柴　毅，紀(jì)晨星，祝新根△，高子云

(1上饒市人民醫(yī)院神經(jīng)外科，江西 上饒 334000； 2南昌大學(xué)第二附屬醫(yī)院神經(jīng)外科，江西 南昌 330006)

1　外泌體概述

外泌體是直徑在30～100 nm的胞外囊泡，通過細(xì)胞被釋放到細(xì)胞外液中[1]。它們存在于生物體液中，諸如血液和腦脊液。外泌體攜帶有DNA、RNA、蛋白質(zhì)和脂質(zhì)等。由于外泌體的微泡結(jié)構(gòu)為其內(nèi)在的小分子提供了一個(gè)安全穩(wěn)定的環(huán)境，同時(shí)這些信號(hào)小分子利用循環(huán)系統(tǒng)在胞間信號(hào)交換發(fā)揮重要作用，這讓外泌體表現(xiàn)出一個(gè)成熟、穩(wěn)定的信號(hào)傳輸系統(tǒng)[2]。研究發(fā)現(xiàn)，外泌體中的mRNA 和microRNA(miRNA)可從一個(gè)細(xì)胞轉(zhuǎn)移到另一個(gè)細(xì)胞中，并在新的細(xì)胞中發(fā)揮功能[3]。因此，外泌體是理想的細(xì)胞間信息傳遞媒介[4]。外泌體通過如下3種方式進(jìn)行細(xì)胞間信息傳遞：膜融合后內(nèi)容物釋放、膜表面信號(hào)分子以及信號(hào)分子胞外釋放。細(xì)胞可通過分泌外泌體對(duì)各種刺激作出應(yīng)答，在腫瘤組織中，腫瘤局部缺氧可以促進(jìn)乳腺癌細(xì)胞釋放更多的外泌體進(jìn)入腫瘤微環(huán)境，進(jìn)而促進(jìn)腫瘤細(xì)胞的生存和侵襲[5]。外泌體含有豐富的生物信息物質(zhì)，具有在細(xì)胞間傳遞生物信息的獨(dú)特功能[6]，新近研究發(fā)現(xiàn)外泌體在機(jī)體各種重要生理及病理生理過程中發(fā)揮著越來越重要的作用。

2　外泌體對(duì)缺血組織的保護(hù)作用

研究發(fā)現(xiàn)，血管內(nèi)皮細(xì)胞在受到低氧刺激時(shí)能反應(yīng)性地增加外泌體分泌，這些受低氧刺激分泌的外泌體，可被鄰近細(xì)胞攝取，或通過循環(huán)傳輸被遠(yuǎn)處細(xì)胞攝取[6]。研究發(fā)現(xiàn)人體多種臟器，如心臟[7]和腦[8]等器官都能通過遠(yuǎn)程缺血預(yù)處理提高缺血耐受能力。Vicencio等[7]發(fā)現(xiàn)，在缺血處理后，血漿內(nèi)外泌體大量增加，這些內(nèi)源性外泌體通過外泌體表面膜蛋白，如熱休克蛋白 (heat shock protein，HSP)70能夠呈遞信號(hào)給心臟組織，激活心肌細(xì)胞膜表面的Toll樣受體4，活化細(xì)胞外信號(hào)調(diào)節(jié)激酶和p38絲裂原活化蛋白激酶信號(hào)通路，誘導(dǎo)HSP27磷酸化，從而提高心肌對(duì)于缺血再灌注損傷的耐受，達(dá)到心肌保護(hù)作用。循環(huán)內(nèi)皮祖細(xì)胞(endothelial progenitor cells，EPCs)分泌的外泌體，通過轉(zhuǎn)運(yùn)磷脂酰肌醇3激酶/蛋白激酶B信號(hào)通路相關(guān)mRNA，激活血管內(nèi)皮細(xì)胞受體，并促進(jìn)內(nèi)皮細(xì)胞的增殖和成管[9]，同時(shí)，EPCs來源外泌體內(nèi)攜帶有促血管生成miRNA-126和miRNA-296，能上調(diào)血管生成因子，促進(jìn)血管內(nèi)皮細(xì)胞的增殖和分化[10]，從而發(fā)揮其抗凋亡作用。通過進(jìn)行基因芯片分析中風(fēng)組與非中風(fēng)組動(dòng)物的腦血管內(nèi)皮細(xì)胞與神經(jīng)祖細(xì)胞所分泌的外泌體中成分，發(fā)現(xiàn)2組外泌體內(nèi)的蛋白質(zhì)和miRNA均有大幅不同[11]，這提示中風(fēng)能夠改變腦血管內(nèi)皮細(xì)胞與神經(jīng)祖細(xì)胞外泌體內(nèi)成分。此外，缺血組神經(jīng)祖細(xì)胞分泌的外泌體能夠促進(jìn)內(nèi)皮細(xì)胞的遷移和成管，反之，缺血組內(nèi)皮細(xì)胞外泌體能夠提高神經(jīng)祖細(xì)胞的增殖與分化能力。這說明了卒中后腦血管內(nèi)皮細(xì)胞與神經(jīng)祖細(xì)胞能通過分泌外泌體，相互協(xié)調(diào)和促進(jìn)神經(jīng)發(fā)生與血管形成，從而發(fā)揮對(duì)缺血組織的保護(hù)作用。

