TREM2在小胶质细胞介导的帕金森病神经炎症中的作用研究

发布时间：2018-01-01 00:25

本文关键词：TREM2在小胶质细胞介导的帕金森病神经炎症中的作用研究　出处：《南方医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：帕金森病(Parkinson's disease, PD)是常见的中枢神经系统退行性疾病,由于早期黑质致密部的多巴胺(dopamine, DA)能神经元死亡而出现多巴胺耗竭,导致出现以运动迟缓、静止性震颤、肌强直等为特征的运动障碍。PD也会出现很多非运动症状,如轻度认知功能障碍(Mild cognitive impairment in Parkisnon's disease, PD-MCI)、痴呆、情绪和精神障碍等。中脑黑质DA能神经元缺失、α-突触核蛋白(a-synuclein)沉积和路易小体形成是PD的主要病理特征。目前PD的治疗是以增加DA浓度及直接的DA受体激动剂为主的症状性治疗。疾病修饰治疗可减缓神经变性或遏制疾病的进展,但目前PD的疾病修饰治疗仍有待进一步探索。因此,阐明PD的发病机制将有助于疾病修饰治疗的发展。由于大部分PD患者为散发性,很可能由遗传因素和环境因素相互作用而导致。自从20年前在PD患者中首次报道小胶质细胞活化介导的神经炎症,PD神经炎症成为了研究的焦点。众所周知,小胶质细胞同时具有细胞毒性及细胞保护性功能。小胶质细胞既可活化为致炎的M1型(或过度活化型),也可活化为抗炎的M2型。M1型小胶质细胞可产生并释放多种致炎因子,促进清除中枢神经系统的病原体如伐-突触核蛋白,但过度活化的小胶质细胞可因产生过多具有神经毒性的细胞因子,反而造成神经元损伤继而加重PD。相反,M2型小胶质细胞可产生抗炎因子、抑制炎症反应、促进组织修复。因此,通过调控小胶质细胞的亚型分化,促进小胶质细胞向抗炎的M2型小胶质细胞转化,将可能为PD提供新的治疗策略,但目前小胶质细胞亚型分化的调控机制尚未明确。髓样细胞表达的触发受体-2 (triggering receptor expressed on myeloid cells 2, TREM2)是表达于小胶质细胞表面的受体,可减少小胶质细胞介导的炎症因子导致的促炎反应,并促进小胶质细胞对神经元碎片及细菌的吞噬作用。最近一项大型的GWAS研究表明TREM2基因突变与PD、AD、FTD等发病风险增加有关。TREM2可能是调节PD中小胶质细胞M1/M2亚型平衡的重要因素。本研究将通过临床研究PD患者外周血中TREM2表达水平与对照组的差异,并建立PD小鼠模型初步探讨TREM2及其下游神经炎症因子的表达水平,进一步通过TREM2-shRNA及wtTREM2慢病毒转染小胶质细胞以使TREM2表达沉默或过表达,然后分析TREM2表达水平对小胶质细胞M1/M2亚型分化的影响,以探讨TREM2对小胶质细胞亚型分化的调控作用,以阐明TREM2在小胶质细胞介导的神经炎症中的作用。第一章可溶性TREM2在PD及PD认知障碍中的表达水平研究目的：研究sTREM2在PD患者和正常对照者间的表达差异,并探讨sTREM2对PD认知功能障碍的影响。方法：研究纳入114例PD患者和120例健康对照者,收集一般临床资料,采用统一帕金森病评分量表(Unified Parkinson Disease Rating Scale, UPDRS)第1U部分评估PD患者的运动功能,通过MoCA(Montreal Cognitive Assessment, MoCA)量表评估整体认知功能、韦氏智力和韦氏记忆量表评估执行功能、记忆、注意力及视空间空能。进一步根据PD轻度认知功能障碍的诊断标准,将PD组分为PD认知正常组(PD patients with normal cognition, PD-CN组)和PD轻度认知功能障碍组(PD patients with cognitive impairment,PD-MCI组),其中PD-CN组66例,PD-MCI组48例。并采集PD组及对照组的外周血,用ELISA法检测外周血中sTREM2的表达水平,分析各组外周血中sTREM2表达水平的差异,并进一步探讨sTREM2在PD认知功能中的作用。结果：①114例PD思看中,PD-MCI思者48例,占42.11%。PD-MCI组的受教育水平较PD-CN组低(P0.05)；运动功能(UPDRS-III评分、H-Y分级)及整体认知功能(MMSE评分及MoCA评分)均较PD-CN组更差,差异有统计学意义(P0.05)；②PD患者外周血中sTREM2表达水平显著高于健康对照组,差异有统计学意义(P0.001)；③健康对照组、PD-CN组和PD-MCI组三组间外周血sTREM2表达水平有显著性差异(P0.001), PD-MCI组患者外周血中的sTREM2表达水平显著高于PD-CN组,差异有统计学意义(P0.001)；④PD患者外周血中的sTREM2水平与MoCA评分呈中度负相关(r=-0.464,P0.001);⑤PD患者外周血中的sTREM2水平升高与受教育年限水平低(P=0.023)、相似性量表分(P=-0.015)及再认量表分(P=-0.018)下降相关。结论：PD患者外周血的sTREM2表达水平升高与PD患者受教育年限低、全面认知功能下降、执行功能及记忆功能下降有关,外周血的sTREM2表达水平可能有助于监测PD及PD认知障碍的病情进展,表明TREM2可能参与了PD及PD认知功能障碍的发生发展,但内在机制仍有待进一步研究。第二章TREM2对MPTP诱导的PD小鼠模型神经炎症的影响目的：分析MPTP诱导的PD小鼠模型和正常对照小鼠脑组织中TREM2、促炎因子及抗炎因子的表达水平差异,探讨TREM2在PD小鼠神经炎症中的作用。方法：通过C57BL/6J小鼠小剂量长期腹腔注射MPTP和丙磺舒建立PD慢性模型,另设丙磺舒对照组和生理盐水对照组。建立PD模型后,取各组小鼠中脑、皮层、海马、纹状体等部位脑组织,通过酪氨酸羟化酶(tyrosine hydroxylase, TH)染色观察中脑DA能神经元死亡情况,并采用荧光定量PCR方法检测：rREM2及炎症因子IL-6、IL-1β、TNF-a基因的mRNA表达水平,进一步通过western blot检测TREM2及抗炎因子Arginase-1蛋白的表达水平,最后应用单因素方差分析比较三组间DA能神经元缺失、TREM2、炎症因子及抗炎因子的表达水平差异。结果：①MPTP模型组中TH染色阳性DA能神经元数目较丙磺舒对照组及生理盐水对照组显著减少(P0.001),MPTP组小鼠中脑TH+神经元数目较生理盐水对照组减少39.7%：②MPTP模型组、丙磺舒对照组及生理盐水对照组之间中脑及皮层中TREM2、 IL-1β、IL-6, TNF-a基因的mRNA相对表达水平有显著性差异(P0.05),其中MPTP组表达水平最高,差异有统计学意义(P0.05)；③MPTP模型组、丙磺舒对照组及生理盐水对照组三组间中脑、皮层、纹状体及海马组织中TREM2及抗炎因子Arginase-1蛋白的表达水平有显著性差异(P0.05),其中MPTP组表达水平高于丙磺舒对照组及生理盐水对照组,差异有统计学意义(P0.05)；结论：MPTP诱导的PD小鼠模型中脑、纹状体、海马及皮层等部位脑组织均出现TREM2及抗炎因子Arginase-1蛋白表达增加,伴有中脑DA能神经元缺失及显著的神经炎症,提示TREM2与PD中脑DA能神经元缺失及神经炎症有密切的联系,可能是PD出现神经炎症的情况下为了阻止炎症反应而出现的代偿性升高,但其确切机制有待进一步阐明。第三章TREM2对小胶质细胞亚型分化的调节作用目的：初步探讨TREM2是否在小胶质细胞M1/M2亚型分化中发挥调控作用。方法：选用BV2细胞作为小胶质细胞模型,用TREM2-shRNA'慢病毒感染小胶质细胞使TREM2沉默表达,wtTREM2'慢病毒使小胶质细胞过表达TREM2,并分别设阴性对照病毒感染组,感染96小时后通过免疫荧光、荧光定量PCR及western blot验证感染效率。各组慢病毒感染后根据加入M1型小胶质细胞诱导剂(IL-4+IL-13)或M2型小胶质细胞诱导剂(LPS+IFN-y)的不同,再将小胶质细胞分为3组：对照组、IL-4 (1 Ong/ml)+IL-13(10ng/ml)组、LPS(100ng/ml)) +IFN-y (1 Ong/ml)组。