LncRNA GAS5在小胶质细胞极化中的作用和机制研究

发布时间：2018-05-19 18:46

本文选题：小胶质细胞 + M2型极化　；参考：《第二军医大学》2016年博士论文

【摘要】：研究目的:多发性硬化症(Multiple Sclerosis,MS)是一种主要累及中枢神经系统白质的炎性脱髓鞘性疾病,临床表现为全身多系统的神经功能障碍。MS在发病初期尚可在治疗后缓解,但后期转变为继发进展型,进行性恶化不再缓解。目前,进展型MS尚缺乏确切有效的治疗手段,寻找合适的治疗策略和新靶点至关重要。在目前的研究和治疗中,中枢神经系统的炎症调控一直是控制MS发生、发展的关键。最近的研究表明,MS不仅是一种中枢神经系统自身免疫性疾病,也是神经退行性疾病,退行性改变与炎症性反应相互促进加重疾病的进展。小胶质细胞作为中枢神经系统的固有免疫细胞以及联结固有免疫和获得性免疫的桥梁,不仅影响了MS的炎症进程,而且在MS的神经退行性改变中也发挥重要作用。小胶质细胞在外界不同因子刺激下激活后可表现为两种不同的功能状态,一种状态下大量分泌炎性因子和毒性物质,发挥促炎和清除异物作用;另一种功能状态下则大量分泌抗炎因子和神经保护性物质,发挥抗炎和促修复作用。这两种功能状态分别称为M1型/经典型和M2型/非经典型极化。阐明小胶质细胞M1型与M2型的极化机制是调控其功能的重要着力点,而目前对M2型极化的调控机制了解更少,亟待进一步研究。长链非编码RNA(long non-coding RNAs,lnc RNAs)是一类转录本长度大于200bp,没有蛋白编码功能的RNA分子。近年来随着芯片和测序技术的进步,人们发现lnc RNAs不仅被广泛转录,而且在几乎所有生物学过程中都存在相应的表达变化,而大量的相关研究也使得越来越多有功能的lnc RNAs被发现。随着功能研究的深入,lnc RNAs对环境变化高度敏感、广泛存在、作用方式多样等特性得到公认,被认为是表观遗传调控机制的重要承担者。在中枢神经系统中,lnc RNAs不仅表达于各种类型的脑细胞中,而且脑中表达谱占所有已发现的lnc RNAs的比例可能是所有器官中最高的,可能具有较其他系统更为重要的作用,而对lnc RNAs在小胶质细胞功能和MS的疾病进展中发挥何种作用,目前还不清楚。研究方法:本研究拟采用lnc RNAs芯片比较分析静息型与M2型小胶质细胞的lnc RNAs表达谱,通过差异表达分析和共表达分析等生物信息学手段寻找可能在小胶质细胞M2型极化调控中具有作用的lnc RNAs。针对特定筛选出的分子,将综合运用分子生物学、细胞生物学、形态学和行为学等研究手段进一步阐明其作用形式与分子机制,以期深入认识lnc RNAs在小胶质细胞极化过程中的作用。研究结果:我们通过芯片筛选发现,总共有120条lnc RNAs在小胶质细胞向M2型极化过程中存在表达变化,其中lnc RNA growth arrest-specific 5(GAS5)在IL-4刺激后的M2型小胶质细胞中表达明显下调。在体外原代培养的小胶质细胞中,通过过表达与干扰实验我们发现GAS5可以抑制Ym-1、Fizz-1、CD206、IGF-1等M2型小胶质细胞的标志物表达,同时增加TNF-α、IL-1β等M1型炎性因子的分泌。GAS5基因修饰的小胶质细胞条件上清显著影响共培养的少突胶质细胞前体细胞(oligodendrocyte precursor cells,OPCs)的凋亡、分化和神经元的突起生长。在体内,将GAS5基因修饰的小胶质细胞移植入MS的动物模型EAE小鼠可影响疾病进展。干扰GAS5的小胶质细胞移植入溶血软磷脂(lysophosphatidylcholine,LPC)诱导的局部脱髓鞘模型小鼠可促进髓鞘再生。通过RNA-IP、RNA-pull down和Ch IP等实验进一步研究发现,GAS5通过与PRC2结合,调节控制极化的关键转录因子IRF4的转录。此外,我们发现GAS5在老年小鼠小胶质细胞中的表达较青年小鼠明显升高。在MS病人脑片中,我们发现GAS5在倾向于M1型的阿米巴样的小胶质细胞中表达更高。我们的结果表明lnc RNAs参与了对小胶质细胞极化的调控,GAS5在小胶质细胞极化调控中发挥重要作用,其作用是通过调节控制极化的关键转录因子IRF4的转录实现的。此外,我们的研究也部分揭示了老年小鼠中枢神经系统小胶质细胞M2型极化障碍的原因。结论:通过本研究,我们可得到如下结论:1、Lnc RNAs参与了小胶质细胞极化调控过程,其中lnc RNA GAS5抑制了小胶质细胞向M2型极化并促进其向M1型极化;2、GAS5过表达或干扰的小胶质细胞显著影响MS的动物模型EAE小鼠的疾病进展和LPC模型小鼠髓鞘再生过程,显示GAS5在体内小胶质细胞功能发挥中的重要作用;3、GAS5主要通过结合体内主要抑制性复合物PRC2抑制IRF4的转录,从而抑制小胶质细胞向M2型极化。就我们所知,本工作首次揭示了一个lnc RNA分子在小胶质细胞中的功能,也是第一次证明了一个lnc RNA通过影响免疫反应参与EAE和MS的发病过程。
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.51

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