【摘要】：研究目的:腦卒中是引起人類(lèi)死亡的三大原因之一,其高發(fā)病率、高死亡率、高致殘率給社會(huì)和人民生活帶來(lái)了巨大的負(fù)擔(dān)。缺血性腦卒中由血管堵塞引起,約占腦卒中的80%以上。腦缺血涉及的機(jī)制十分復(fù)雜,如:興奮性毒性、鈣超載、氧化應(yīng)激、炎癥反應(yīng)及凋亡等。其中炎癥反應(yīng)是引起神經(jīng)元損傷的重要因素。小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)固有的免疫細(xì)胞,參與抗原呈遞、吞噬及分泌促炎/抗炎因子等。眾多研究表明小膠質(zhì)細(xì)胞激活在中風(fēng)后對(duì)腦組織損傷起到雙向作用,這與其極化分型相關(guān)。小膠質(zhì)細(xì)胞可以極化為經(jīng)典激活的促炎的M1型(classically actived microglia)和選擇性激活的抗炎的M2型(alternatively actived microglia)。而在腦缺血發(fā)病進(jìn)程中,小膠質(zhì)細(xì)胞的激活與極化與神經(jīng)凋亡或修復(fù)有密切關(guān)系。因此,我們不應(yīng)該簡(jiǎn)單地抑制小膠質(zhì)細(xì)胞的激活,而應(yīng)該促進(jìn)小膠質(zhì)細(xì)胞向具有神經(jīng)保護(hù)作用的M2型轉(zhuǎn)換,從而為缺血性腦卒中的治療策略提供指導(dǎo)。單核細(xì)胞遷移抑制因子(monocyte locomotion inhibitory factor,MLIF)是一種由溶組織阿米巴分泌的、具有熱穩(wěn)定性的活性五肽。本課題組在前期研究中發(fā)現(xiàn)MLIF可以減少M(fèi)CAO大鼠和小鼠模型的腦梗死面積,并可以通過(guò)作用于真核翻譯延長(zhǎng)因子1A1(eukaryotic elongation factor1A1,eEF1A1)上調(diào)bEnd3細(xì)胞中血管內(nèi)皮一氧化氮合酶(eNOS)的表達(dá),降低氧糖剝奪(oxygen and glucose deprivation,OGD)OGD和ox-LDL誘導(dǎo)的粘附分子ICAM-1、VCAM-1,發(fā)揮腦缺血血管保護(hù)作用。另外,我們還發(fā)現(xiàn)MLIF可以作用于eEF1A2而抑制由OGD誘導(dǎo)的神經(jīng)細(xì)胞SH-SY5Y的凋亡發(fā)揮腦缺血神經(jīng)保護(hù)作用。因此,本課題組在前期研究的基礎(chǔ)上,進(jìn)一步研究MLIF對(duì)小膠質(zhì)細(xì)胞激活與極化的調(diào)控作用,闡明MLIF在腦缺血中的神經(jīng)保護(hù)作用的抗炎機(jī)制,為腦缺血的防治提供新思路。方法與結(jié)果:1、建立BV2小膠質(zhì)細(xì)胞體外缺氧模型,采用Conditioned Medium Transfer System方法,研究體外腦缺血模型中小膠質(zhì)細(xì)胞對(duì)神經(jīng)細(xì)胞的影響。將BV2小膠質(zhì)細(xì)胞正常培養(yǎng)或OGD后的培養(yǎng)上清(conditioned medium,CM)分別加入正�；蛘逴GD處理的SH-SY5Y神經(jīng)細(xì)胞中,采用MTT檢測(cè)細(xì)胞的生存率。結(jié)果顯示,添加BV2細(xì)胞OGD后CM使SY5Y細(xì)胞的生存率下降,給予MLIF處理的CM后,提高了SY5Y細(xì)胞的生存率。2、利用Real-time PCR及ELISA檢測(cè)MLIF在缺氧條件下對(duì)BV2小膠質(zhì)細(xì)胞極化的調(diào)控作用。結(jié)果顯示缺氧誘導(dǎo)了BV2小膠質(zhì)細(xì)胞M1 markers(CD11b、CD32、cd86、inos、il-1β、tnf-a)mrna或蛋白的表達(dá)顯著升高,給予mlif處理后明顯下調(diào);缺氧條件下,m2markers(arg-1、cd206、fizz-1、ym-1、il-4、il-10)mrna或蛋白的表達(dá)在給予mlif處理后顯著上調(diào)。表明mlif促進(jìn)ogd誘導(dǎo)的小膠質(zhì)細(xì)胞m1型向m2型轉(zhuǎn)化。3、采用mcao法制造大鼠、小鼠腦缺血模型。tunel染色檢測(cè)大鼠缺血腦組織的細(xì)胞的凋亡情況,采用he染色和透射電鏡技術(shù)觀察大鼠缺血腦組織的病理顯微結(jié)構(gòu)及超顯微結(jié)構(gòu)的改變;real-timepcr與elisa檢測(cè)大鼠缺血腦組織的激活的小膠質(zhì)細(xì)胞m1markers及m2markers的表達(dá)。利用免疫熒光方法檢測(cè)大鼠腦組織小膠質(zhì)細(xì)胞的激活情況及小鼠缺血腦組織的激活的小膠質(zhì)細(xì)胞m1markercd16/32及m2markercd206的表達(dá)。結(jié)果顯示,mlif可抑制大鼠缺血腦組織細(xì)胞凋亡、改善大鼠缺血腦組織在顯微結(jié)構(gòu)及超顯微結(jié)構(gòu)上的病理改變,并可以抑制大鼠腦缺血后小膠質(zhì)細(xì)胞的激活,抑制大、小鼠腦缺血后m1型markers的表達(dá),促進(jìn)m2型markers的表達(dá),從而促進(jìn)小膠質(zhì)細(xì)胞從m1型向m2型轉(zhuǎn)化。4、利用westernblot實(shí)驗(yàn)檢測(cè)可能與mlif相關(guān)的nf-κb及mapk通路,結(jié)果顯示mlif可以抑制ogd誘導(dǎo)的nf-κb通路的激活,而對(duì)mapk通路沒(méi)有顯著影響。