3　外泌體與神經(jīng)保護(hù)

短暫或更為持久的局灶性腦缺血引起的腦卒中涉及一系列病理生理反應(yīng)，其中大腦損傷會(huì)產(chǎn)生并累積相關(guān)毒性蛋白，它們的降解與Nedd4家族相互作用蛋白1(Nedd4 family interacting protein 1，Ndfip1)和泛素連接酶Nedd4的相互作用相聯(lián)系[12]。 Putz等[13]發(fā)現(xiàn)神經(jīng)元來源的外泌體內(nèi)含有Ndfip1和Nedd4，Ndfip1與泛素連接酶Nedd4能發(fā)揮損傷后移除毒性蛋白的功能。Bianco等[14]已經(jīng)證實(shí)在損傷的神經(jīng)組織中，胞外ATP水平升高，使小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞釋放外泌體，同時(shí)這些外泌體包含炎癥細(xì)胞因子白細(xì)胞介素1β，誘導(dǎo)炎癥反應(yīng)。在此應(yīng)激條件下，星形膠質(zhì)細(xì)胞會(huì)釋放含有突觸小泡相關(guān)的具有神經(jīng)元特異性的磷酸蛋白質(zhì)——突觸蛋白的外泌體。同時(shí)，少突膠質(zhì)細(xì)胞來源外泌體內(nèi)含髓磷脂和應(yīng)激蛋白質(zhì)。外泌體通過遞送這些mRNA和蛋白質(zhì)，在損傷神經(jīng)再生方面發(fā)揮重要功能。

Zhang等[15]發(fā)現(xiàn)間充質(zhì)干細(xì)胞(mesenchymal stem cells，MSCs)來源外泌體可通過減輕炎癥反應(yīng)并且促進(jìn)血管新生和神經(jīng)發(fā)生，從而有效促進(jìn)創(chuàng)傷性顱腦損傷(traumitic brain injury,TBI)大鼠的功能恢復(fù)；Xin等[16]的研究結(jié)果同樣表明，MSCs來源外泌體可通過促進(jìn)神經(jīng)血管再生來改善神經(jīng)功能。其后，又陸續(xù)有研究證明[17-18]MSCs來源外泌體在腦卒中與腦損傷動(dòng)物模型中的治療效益，并發(fā)現(xiàn)其有明顯改善運(yùn)動(dòng)協(xié)調(diào)的功能和大幅度保留空間學(xué)習(xí)的能力。

4　外泌體與神經(jīng)血管單元

神經(jīng)血管單元是由內(nèi)皮細(xì)胞、血管平滑肌、神經(jīng)膠質(zhì)細(xì)胞、神經(jīng)元及相關(guān)組織基質(zhì)蛋白組成的[19],卒中后的內(nèi)源性腦修復(fù)涉及到神經(jīng)血管單元及神經(jīng)干細(xì)胞一系列高度精密的交叉式反應(yīng)，如血管再生和神經(jīng)發(fā)生等，它們共同協(xié)調(diào)神經(jīng)修復(fù)[11]。外泌體在這一系列過程中發(fā)揮巨大作用。

