孫爭宇，顧麗紅，李 林

少突膠質(zhì)細(xì)胞體外培養(yǎng)方法的研究進(jìn)展①

孫爭宇，顧麗紅，李 林

尋求純度高、簡便易行的體外培養(yǎng)少突膠質(zhì)細(xì)胞的方法具有非常重要的應(yīng)用價(jià)值。本文綜述大鼠皮層膠質(zhì)細(xì)胞混合培養(yǎng)、少突膠質(zhì)前體細(xì)胞增殖純化、少突膠質(zhì)細(xì)胞誘導(dǎo)分化及細(xì)胞體系各發(fā)育分化階段細(xì)胞表面抗原的鑒定等體外培養(yǎng)方法。

少突膠質(zhì)細(xì)胞；少突膠質(zhì)前體細(xì)胞；細(xì)胞培養(yǎng)；綜述

少突膠質(zhì)細(xì)胞(oligodendrocyte,OL)是中樞神經(jīng)系統(tǒng)的髓鞘形成細(xì)胞，起源于胚胎神經(jīng)管腹側(cè)的神經(jīng)上皮細(xì)胞，在胚胎發(fā)生過程中從運(yùn)動神經(jīng)元前體區(qū)(motor neuron progenitor domain, MNPD)遷移出[1]，胚胎發(fā)生末期聚集于邊緣區(qū)并開始分化；隨著中樞神經(jīng)系統(tǒng)的發(fā)育，逐步遷移到白質(zhì)，并進(jìn)一步增殖、分化；成熟的OL可包繞神經(jīng)纖維的軸突并形成髓鞘，在促進(jìn)神經(jīng)元功能電位的正確快速傳導(dǎo)和支持軸突存活中發(fā)揮著重要作用[2]。OL在發(fā)育過程中，大致經(jīng)歷了少突膠質(zhì)祖細(xì)胞、少突膠質(zhì)前體細(xì)胞(oligodendrocyte precursor cells,OPC)、未成熟OL和成熟OL幾個(gè)階段[3]，其形態(tài)、功能和表達(dá)產(chǎn)物呈現(xiàn)連續(xù)漸變過程，各發(fā)育階段之間沒有嚴(yán)格界限。越來越多的研究表明，OL異常在中樞神經(jīng)系統(tǒng)脫髓鞘病變[4]、神經(jīng)元損傷[5]、精神疾病等的發(fā)病機(jī)制中起著重要作用[6-8]。OL可能成為逆轉(zhuǎn)神經(jīng)精神疾病(抑郁癥、孤獨(dú)癥、精神分裂癥等)認(rèn)知障礙的潛在治療靶點(diǎn)[9]。精神分裂癥患者OL密度及數(shù)量下降，而且細(xì)胞內(nèi)出現(xiàn)異染色質(zhì)及異常線粒體聚集等凋亡現(xiàn)象[10]。

將體外培養(yǎng)的OL移植入髓鞘形成障礙或脫失的中樞神經(jīng)系統(tǒng)內(nèi)行細(xì)胞替代治療[11]，或應(yīng)用一些藥物促進(jìn)髓鞘的形成和再生，有利于脫髓鞘病灶的修復(fù)以改善腦白質(zhì)病變[12-13]，是近年來的研究熱點(diǎn)。而體外純化和培養(yǎng)OL也是了解該細(xì)胞生長特性、對缺氧耐受性、某些特殊因子的表達(dá)及其內(nèi)部信號傳遞、揭示髓鞘形成和再生機(jī)制等研究的重要前提[14]。尋求一種純化度高、簡便易行的培養(yǎng)OL方法，具有非常重要的應(yīng)用價(jià)值。

1 大鼠皮層膠質(zhì)細(xì)胞混合培養(yǎng)

大多數(shù)研究者選取出生后0～3 d的新生Sprague-Dawley大鼠[15-17]，雌雄不限；也有研究者選取孕14～18 d的胎鼠[18]，或出生后0～2 d新生C57B6小鼠[19]。OPC培養(yǎng)在種屬之間的差異尚不十分清楚，小鼠OPC對胰酶消化較敏感，成活率較Sprague-Dawley大鼠低[20]。有研究表明[21]，成年大鼠中樞神經(jīng)系統(tǒng)中絕大部分為成熟OL，喪失遷移及增殖能力，但也存在OPC；而新生大鼠出生后約1周，OL即逐漸成熟。髓鞘化發(fā)生的高峰期約在哺乳期結(jié)束時(shí)，即出生后第3周，因此可采用72 h內(nèi)的新生Sprague-Dawley大鼠皮層進(jìn)行OL原代培養(yǎng)，且多采用出生24 h內(nèi)的，這時(shí)處在前體細(xì)胞充分發(fā)育后，髓鞘形成活躍期之前。也有研究者取雙側(cè)視神經(jīng)進(jìn)行OL培養(yǎng)[22-23]。