感染96小时后各组分别加入相应浓度的M1/M2型诱导剂,24小时后通过免疫荧光、western blot及荧光定量PCR检测小胶质细胞上TREM2及抗炎因子Arginase-1的表达水平,并采用Griess法检测炎症因子NO的表达水平。结果：①TREM2-shRNA慢病毒及scramble TREM2阴性对照病毒、wtTREM2慢病毒及vector阴性对照病毒感染小胶质细胞后,免疫荧光结果表明TREM2-shRNA'陧病毒及scramble TREM2阴性对照病毒、vector阴性对照病毒在小胶质细胞中的感染效率均达90%以上,wtTREM2慢病毒的感染效率达80%以上,可保证后续研究的进行；②慢病毒感染小胶质细胞后,TREM2-shRNA组TREM2及M2型小胶质细胞标志物Arginase-1蛋白的表达水平较scramble TREM2组显著下降(P0.05),相反,wtTREM2组TREM2及Arginase-1蛋白的表达水平较vector组显著升高(P0.05)：③慢病毒感染小胶质细胞并利用M1/M2型小胶质细胞诱导剂后,IL-4+IL-13(M2型小胶质细胞诱导剂)组的TREM2及M2型小胶质细胞标志物Arginase-1表达水平较对照组升高,M1型小胶质细胞标志物NO表达减少(P0.05),而LPS+IFN-γ(M1型小胶质细胞诱导剂)组恰好相反,TREM2及Arginase-1表达水平较对照组降低,NO表达增加(P0.05),表明小胶质细胞在M1型/M2型诱导剂下出现了亚型转化；④沉默TREM2表达后,TREM2及M2型小胶质细胞标志物Arginase-1表达水平显著下降,而使用M2型小胶质细胞诱导剂IL-4/IL-13可诱导其表达增加；相反,TREM2过表达后,TREM2及Arginase-1表达水平显著增加,而使用M1型小胶质细胞诱导剂LPS/IFN-γ可诱导其表达下降；表明TREM2可促进小胶质细胞从M1型向M2型转化。结论：(1)在没有使用M1型诱导剂的情况下,TREM2沉默表达即可减弱小胶质细胞向M2型分化的功能,表明TREM2在M2型小胶质细胞分化中发挥着重要的作用,可能参与了PD神经炎症的发病机制；(2) TREM2沉默表达可抑制小胶质细胞向M2型分化而加重M1型小胶质细胞介导的炎症反应,相反,TREM2过表达可诱导小胶质细胞向M2型转化,抑制M1型小胶质细胞介导的炎症反应并产生抗炎因子,表明TREM2在PD神经炎症中发挥保护作用,但TREM2如何调节小胶质细胞M1/M2亚型分化的具体分子机制有待进一步阐明。
[Abstract]:Parkinson's disease (Parkinson's disease PD) is a common neurodegenerative disease of the central nervous system, due to the early substantia nigra pars compacta (dopamine, DA) to the death of neurons and dopamine depletion, resulting in bradykinesia, resting tremor, rigidity and other characteristics of movement disorders.PD will also be a lot of non motor symptoms such as, mild cognitive impairment (Mild cognitive impairment in Parkisnon's disease, PD-MCI), dementia, emotional and mental disorders. The substantia nigra DA neurons lack of alpha synuclein (a-synuclein) deposition and Louis body formation is the main pathological features of PD. The PD treatment is symptomatic treatment to increase the concentration of DA and direct DA receptor agonist based. Progress of disease modifying therapy can alleviate nerve degeneration or prevent disease, but the modified PD disease treatment remains to be further explored for. This, to elucidate the mechanism of PD will contribute to the development of disease modifying therapy. Because most PD patients are sporadic, probably by the interaction of genetic and environmental factors caused. Since 20 years ago in PD were reported for the first time in the activation of microglia mediated neuroinflammation, PD nerve inflammation has become a research focus. As everyone knows, microglia also have cell toxicity and cell protective function. Microglia can be activated as M1 type of inflammatory (or excessive activation), can also be activated as anti-inflammatory M2 type.M1 type of microglia can produce and release a variety of inflammatory factors, promote the removal of the central nervous system the pathogens such as cutting - synuclein, but excessive activation of microglial cells by cytokines produced more neurotoxic, but cause neuronal injury and aggravation of PD. instead of M2 type of microglia can produce anti-inflammatory Factor, inhibit inflammation, promote tissue repair. Therefore, the subtype differentiation and regulation of microglial cells, promote the transformation of microglia to anti-inflammatory M2 microglial cells may provide a new therapeutic strategy for PD, but the regulation mechanism of microglia subtype differentiation is not clear. The expression of myeloid cell triggering receptor (triggering receptor expressed -2 on myeloid cells 2, TREM2) is expressed in microglia cell surface receptors, proinflammatory response can reduce inflammatory