5、利用蛋白間pull-down技術(shù),研究mlif在bv2小膠質(zhì)細(xì)胞中的結(jié)合蛋白。設(shè)置生物素標(biāo)記的mlif組和陰性對(duì)照組,完成pull-down實(shí)驗(yàn)后,通過(guò)sds-page及考馬斯亮藍(lán)染色后在50kda處出現(xiàn)了一條清晰條帶,經(jīng)過(guò)westernblot驗(yàn)證,結(jié)果表明mlif在bv2小膠質(zhì)細(xì)胞中的特異性結(jié)合蛋白為eef1a1。6、采用sirna將eef1a1基因敲減后,研究mlif在缺氧條件下對(duì)bv2小膠質(zhì)細(xì)胞極化的調(diào)控作用。結(jié)果顯示,sirna轉(zhuǎn)染的bv2小膠質(zhì)細(xì)胞缺氧后m1markers(cd11b、cd32、inos、il-1β、tnf-α)mrna或蛋白的表達(dá)較陰性對(duì)照組顯著升高,m2markers(arg-1、cd206、il-4、il-10)mrna或蛋白的表達(dá)較陰性對(duì)照組顯著降低,而mlif處理后細(xì)胞m1markers表達(dá)降低、m2markers的表達(dá)明顯升高,小膠質(zhì)細(xì)胞向m2型極化;在eef1a1敲減后bv2小膠質(zhì)細(xì)胞中mlif對(duì)缺氧誘導(dǎo)的小膠質(zhì)細(xì)胞極化分型的調(diào)控作用減弱,且mlif對(duì)nf-κb通路的抑制作用減弱,提示eef1a1在mlif抑制缺氧誘導(dǎo)的小膠質(zhì)細(xì)胞極化分型中發(fā)揮重要的調(diào)控作用;同時(shí)結(jié)果還顯示eef1a1敲減后,缺氧誘導(dǎo)小膠質(zhì)細(xì)胞中m1markers表達(dá)較缺氧對(duì)陰性對(duì)照組具有顯著的上調(diào)作用、m2markers表達(dá)較缺氧對(duì)陰性對(duì)照組具有顯著的下調(diào)作用,提示eef1a1對(duì)缺氧誘導(dǎo)的小膠質(zhì)細(xì)胞極化具有一定的調(diào)控作用。結(jié)論:1.體內(nèi)外腦缺血模型中,mlif通過(guò)降低激活的小膠質(zhì)細(xì)胞m1markers、升高激活的小膠質(zhì)細(xì)胞M2 markers,調(diào)控小膠質(zhì)細(xì)胞向M2型轉(zhuǎn)化從而發(fā)揮抗炎作用,進(jìn)而發(fā)揮腦缺血神經(jīng)保護(hù)作用。2.MLIF通過(guò)與BV2小膠質(zhì)細(xì)胞內(nèi)eEF1A1蛋白作用,抑制NF-κB通路從而調(diào)控小膠質(zhì)細(xì)胞在缺氧條件下由M1型向M2型轉(zhuǎn)化,發(fā)揮抗炎神經(jīng)保護(hù)作用。同時(shí)提示eEF1A1可能是調(diào)控腦缺血中巨噬細(xì)胞/小膠質(zhì)細(xì)胞極化的一個(gè)新靶點(diǎn)。
[Abstract]:Research purposes : Stroke is one of the three causes of human death . Its high incidence , high mortality rate and high disability rate bring enormous burden on society and people ' s life . The mechanism of cerebral ischemia is complicated , such as excitotoxicity , calcium overload , oxidative stress , inflammatory reaction and apoptosis . In this study , we found that MLIF can reduce the expression of endothelial nitric oxide synthase ( eNOS ) in cerebral ischemia model , reduce the expression of vascular endothelial nitric oxide synthase ( eNOS ) induced by OGD , decrease oxygen and glucose deprivation ( OGD ) OGD and ox - LDL - induced expression of vascular endothelial nitric oxide synthase ( eNOS ) . expression of m1marksd16 / 32 and m1marksd206 in ischemic brain tissue of rats was studied by means of immuno - fluorescence method . Conclusion : 1 . In vivo external cerebral ischemia model , mLIF plays an important role in the inhibition of hypoxia - induced microglial cell polarization , and suggests eef1a1 plays an important role in inhibiting hypoxia - induced microglial cell polarization .
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R743.3
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本文編號(hào)：2138285