在無菌條件下取大腦，解剖顯微鏡下剔除腦膜及血管，分離出大腦皮層是取材的關(guān)鍵步驟。原代細(xì)胞培養(yǎng)常用胰酶消化法結(jié)合物理吹打法獲得單細(xì)胞懸液[24]，但在實(shí)際操作過程中，可能因胰酶消化過度或吹打力度不均，導(dǎo)致細(xì)胞產(chǎn)量過低。胰蛋白酶的濃度多選取0.125%～0.25%[25]，37℃孵育15～25 min，用膠質(zhì)細(xì)胞基本培養(yǎng)液終止消化，如含10%胎牛血清的DMEM/F12培養(yǎng)基[26]，或含15%胎牛血清的高糖DMEM培養(yǎng)基[27]。機(jī)械吹打時(shí)，有研究者采用平滑管口[28]或經(jīng)火焰拋光的細(xì)頭吸管[29]分層輕柔吹打，以達(dá)到離散細(xì)胞的目的，在很大程度上減少吹打所致的機(jī)械性損傷。制備成細(xì)胞數(shù)為5×106/ml的細(xì)胞懸液，種植于預(yù)先用0.1 mg/ml多聚賴氨酸包被的培養(yǎng)瓶中，置于37℃、5%CO2培養(yǎng)箱中，24 h后換全液，之后可每3天換半液或每隔1天換全液，培養(yǎng)7～8 d。

2 OPC增殖、純化及OL誘導(dǎo)分化

OPC增殖、發(fā)育分化和髓鞘形成過程受細(xì)胞內(nèi)部性質(zhì)、細(xì)胞生存時(shí)間周期和來源于其他細(xì)胞(神經(jīng)元、間充質(zhì)干細(xì)胞、施萬細(xì)胞、心肌細(xì)胞等)信號分子等因素的影響[30]?；旌夏z質(zhì)細(xì)胞生長7～8 d時(shí)可進(jìn)行OPC的純化[31]。有研究發(fā)現(xiàn)[32]，小鼠OPC與心肌細(xì)胞共培養(yǎng)時(shí)，心肌細(xì)胞條件培養(yǎng)基中磷酸化的磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)和細(xì)胞外信號調(diào)節(jié)激酶(extracellular signal-regulated kinase,ERK)可以促進(jìn)OPC的增殖，且共培養(yǎng)比OPC單獨(dú)培養(yǎng)表現(xiàn)出更高的增殖能力。而將來源于外胚層神經(jīng)嵴的間充質(zhì)干細(xì)胞與OPC共培養(yǎng)，可以促進(jìn)OPC的分化和成熟，能夠增加OL的數(shù)量和突起長度[33]。腦微血管內(nèi)皮細(xì)胞(microvascular endothelial cell, MVEC)能夠減少OPC的凋亡[34]。MVEC條件培養(yǎng)液釋放胞外囊泡可以促進(jìn)OPC的增殖和遷移，促進(jìn)軸突髓鞘修復(fù)。血小板源性生長因子(platelet-derived growth factor,PDGF)、堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor,bFGF)、神經(jīng)營養(yǎng)因子3(neurotrophin 3,NT3)和胰島素樣生長因子1(insulin-like growth factor 1,IGF-1)等可促進(jìn)OPC增殖[35-37]。甲狀腺素、雌激素、孕激素、糖皮質(zhì)激素、視黃酸以及細(xì)胞外基質(zhì)相關(guān)分子等都對OPC的發(fā)育分化有重要作用，而外環(huán)境因素需通過調(diào)節(jié)細(xì)胞內(nèi)部的因子來發(fā)揮作用[38]。有研究發(fā)現(xiàn)[39]，P21激酶3(p21-activated kinase 3,PAK3)在OPC中高表達(dá)，可能是OPC分化的一個(gè)新的調(diào)節(jié)因子。在OPC培養(yǎng)過程中添加10 ng/ml bFGF，可明顯提高OPC的產(chǎn)出率和純度；更高濃度的bFGF則減弱其促增殖作用[40]。經(jīng)β-淀粉樣蛋白處理過的OL出現(xiàn)了細(xì)胞凋亡，可能是通過提高Dp5的表達(dá)、氧化應(yīng)激等機(jī)制參與誘導(dǎo)OL的死亡[41]。