4.1外泌體與神經(jīng)再生神經(jīng)元與膠質(zhì)細(xì)胞來源外泌體能夠調(diào)節(jié)神經(jīng)元和膠質(zhì)細(xì)胞之間的細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)，影響軸突生長(zhǎng)和髓鞘形成[20-21]。皮質(zhì)神經(jīng)元外泌體攜帶有L1細(xì)胞黏附分子和谷氨酸受體2/3亞基，胞內(nèi)Ca2+水平增加會(huì)促進(jìn)外泌體的分泌[22-24]。當(dāng)去極化神經(jīng)元的神經(jīng)突分泌外泌體時(shí)，會(huì)使外泌體內(nèi)富含微管相關(guān)蛋白1b和神經(jīng)突重塑相關(guān)的靶向miRNA[23-24]。此外，神經(jīng)元來源外泌體內(nèi)有α-氨基-3-羥基-5-甲基-4-異惡唑丙酸(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid，AMPA)受體，AMPA受體的激活有助于腦卒中后運(yùn)動(dòng)功能的復(fù)原[25]。在成人中樞神經(jīng)系統(tǒng)中，抑制神經(jīng)元第10號(hào)染色體缺失的磷酸酶和張力蛋白同源物(phosphatase and tensin homolog deleted on chromosome 10，PTEN)信號(hào)與減少星形膠質(zhì)細(xì)胞疤痕，有助于提高脊柱損傷及腦卒中后軸突出芽[26-27]。Goncalves等[28]發(fā)現(xiàn)，視黃酸受體β激動(dòng)劑能使神經(jīng)元細(xì)胞分泌富含PTEN蛋白的外泌體，這些外泌體能抑制皮質(zhì)神經(jīng)元PTEN信號(hào)通路；轉(zhuǎn)運(yùn)PTEN蛋白至膠質(zhì)細(xì)胞，可抑制膠質(zhì)細(xì)胞的增殖，對(duì)神經(jīng)元及膠質(zhì)細(xì)胞產(chǎn)生雙重影響。Morel等[29]進(jìn)一步證實(shí)，皮質(zhì)神經(jīng)元外泌體轉(zhuǎn)運(yùn)mi-RNA-124至膠質(zhì)細(xì)胞，提高谷氨酸轉(zhuǎn)運(yùn)體1的表達(dá)，從而調(diào)節(jié)膠質(zhì)細(xì)胞功能。這些數(shù)據(jù)表明，神經(jīng)元來源外泌體通過轉(zhuǎn)運(yùn)囊泡內(nèi)活性物質(zhì)，并與膠質(zhì)細(xì)胞胞間信號(hào)轉(zhuǎn)導(dǎo)，從而調(diào)節(jié)突觸和軸突重塑。

4.2外泌體與血管新生Notch信號(hào)通路與血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor，VEGF)信號(hào)通路的相互作用對(duì)介導(dǎo)血管再生和維持腦血管屏障完整至關(guān)重要[30-31]。激活腦血管內(nèi)皮細(xì)胞與周細(xì)胞內(nèi)Notch通路，可促使腦血管再生并維持腦血管屏障的完整性[32]。Yamamoto等[32]發(fā)現(xiàn)，由小鼠腦血管內(nèi)皮細(xì)胞分泌的外泌體，能增加受體細(xì)胞周細(xì)胞內(nèi)VEGF-B的mRNA與蛋白表達(dá)水平，通過VEGF-B與受體VEGFR-1介導(dǎo)血管再生[33-34]。研究發(fā)現(xiàn)人腦微血管內(nèi)皮細(xì)胞與人臍靜脈內(nèi)皮細(xì)胞來源的外泌體內(nèi)含Delta-like ligand 4(Dll4)蛋白，Dll4 能刺激周細(xì)胞上的Notch3受體，并保護(hù)腦血管結(jié)構(gòu)穩(wěn)定及調(diào)節(jié)血管再生[35-36]。以上結(jié)果表明，腦血管內(nèi)皮細(xì)胞外泌體通過傳遞生物信號(hào)至周細(xì)胞，并通過VEGF和Notch信號(hào)通路介導(dǎo)血管再生和維持腦血管屏障完整。

Haqqani等[37]發(fā)現(xiàn)，人腦微血管內(nèi)皮細(xì)胞來源的外泌體內(nèi)至少含有1 179種蛋白，其中包括約60種標(biāo)記蛋白，如Alix、TSG101、CD81和CD9等，同時(shí)，他們進(jìn)一步發(fā)現(xiàn)，這些外泌體能夠攜帶大分子蛋白穿越血腦屏障，其中包括轉(zhuǎn)鐵蛋白受體、胰島素受體、LRP、LDL和TMEM30A等。此外，Skog等[38]發(fā)現(xiàn)，膠質(zhì)母細(xì)胞瘤分泌的外泌體富含促血管生成蛋白，而腦微血管內(nèi)皮細(xì)胞可以吸收這些外泌體，從而誘導(dǎo)血管新生。

