神经元可溶性FasL介导缺血性脑卒中后M1型小胶质细胞极化及机制研究

发布时间：2018-04-20 22:28

本文选题：缺血性脑卒中 + 炎症反应　；参考：《南京大学》2016年博士论文

【摘要】：研究背景：急性缺血性脑卒中是全球致死率、致残率最高的疾病之一,给家庭和社会造成巨大负担。然而,除了早期静脉溶栓外,目前临床上尚缺乏缺血性脑卒中的有效治疗方法。缺血性脑卒中后的免疫炎症反应贯穿了疾病发生发展的整个病理过程,是影响缺血性脑卒中预后的重要因素。脑内固有小胶质细胞构成了脑内的第一道免疫防线,急性脑缺血发生后,小胶质细胞可被迅速激活并极化为两种不同的表型,即促炎的M1型(经典活化型)和抑炎的M2型(替代活化型),发挥组织损伤和神经保护双重作用。因此,调控小胶质细胞的不同表型及功能是减轻缺血性脑损伤、改善卒中预后的重要靶点,也是近年来卒中领域的研究热点。神经元可以通过接触依赖和非接触依赖途径调控小胶质细胞的活化和功能而维持中枢神经系统稳态,但具体的效应分子和机制有待进一步研究。既往研究发现,FasL能够直接诱导卒中后炎症反应,活化小胶质细胞而加重缺血性脑损伤。在本论文中,我们进一步明确了FasL是否能够调控活化后小胶质细胞的表型和功能,并探讨了可溶性FasL (soluble FasL, sFasL)是否参与介导了缺血性脑损伤后神经元与小胶质细胞间的相互作用,及JAK2/STAT3和NF-κB信号转导通路参与其中的作用机制。此外,我们进一步探讨了sFasL是否影响了不同表型小胶质细胞对缺血损伤后神经元的凋亡。研究方法：体内实验部分,分别对C57BL/6J小鼠及B6 Smn.C3H-FasLgld (Gld)小鼠进行急性大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)模型,在再灌注后24小时、72小时,采用实时定量PCR和免疫荧光染色的方法检测小胶质细胞向M1/M2表型极化的程度,同时对小鼠进行了神经行为功能评分。体外实验部分,分别培养两种基因型(WT/Gld)小鼠来源的原代神经元和小胶质细胞,建立了神经元和小胶质细胞共培养体系,并制备氧糖剥夺(oxygen glucose deprivation, OGD)模型,随后收集OGD后上清,一方面利用ELISA检测sFasL的表达,另一方面制备神经元条件性培基处理小胶质细胞,检测小胶质细胞向M1/M2表型极化的程度；同时,利用外源性sFasL直接刺激小胶质细胞,探索sFasL对小胶质细胞表型的调控作用；利用WB检测了JAK2/STAT3和NF-κB通路上相关蛋白的的表达情况；在部分实验中,使用sFasL中和抗体抑制其功能,AG490和JSH-23分别抑制JAK2/STAT3和NF-κB信号通路的活化。最后,分别培养两种基因型小鼠来源的原代小胶质细胞,使用LPS加IFN-y和IL-4分别诱导M1/M2型小胶质细胞极化,并收集小胶质细胞条件性培基处理OGD后神经元；利用MTT检测神经元活力,LDH分泌试验检测细胞毒性以及钙黄绿素-AM和PI染色评估神经元存活和凋亡。结果：(1)体内实验提示,在MCAO 24h和72h后,与野生型小鼠(WT)相比,FasL突变(Gld)小鼠脑梗死周边区神经元凋亡减少,神经功能缺损有所改善。(2)在MCAO 24h和72h后,Gld小鼠脑梗死周边区小胶质细胞M2型极化增多而M1型极化减少。(3)体外实验提示,神经元OGD 6h后条件性培基会诱导小胶质细胞向M1型极化,并分泌更多的炎症介质。(4)神经元OGD 6h后上清中sFasL表达水平增加；而使用sFasL中和抗体或Gld来源的神经元OGD后条件性培基处理,会显著减少M1型小胶质细胞的极化程度。(5)外源性sFasL刺激小胶质细胞M1型极化增多,炎症反应增强,同时p-JAK2 p-STAT3蛋白表达水平上调,p50/p65核转移增加,p-IκBα增多；而抑制JAK2/STAT3或NF-κB信号通路会阻断sFasL介导小胶质细胞的炎症效应。(6)M1型小胶质细胞条件性培基促进OGD后神经元凋亡,加重细胞毒性：相反,M2型小胶质细胞条件性培基减少OGD后神经元凋亡,减轻细胞毒性而发挥保护功能。(7) FasL突变后sFasL的丢失会显著减轻M1型小胶质细胞条件性培基诱导的OGD后神经元损伤。结论：(1) FasL突变减少卒中后脑梗死周边区神经元凋亡及M1型小胶质细胞极化,并能有效改善小鼠运动感觉功能。(2)缺血损伤后神经元分泌的sFasL对诱导M1型小胶质细胞的极化及炎症效应发挥着重要的作用；JAK2/STAT3/NF-κB信号转导通路的相继活化参与介导了sFasL诱导的小胶质细胞炎症反应。(3)抑制sFasL可以减轻M1型小胶质细胞诱导的缺血后神经元损伤。
[Abstract]:Background: acute ischemic stroke is one of the global mortality rates, one of the most disabling diseases, causing great burden to the family and society. However, in addition to early venous thrombolysis, there is a lack of effective treatment for ischemic stroke in the clinic. The whole pathological process is an important factor affecting the prognosis of ischemic stroke. The intrinsic microglia in the brain constitutes the first immune defense in the brain. After acute cerebral ischemia, the microglia can be quickly activated and polarized into two different phenotypes, the proinflammatory M1 type (classical activation type) and the anti inflammatory M2 type (substitute for activation type). It is also a hot spot in the field of stroke in recent years, which can regulate the activation and function of microglia through contact dependence and non-contact dependent pathway. It is necessary to further study the homeostasis of the central nervous system, but the specific molecules and mechanisms need to be further studied. Previous studies have found that FasL can directly induce post stroke inflammatory response, activate microglia and aggravate ischemic brain damage. In this paper, we further clarify whether FasL can regulate the phenotype and work of activated microglia after activation. We can, and explore whether the soluble FasL (soluble FasL, sFasL) is involved in mediating the interaction between neurons and microglia after ischemic brain injury, and the mechanism of JAK2/STAT3 and NF- kappa B signal transduction pathway involved. In addition, we further explore whether sFasL affects the ischemic lesion of different phenotypic microglia. Apoptosis of neurons after injury. Research methods: in the experimental part of the body, the acute middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO) model of C57BL/6J mice and B6 Smn.C3H-FasLgld (Gld) mice was modeled respectively. 