factors lead to microglia mediated phagocytosis, and promote microglia to neurons and bacterial debris. A recent study showed that large GWAS PD and AD, TREM2 gene mutation, FTD increased risk of.TREM2 may be an important factor in the regulation of microglia in PD M1/M2 subtype balance. This will be through the clinical study of PD in peripheral blood of patients with TREM The difference between the 2 expression levels compared with the control group, and the establishment of a mouse model of PD to investigate the expression level of TREM2 and its downstream nerve inflammation factor, further TREM2-shRNA and wtTREM2 lentiviral transfection of microglia by silencing TREM2 expression or overexpression, and then analyze the influence of TREM2 expression level on microglia M1/M2 subtype differentiation, to to discuss the effects of TREM2 on microglia subtype differentiation regulation, to elucidate the role of TREM2 in microglia mediated neuroinflammation. Objective to study the expression of in the first chapter of soluble TREM2 in PD and PD cognitive impairment: a study of sTREM2 in the differential expression of PD between patients and normal controls, and to investigate the effect of sTREM2 PD of cognitive dysfunction. Methods: the study included 114 PD patients and 120 healthy controls, collected clinical data, using the unified Parkinson's Disease Rating Scale (Unified Parkinson Disease Rating Scale, UPDRS) assessment of motor function in patients with PD and part 1U, through MoCA (Montreal Cognitive Assessment, MoCA) scale to assess the overall cognitive function, intelligence and Wechsler Memory Scale assessment of executive function, memory, attention and visual space can be empty. Further according to the PD diagnostic criteria of mild cognitive impairment. The PD group was divided into PD normal group (PD patients with cognitive normal cognition, PD-CN group) and PD group (PD patients MCI with cognitive impairment, PD-MCI group), including 66 cases of PD-CN group, PD-MCI group of 48 cases. Peripheral blood samples were collected in PD group and control group, the expression level of sTREM2 detection blood samples were analyzed by ELISA method. The expression of sTREM2 in peripheral blood of different levels, and to further explore the role of sTREM2 in PD cognitive function. Results: 114 cases of PD, 48 cases of fancy, in patients with PD-MCI, accounted for 42.11%.PD-MCI group The education level was lower than that of PD-CN group (P0.05); motor function (UPDRS-III score, H-Y grade) and overall cognitive function (MMSE score and MoCA score) were compared with those in PD-CN group, the difference was statistically significant (P0.05); the PD in peripheral blood of patients with the expression level of sTREM2 was significantly higher than that in healthy controls, there are statistically significant difference (P0.001); the healthy control group, PD-CN group and PD-MCI group between the three groups of peripheral blood sTREM2 expression level had significant difference (P0.001), the expression level of PD-MCI group in peripheral blood of patients with sTREM2 was significantly higher than that of PD-CN group, the difference was statistically significant (P0.001); the patients with PD the blood sTREM2 level and MoCA score were negatively correlated (r=-0.464, P0.001); the PD in peripheral blood of patients with elevated levels of sTREM2 and low level of Education (P=0.023), similarity scale (P=-0.015) and recognition scale (P=-0.018) decreased. Conclusion: Patients with PD The