the disease, but the later stage is transformed into progressive progression and progressive deterioration no longer remission. Currently, progressing type MS is still lacking in effective treatment and finding appropriate therapeutic strategies and targets. In the current research and treatment, the regulation of inflammation in the central nervous system has been the key to control the development of MS. Recent studies have shown that MS is not only an autoimmune disease of the central nervous system, but also a neurodegenerative disease. Disease, degenerative changes and inflammatory reactions promote the progress of the disease. Microglia, as an inherent immune cell of the central nervous system and a bridge linking inherent immunity and acquired immunity, not only affects the inflammatory process of MS, but also plays an important role in the neurodegenerative changes of MS. Microglia is in the outside. There are two different functional states under the stimulation of different factors. In one state, a large number of inflammatory factors and toxic substances are secreted, and the effect of proinflammatory and removal of foreign bodies is exerted; the other function states secrete anti-inflammatory factors and neuroprotective substances to play anti-inflammatory and repair effects. These two functional states are called respectively. It is M1 / classic and M2 / non classical polarization. The polarization mechanism of M1 type and M2 type of microglia is an important point to regulate its function, but the regulation mechanism of M2 polarization is less understood and further studied. Long chain non coded RNA (long non-coding RNAs, LNC RNAs) is a class of transcriptional length greater than 200bp, no eggs. RNA molecules of white coding function. In recent years, with the progress of chip and sequencing technology, people have found that LNC RNAs is not only widely transcribed, but also has corresponding expression changes in almost all biological processes, and a large number of related studies have also made more and more functional LNC RNAs found. With the development of functional research, LNC RNAs In the central nervous system, LNC RNAs is not only expressed in various types of brain cells, but also the proportion of all the found LNC RNAs in the brain may be the most important of all organs in the central nervous system. High, may have a more important role than other systems, and what role LNC RNAs plays in microglia function and the progression of MS disease is still unclear. Research methods: This study intends to compare the LNC RNAs expression profiles of resting and M2 microglia by LNC RNAs chips, through differential expression analysis and co expression. LNC RNAs., which may play a role in the M2 polarization regulation of microglia, can be used to further clarify its functional and molecular mechanisms by means of molecular biology, cell biology, morphology and behaviourology, in order to understand LNC RNAs in a small way. The effect in the process of glial cell polarization. Results: we found that there were 120 LNC RNAs in a total of microglia to M2 polarization, in which LNC RNA growth arrest-specific 5 (GAS5) decreased obviously in the M2 microglia microglia after IL-4 stimulation. In the stromal cells, through over expression and interference experiments, we found that GAS5 can inhibit the expression of Ym-1, Fizz-1, CD206, IGF-1 and other M2 microglia, and increase the TNF- a, IL-1 beta and other M1 type inflammatory factors that secrete the oligodendrocyte precursor cells (oligode) of the co cultured oligodendrocytes (oligode). Apoptosis, differentiation, and neurite growth of ndrocyte precursor cells, OPCs. In vivo, the migration of GAS5 gene modified microglia into MS animal model EAE mice can affect the disease progression. The local demyelinating model mice induced by the transplantation of GAS5 microglia into hemolytic soft phospholipid (lysophosphatidylcholine, LPC) can be interfered. Promoting myelin regeneration. Further studies, such as RNA-IP, RNA-pull down and Ch IP, found that GAS5 regulates the transcription of IRF4, a key transcription factor that controls polarization, by binding to PRC2. In addition, we found that the expression of GAS5 in geriatric microglia is significantly higher than that of young mice. In the brain slices of MS patients, we found that GAS5 is tilting. Our results suggest that LNC RNAs is involved in the regulation of microglia polarization, and that GAS5 plays an important role in the polarization regulation of microglia, and its role is to regulate the transcription of IRF4, a key transcriptional factor that controls polarization. Furthermore, our research is also part of the study. The causes of M2 polarization disorder in the microglia in the central nervous system of the aged mice were revealed. Conclusion: through this study, we can get the following conclusions: 1, Lnc RNAs is involved in the polarization regulation of microglia, in which LNC RNA GAS5 inhibits the polarization of microglia to M2 type and promotes its polarization to M1 type; 2, GAS5 overexpression or interference is small. Glial cells significantly affect the progression of MS animal model EAE mice and the process of myelin regeneration in LPC model mice, which shows that GAS5 plays an important role in the function of microglia in the body; 3, GAS5 inhibits the M2 polarization by combining the major inhibitory complex PRC2 in the body to inhibit the M2 polarization. It is known that this work has revealed the function of a LNC RNA molecule in microglia for the first time, and is the first to demonstrate that a LNC RNA is involved in the pathogenesis of EAE and MS by affecting the immune response.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R744.51

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