在OPC增殖、分離、接種、分化等培養(yǎng)基中加入相應(yīng)的物質(zhì)，可促進(jìn)其增殖、分化。目前常用的培養(yǎng)基配方如下。①OPC混合培養(yǎng)基：DMEM/F12培養(yǎng)基+10%胎牛血清[42]。②OPC增殖培養(yǎng)基：DMEM/F12+1%N2+15%B104條件培養(yǎng)基[43]；或DMEM/F12+10 ng/ml PDGF+10 ng/ml bFGF+0.5%胎牛血清[35]。③OPC分離培養(yǎng)基：DMEM/F12+0.01%EDTA+0.2 mg/ml DNA酶+5μg/ml胰島素。④OPC接種培養(yǎng)基：50%OPC增殖培養(yǎng)基+50%混合培養(yǎng)基。⑤OPC純化培養(yǎng)基：無糖DMEM+1%Lactate(0.5 mol/L)+1%N2+15%B104條件培養(yǎng)基。⑥OPC分化培養(yǎng)基：DMEM/F12+0.8μg/ml亞硒酸鈉+ 16.1μg/ml腐胺+50μg/ml轉(zhuǎn)鐵蛋白+5μg/ml胰島素+0.4μg/ml甲狀腺素。

B104條件培養(yǎng)基具有促細(xì)胞增殖的效應(yīng)[44]，c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)通過磷酸化促進(jìn)OPC的增殖，其作用與商品化生長因子(PDGF-AA、bFGF)的效應(yīng)相似[45]，應(yīng)用B104培養(yǎng)基代替商品化的細(xì)胞生長因子，花費(fèi)較低。進(jìn)一步研究發(fā)現(xiàn)[46]，B104條件培養(yǎng)基促進(jìn)OPC增殖具有濃度依賴性特征。15%B104條件培養(yǎng)基具有最佳的促進(jìn)OPC增殖效應(yīng)，而更高濃度則抑制OPC增殖?？赡苁且?yàn)锽104條件培養(yǎng)基是培養(yǎng)B104細(xì)胞的陳舊培養(yǎng)基，其中除含有有益于OPC增殖的物質(zhì)外，也含有大量的細(xì)胞代謝廢物。

有研究者通過神經(jīng)細(xì)胞化學(xué)限定性培養(yǎng)基思路，列出了可用于OL體外培養(yǎng)的無血清培養(yǎng)基配方[47-48]。該培養(yǎng)基以DMEM和F12為基礎(chǔ)，通過添加一系列維生素、激素及微量元素等成分而成[42]。但是該配方可能不是專門針對OL的體外生長，用這一配方培養(yǎng)神經(jīng)細(xì)胞、星形膠質(zhì)細(xì)胞，也會增殖和生長。

體外培養(yǎng)OL接種后，一般仍先用有血清培養(yǎng)液，在獲得足夠的細(xì)胞數(shù)量后，換為無血清培養(yǎng)液的化學(xué)條件培養(yǎng)基，可使體外培養(yǎng)條件下OPC定向分化為成熟OL，細(xì)胞形態(tài)呈現(xiàn)漸變過程。楊凱等[49]發(fā)現(xiàn)，DMEM/F12較DMEM/高糖培養(yǎng)基更適于OL培養(yǎng)，在一定范圍內(nèi)，獲取的OL數(shù)量隨細(xì)胞接種密度成梯度增多，接種密度在(4×104～8×104)/cm2范圍時(shí)，有利于觀察細(xì)胞生長情況。

OPC原代培養(yǎng)是一種共培養(yǎng)狀態(tài)[50]，和星形膠質(zhì)細(xì)胞一起生長，需把握好細(xì)胞分離的時(shí)機(jī)。星形膠質(zhì)細(xì)胞層可以主動分泌一些細(xì)胞因子，促進(jìn)OPC的存活和增殖，為OPC的遷移和增殖提供良好的環(huán)境，且不抑制其分化[51]。根據(jù)星形膠質(zhì)細(xì)胞和OL的生長時(shí)間差異、細(xì)胞生長方式及細(xì)胞對培養(yǎng)層黏附等特性的不同[52]，星形膠質(zhì)細(xì)胞貼壁更快且更緊密，可采用兩次恒溫?fù)u床振蕩分離純化法、差速貼壁法、胰酶消化法，結(jié)合條件限定培養(yǎng)基培養(yǎng)，可獲取并鑒定出高純度的大鼠OL[53]。分離時(shí)間過早則存活能力差，分離時(shí)間過晚則影響OPC的分化。

體外純化OPC，常用的方法包括植塊培養(yǎng)法和分離細(xì)胞培養(yǎng)法。分離細(xì)胞培養(yǎng)法又因白質(zhì)組織和灰質(zhì)組織兩種材料來源而分為密度梯度離心法[54]和恒溫?fù)u床處理法[55]兩種。恒溫?fù)u床處理法分離OL的主要缺點(diǎn)是必須經(jīng)過一定時(shí)間的原代培養(yǎng)期。