5　外泌體在腦卒中的研究進(jìn)展

近年來，隨著對(duì)外泌體研究的深入，已證實(shí)外泌體調(diào)控的細(xì)胞間信息交流過程也廣泛參與了腦血管系統(tǒng)的生理、病理過程，并在腦卒中疾病發(fā)生發(fā)展機(jī)制中扮演著重要的角色。Chen等[39]通過收集急性腦卒中病人的血樣本后分析發(fā)現(xiàn)，與陰性對(duì)照組相比，缺血組外泌體內(nèi)miRNA-223含量顯著上調(diào)，并且外泌體內(nèi)miRNA-223的表達(dá)量與急性腦卒中的發(fā)生、嚴(yán)重程度及短期預(yù)后相關(guān)。血漿內(nèi)皮細(xì)胞來源的外泌體(plasma endothelial cell-derived exosomes，EDEs)富含內(nèi)皮細(xì)胞蛋白血管細(xì)胞黏附分子1(vascular cell adhesion molecule-1，VCAM-1)和內(nèi)皮型一氧化氮合酶；具有內(nèi)皮細(xì)胞特性，可以黏附、募集單核細(xì)胞引起炎癥反應(yīng)，提示EDEs在細(xì)胞炎癥反應(yīng)中起重要作用；有研究發(fā)現(xiàn)腦血管疾病的病人EDEs內(nèi)VCAM-1、血管性血友病因子、血小板源生長(zhǎng)因子BB、血管生成素1、賴氨酰氧化酶2以及腦血管選擇性蛋白葡萄糖載體1等較對(duì)照組顯著增高[40-42]，這表明EDEs與腦血管疾病有著某種潛在聯(lián)系。

目前，體內(nèi)外實(shí)驗(yàn)[16-17]均已證實(shí)MSCs外泌體在腦卒中后腦重塑中的治療效益,外泌體能夠呈遞MSCs內(nèi)的miRNA至受體細(xì)胞。目前已發(fā)現(xiàn)有超過700種miRNA在MSCs分泌的外泌體中，這些miRNA能與RNA誘導(dǎo)沉默復(fù)合體組件AGO2結(jié)合[11,43]。 Xin等[16-17]發(fā)現(xiàn)，在缺血腦組織中miRNA-133b水平顯著降低，然而，miRNA-133b能靶向調(diào)節(jié)結(jié)締組織生長(zhǎng)因子(connective tissue growth factor，CTGF)和Ras基因家族成員A，從而促進(jìn)軸突生長(zhǎng)；同時(shí)，他們發(fā)現(xiàn)MSCs治療能上調(diào)缺血腦組織中的miRNA-133b水平，并且，體內(nèi)實(shí)驗(yàn)表明，MSCs與缺血腦細(xì)胞共同培養(yǎng)會(huì)促進(jìn)MSCs分泌富含miRNA-133b的外泌體；此外，他們運(yùn)用慢病毒轉(zhuǎn)染技術(shù)分別制造miRNA-133b高、低表達(dá)的特異性外泌體，并發(fā)現(xiàn)相比于單純給予未處理的MSCs外泌體，高表達(dá)和低表達(dá)miRNA-133b，分別能促進(jìn)和抑制大鼠軸突重建與神經(jīng)功能的恢復(fù)；高表達(dá)miRNA-133b外泌體抑制星形膠質(zhì)細(xì)胞內(nèi)CTGF表達(dá)，并促進(jìn)皮質(zhì)神經(jīng)元神經(jīng)突生長(zhǎng)[44]。我們課題組[45]通過體外實(shí)驗(yàn)發(fā)現(xiàn)，人臍靜脈內(nèi)皮細(xì)胞來源的外泌體能改變?nèi)毖跎窠?jīng)細(xì)胞內(nèi)相關(guān)凋亡蛋白Bax、caspase-3、Bcl-2的表達(dá)，同時(shí)促進(jìn)細(xì)胞增殖、侵襲和遷移，從而協(xié)助神經(jīng)細(xì)胞抵抗缺血再灌注損傷。另外，腦卒中會(huì)誘導(dǎo)外周血的免疫抑制，從而抑制神經(jīng)功能的恢復(fù)[46-47]，研究發(fā)現(xiàn)，MSCs外泌體除了與腦細(xì)胞相互作用，還能在外周血中與自然殺傷細(xì)胞和淋巴細(xì)胞相互作用，從而減弱缺血后的免疫抑制[48]。

6　小結(jié)與展望

腦卒中后，外泌體在神經(jīng)保護(hù)和神經(jīng)功能修復(fù)這一系列過程中涉及到多種胞間信號(hào)通路的精密調(diào)控，神經(jīng)血管單元的重建對(duì)神經(jīng)功能修復(fù)有著舉足輕重的影響；外泌體通過胞間信號(hào)溝通，靶向調(diào)節(jié)多種胞間信號(hào)通路，在調(diào)節(jié)腦卒中后的神經(jīng)功能恢復(fù)中，具有巨大潛能。然而，有關(guān)腦卒中領(lǐng)域中外泌體的研究仍存在許多未知性，例如，腦卒中后，腦實(shí)質(zhì)細(xì)胞或者遠(yuǎn)程器官如何通過細(xì)胞信號(hào)通路影響外泌體的分泌以及其內(nèi)物質(zhì)的改變，外泌體如何調(diào)控受體細(xì)胞內(nèi)源性基因的表達(dá)等，這些問題都等待著我們一一探究。

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