high expression level of peripheral blood sTREM2 in patients with PD years of education is low, overall decline in cognitive function, executive function and memory function decline, progress may help monitor PD and cognitive impairment of PD disease of peripheral blood sTREM2 expression showed that TREM2 may be involved in the development of cognitive dysfunction in PD and PD. But the internal mechanism still need further research. The second chapter TREM2 on MPTP induced mouse model of PD nerve inflammation Objective: TREM2 analysis of PD mice model induced by MPTP and normal mouse brain tissue, inflammatory cytokines and anti-inflammatory due to the expression level of sub differences, to explore the role of TREM2 in PD mice. Methods in neuroinflammation PD: to establish a model of chronic C57BL/6J mice by long-term low-dose intraperitoneal injection of MPTP and probenecid, another probenecid control group and saline control group. After the establishment of PD model, the mice in each group, The cerebral cortex, hippocampus, striatum and other parts of the brain, by tyrosine hydroxylase (tyrosine hydroxylase, TH) staining observed in the midbrain DA neurons death, and using fluorescent quantitative PCR method to detect rREM2 and inflammatory cytokines IL-6, IL-1 beta, TNF-a mRNA gene expression level, further through the expression level of Western blot detection of TREM2 and anti-inflammatory factor Arginase-1 protein, single factor variance analysis finally the application of comparison between the three groups of DA neurons lack TREM2, expression of inflammatory and anti-inflammatory factors. Results: the MPTP in the model group TH staining DA positive neurons compared with the number of probenecid control group and saline control group significantly decreased (P0.001), the number of midbrain TH+ neurons. Mice in group MPTP compared with the saline control group reduced 39.7%: the MPTP model group, probenecid between the control group and saline control group of midbrain and cortical layer TREM2, IL-1 beta, I L-6, the relative expression of TNF-a gene mRNA level had significant difference (P0.05), in MPTP group, the highest expression level, the difference was statistically significant (P0.05); the MPTP model group, probenecid control group and saline control group between the three groups in the midbrain, cortex, striatum and hippocampus tissue in the expression level of TREM2 and anti-inflammatory factor Arginase-1 the protein had significant difference (P0.05), the group MPTP expression level was higher than probenecid control group and saline control group, the difference was statistically significant (P0.05); conclusion: mesencephalic PD mice model induced by MPTP, striatum, cerebral cortex and hippocampus and other parts are TREM2 and anti-inflammatory factor Arginase-1 protein expression increased with midbrain DA neurons, and lack of significant nerve inflammation, suggesting that TREM2 and PD in DA is closely related to neuronal loss and nerve inflammation, PD may be the case for nerve inflammation Compensatory to prevent inflammation and increased, but the exact mechanism remains to be clarified. The third chapter TREM2 regulation of microglia subtype differentiation Objective: To investigate whether TREM2 play a regulatory role in microglia M1/M2 subtype differentiation. Methods: using BV2 cells as microglia with TREM2-shRNA'model. Slow virus infection of microglia TREM2 to silence the expression of TREM2 over expression of microglia wtTREM2' lentivirus, and negative