本文選題：缺血性腦卒中 + 炎癥反應(yīng)�。� 參考：《南京大學(xué)》2016年博士論文

【摘要】：研究背景：急性缺血性腦卒中是全球致死率、致殘率最高的疾病之一,給家庭和社會造成巨大負(fù)擔(dān)。然而,除了早期靜脈溶栓外,目前臨床上尚缺乏缺血性腦卒中的有效治療方法。缺血性腦卒中后的免疫炎癥反應(yīng)貫穿了疾病發(fā)生發(fā)展的整個病理過程,是影響缺血性腦卒中預(yù)后的重要因素。腦內(nèi)固有小膠質(zhì)細(xì)胞構(gòu)成了腦內(nèi)的第一道免疫防線,急性腦缺血發(fā)生后,小膠質(zhì)細(xì)胞可被迅速激活并極化為兩種不同的表型,即促炎的M1型(經(jīng)典活化型)和抑炎的M2型(替代活化型),發(fā)揮組織損傷和神經(jīng)保護(hù)雙重作用。因此,調(diào)控小膠質(zhì)細(xì)胞的不同表型及功能是減輕缺血性腦損傷、改善卒中預(yù)后的重要靶點,也是近年來卒中領(lǐng)域的研究熱點。神經(jīng)元可以通過接觸依賴和非接觸依賴途徑調(diào)控小膠質(zhì)細(xì)胞的活化和功能而維持中樞神經(jīng)系統(tǒng)穩(wěn)態(tài),但具體的效應(yīng)分子和機(jī)制有待進(jìn)一步研究。既往研究發(fā)現(xiàn),FasL能夠直接誘導(dǎo)卒中后炎癥反應(yīng),活化小膠質(zhì)細(xì)胞而加重缺血性腦損傷。在本論文中,我們進(jìn)一步明確了FasL是否能夠調(diào)控活化后小膠質(zhì)細(xì)胞的表型和功能,并探討了可溶性FasL (soluble FasL, sFasL)是否參與介導(dǎo)了缺血性腦損傷后神經(jīng)元與小膠質(zhì)細(xì)胞間的相互作用,及JAK2/STAT3和NF-κB信號轉(zhuǎn)導(dǎo)通路參與其中的作用機(jī)制。此外,我們進(jìn)一步探討了sFasL是否影響了不同表型小膠質(zhì)細(xì)胞對缺血損傷后神經(jīng)元的凋亡。研究方法：體內(nèi)實驗部分,分別對C57BL/6J小鼠及B6 Smn.C3H-FasLgld (Gld)小鼠進(jìn)行急性大腦中動脈阻塞(middle cerebral artery occlusion,MCAO)模型,在再灌注后24小時、72小時,采用實時定量PCR和免疫熒光染色的方法檢測小膠質(zhì)細(xì)胞向M1/M2表型極化的程度,同時對小鼠進(jìn)行了神經(jīng)行為功能評分。體外實驗部分,分別培養(yǎng)兩種基因型(WT/Gld)小鼠來源的原代神經(jīng)元和小膠質(zhì)細(xì)胞,建立了神經(jīng)元和小膠質(zhì)細(xì)胞共培養(yǎng)體系,并制備氧糖剝奪(oxygen glucose deprivation, OGD)模型,隨后收集OGD后上清,一方面利用ELISA檢測sFasL的表達(dá),另一方面制備神經(jīng)元條件性培基處理小膠質(zhì)細(xì)胞,檢測小膠質(zhì)細(xì)胞向M1/M2表型極化的程度；同時,利用外源性sFasL直接刺激小膠質(zhì)細(xì)胞,探索sFasL對小膠質(zhì)細(xì)胞表型的調(diào)控作用；利用WB檢測了JAK2/STAT3和NF-κB通路上相關(guān)蛋白的的表達(dá)情況；在部分實驗中,使用sFasL中和抗體抑制其功能,AG490和JSH-23分別抑制JAK2/STAT3和NF-κB信號通路的活化。最后,分別培養(yǎng)兩種基因型小鼠來源的原代小膠質(zhì)細(xì)胞,使用LPS加IFN-y和IL-4分別誘導(dǎo)M1/M2型小膠質(zhì)細(xì)胞極化,并收集小膠質(zhì)細(xì)胞條件性培基處理OGD后神經(jīng)元；利用MTT檢測神經(jīng)元活力,LDH分泌試驗檢測細(xì)胞毒性以及鈣黃綠素-AM和PI染色評估神經(jīng)元存活和凋亡。結(jié)果：(1)體內(nèi)實驗提示,在MCAO 24h和72h后,與野生型小鼠(WT)相比,FasL突變(Gld)小鼠腦梗死周邊區(qū)神經(jīng)元凋亡減少,神經(jīng)功能缺損有所改善。(2)在MCAO 24h和72h后,Gld小鼠腦梗死周邊區(qū)小膠質(zhì)細(xì)胞M2型極化增多而M1型極化減少。(3)體外實驗提示,神經(jīng)元OGD 6h后條件性培基會誘導(dǎo)小膠質(zhì)細(xì)胞向M1型極化,并分泌更多的炎癥介質(zhì)。(4)神經(jīng)元OGD 6h后上清中sFasL表達(dá)水平增加；而使用sFasL中和抗體或Gld來源的神經(jīng)元OGD后條件性培基處理,會顯著減少M1型小膠質(zhì)細(xì)胞的極化程度。(5)外源性sFasL刺激小膠質(zhì)細(xì)胞M1型極化增多,炎癥反應(yīng)增強(qiáng),同時p-JAK2 p-STAT3蛋白表達(dá)水平上調(diào),p50/p65核轉(zhuǎn)移增加,p-IκBα增多；而抑制JAK2/STAT3或NF-κB信號通路會阻斷sFasL介導(dǎo)小膠質(zhì)細(xì)胞的炎癥效應(yīng)。(6)M1型小膠質(zhì)細(xì)胞條件性培基促進(jìn)OGD后神經(jīng)元凋亡,加重細(xì)胞毒性：相反,M2型小膠質(zhì)細(xì)胞條件性培基減少OGD后神經(jīng)元凋亡,減輕細(xì)胞毒性而發(fā)揮保護(hù)功能。(7) FasL突變后sFasL的丟失會顯著減輕M1型小膠質(zhì)細(xì)胞條件性培基誘導(dǎo)的OGD后神經(jīng)元損傷。結(jié)論：(1) FasL突變減少卒中后腦梗死周邊區(qū)神經(jīng)元凋亡及M1型小膠質(zhì)細(xì)胞極化,并能有效改善小鼠運動感覺功能。(2)缺血損傷后神經(jīng)元分泌的sFasL對誘導(dǎo)M1型小膠質(zhì)細(xì)胞的極化及炎癥效應(yīng)發(fā)揮著重要的作用；JAK2/STAT3/NF-κB信號轉(zhuǎn)導(dǎo)通路的相繼活化參與介導(dǎo)了sFasL誘導(dǎo)的小膠質(zhì)細(xì)胞炎癥反應(yīng)。(3)抑制sFasL可以減輕M1型小膠質(zhì)細(xì)胞誘導(dǎo)的缺血后神經(jīng)元損傷。
[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.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R743.3

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