24 hours, 72 hours after reperfusion, microglia was detected by real time quantitative PCR and immunofluorescence staining. The degree of M1/M2 phenotypic polarization and the neurobehavioral function of mice were evaluated. In vitro, the primary neurons and microglia were cultured in two genotypes (WT/Gld) mice. The co culture system of neurons and microglia was established and the oxygen glucose deprivation (OGD) model was prepared. After collecting the supernatant after OGD, the expression of sFasL was detected by ELISA. On the other hand, the neuron conditioned culture was prepared to treat microglia, and the degree of polarization of microglia to M1/M2 was detected. At the same time, using exogenous sFasL to stimulate microglia directly and explore the regulation effect of sFasL on the phenotype of microglia; and the use of WB to detect the phenotypes of microglia. The expression of related proteins on the JAK2/STAT3 and NF- kappa B pathway was measured. In some experiments, sFasL neutralization antibody was used to inhibit its function and the activation of JAK2/STAT3 and NF- kappa B signaling pathway was inhibited by AG490 and JSH-23 respectively. Finally, the primary microglia from two genotypes of mice were cultured respectively, which were induced by LPS plus IFN-y and IL-4. M1/M2 microglia was polarised and microglia was collected to treat OGD neurons. The activity of neurons was detected by MTT, the cytotoxicity of LDH secretion test and the assessment of neuronal survival and apoptosis were evaluated by calcine green -AM and PI staining. Results: (1) in vivo experiment, after MCAO 24h and 72h, with wild type mice (WT) The neuronal apoptosis in the peripheral region of cerebral infarction in FasL mutant (Gld) mice decreased and the neural function defect improved. (2) after MCAO 24h and 72h, the M2 type polarization of microglia in the peripheral region of cerebral infarction in Gld mice was increased and M1 type polarization decreased. (3) in vitro, the conditioned culture of neuronal OGD 6h was suggested to induce the microglia to the M1 type. More inflammatory mediators were secreted. (4) the expression level of sFasL in the supernatant of neurons after OGD 6h was increased, and the degree of polarization of M1 type microglia could be significantly reduced by using sFasL neutralization antibody or Gld derived neuron OGD conditioned culture. (5) exogenous sFasL stimulated the increase of M1 polarization in microglia and increased inflammatory reaction, and p-JAK. 2 p-STAT3 protein expression level is up, p50/p65 nuclear transfer is increased and p-I kappa B alpha increases; and the inhibition of JAK2/STAT3 or NF- kappa B signaling pathway will block the inflammatory effect of sFasL mediated microglia. (6) M1 type microglia conditioned culture promotes neuronal apoptosis after OGD and aggravates cytotoxicity: conversely, M2 type microglia is conditioned by conditioned culture. (7) the loss of sFasL after FasL mutation and the loss of sFasL can significantly reduce the neuronal damage after OGD induced by M1 microglia. Conclusion: (1) FasL mutation reduces the neuronal decay in the peripheral area of cerebral infarction and the polarization of the M1 microglia, and can effectively improve the small glial cell polarization in the peripheral area of the cerebral infarction. (2) the sFasL secreted by neurons after ischemic injury plays an important role in inducing the polarization and inflammatory effects of M1 microglia; the sequential activation of JAK2/STAT3/NF- kappa B signaling pathway mediates sFasL induced microglia inflammatory response. (3) inhibition of sFasL can reduce the induction of M1 microglia induced by microglia. Neuron injury after ischemia.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.3

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