control virus infection group, 96 h after infection by immunofluorescence and fluorescence quantitative PCR and Western blot to verify the efficiency of infection. The infected according to the M1 type of microglia induced by agent (IL-4+IL-13) or type M2 microglia inducer (LPS+IFN-y) is different, the microglial cells were divided into 3 groups: control group, IL-4 (1 Ong/ml) +IL-13 (10ng/ml) group, LPS (100ng/m L)) +IFN-y (1 Ong/ml) group. After 96 hours of infection in each group were added to the corresponding M1/M2 type concentration of inducer, after 24 hours by immunofluorescence, the expression level of Western blot and fluorescence quantitative PCR detection of microglia on TREM2 and anti-inflammatory factor Arginase-1, and the expression level of Griess was used to detect the inflammatory factor NO. Results: TREM2-shRNA lentivirus and scramble TREM2 negative control virus, wtTREM2 vector lentivirus and negative control virus infection of microglia after immunofluorescence showed that invasion of TREM2-shRNA'virus and scramble virus vector TREM2 negative control, negative control efficiency of infection in microglia are more than 90%, wtTREM2 lentiviral infection efficiency more than 80%, can ensure the follow-up research; the lentivirus infected microglia after TREM2-shRNA, group TREM2 and M2 microglia marker Arginase-1 egg The expression level of scramble TREM2 than the white group decreased significantly (P0.05), on the contrary, the expression level of TREM2 and Arginase-1 protein in wtTREM2 group were higher than those of group vector (P0.05): the lentivirus infected microglia and microglia induced by M1/M2, IL-4+IL-13 (M2 microglia inducer) group TREM2 and M2 type microglia marker expression level of Arginase-1 was higher than the control group, the expression of type M1 microglia marker NO (P0.05), and LPS+IFN- (gamma M1 microglia inducer) Group on the contrary, the expression level of TREM2 and Arginase-1 were lower than the control group, the increased expression of NO (P0.05). That microglia in M1 type /M2 type inducer appeared under the subtype transformation; the expression of TREM2 silencing and M2 type TREM2, microglia marker Arginase-1 expression level was significantly decreased, and the use of M2 type of microglia induced by IL-4/IL-13 Can induce its expression increase; conversely, overexpression of TREM2, the expression level of TREM2 and Arginase-1 increased significantly, while the use of type M1 microglia LPS/IFN- inducer can induce its expression decreased; showed that TREM2 can promote the transformation of microglia from M1 type to M2 type. Conclusion: (1) without the use of M1 type induction agent, TREM2 silencing can weaken microglial cells to differentiate into type M2 functions show that TREM2 plays an important role in the differentiation of M2 in microglia, may be involved in the pathogenesis of PD nerve inflammation; (2) TREM2 silencing could inhibit the expression of microglia to M2 differentiation and inflammation reaction type M1 microglia mediated instead, overexpression of TREM2 can be transformed to M2 induced microglial cells, inhibiting the inflammatory reaction of M1 microglia mediated and produce anti-inflammatory factors, suggest that TREM2 play a protective PD neural inflammation However, the specific molecular mechanism of how TREM2 regulates the differentiation of M1/M2 subtypes in microglia remains to be further elucidated.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742.5

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