楊俊林等[16]利用細(xì)胞分層生長的特點(diǎn)和單克隆形成，簡便地制備出高純度的膠質(zhì)限制前體細(xì)胞，分化成星形膠質(zhì)細(xì)胞和OL，而未分化成神經(jīng)元。段朝霞等[19]根據(jù)星形膠質(zhì)細(xì)胞和OPC系生長時(shí)間的差異、細(xì)胞黏附特性的不同，通過振蕩及差速貼壁法，結(jié)合添加N2、PDGF、bFGF的無血清OL定向培養(yǎng)基，成功獲得高純度的OPC，應(yīng)用巨噬細(xì)胞移動抑制因子(macrophage migration inhibitory factor,MIF)促進(jìn)OPC增殖。盧玉仙等[20]依據(jù)各種膠質(zhì)細(xì)胞黏附性及生長時(shí)間的差異，通過機(jī)械振蕩法和差速貼壁法，從原代培養(yǎng)中獲得純化的OPC，接種于含有PDGF、bFGF的培養(yǎng)液中觀察其增殖情況，再以三碘甲狀腺原氨酸和睫狀神經(jīng)營養(yǎng)因子定向誘導(dǎo)分化，發(fā)現(xiàn)OPC具有良好的增殖能力，沿著少突膠質(zhì)譜系細(xì)胞定向分化，隨有形態(tài)學(xué)及表面表達(dá)抗原的變化。與段朝霞等的研究具有一致性。有研究者提出改良化學(xué)分離傳代法[56]，采用EDTA化學(xué)分離為基礎(chǔ)的機(jī)械振蕩法獲取OPC，并與傳統(tǒng)的振蕩分離法及單用化學(xué)分離法進(jìn)行對比，發(fā)現(xiàn)采用EDTA化學(xué)分離為基礎(chǔ)的機(jī)械振蕩改良傳代法能高效獲取大量高純度體外培養(yǎng)的OPC，所得的OPC純度明顯高于另外兩種分離方法，數(shù)量顯著多于傳統(tǒng)的培養(yǎng)方法，且較其他如抗體免疫篩選法[57-58]簡便、可靠。

發(fā)育過程中調(diào)控OPC分化的分子機(jī)制主要包括細(xì)胞骨架水平、轉(zhuǎn)錄水平、時(shí)空水平以及軸突水平等方面[59-60]，胞內(nèi)轉(zhuǎn)錄因子Sox10、SCIP、Krox24以及大量含螺旋-環(huán)-螺旋(helix-loop-helix,HLH)結(jié)構(gòu)域的轉(zhuǎn)錄因子等發(fā)揮重要作用[61-62]。

3 OL體系鑒定

OL的每個(gè)發(fā)育階段都呈現(xiàn)出不同的形態(tài)變化，主要是從簡單的雙極狀態(tài)向分支復(fù)雜的狀態(tài)轉(zhuǎn)變，表達(dá)不同的細(xì)胞表面抗原，通常采用細(xì)胞免疫熒光進(jìn)行鑒定。免疫熒光特異性A2B5、PDGFR和NG2抗體標(biāo)記OPC；特異性O(shè)1、O4、CNP抗體標(biāo)記未成熟OL；髓鞘堿性蛋白(myelin basic protein,MBP)和髓鞘少突膠質(zhì)細(xì)胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG)抗體標(biāo)記成熟OL[63]；特異性膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)抗體標(biāo)記星形膠質(zhì)細(xì)胞[64-65]。

混合膠質(zhì)細(xì)胞制備成單個(gè)細(xì)胞懸液適當(dāng)稀釋后，應(yīng)用臺盼藍(lán)排除實(shí)驗(yàn)方法計(jì)數(shù)活細(xì)胞個(gè)數(shù)，計(jì)數(shù)時(shí)間為3 min，鏡下觀察死細(xì)胞被染成藍(lán)色，活細(xì)胞為無色透明狀。培養(yǎng)并分離純化的OPC定向分化培養(yǎng)15 d后，細(xì)胞形態(tài)發(fā)生明顯改變，突起增多，呈“分枝”或“蜘蛛網(wǎng)”狀分布于胞體四周，95%左右的細(xì)胞成熟OL特異性抗體MBP和O4染色呈陽性。有研究應(yīng)用恒溫?fù)u床結(jié)合無血清培養(yǎng)的方法純化OL[54]，Galc免疫細(xì)胞化學(xué)法鑒定OL，發(fā)現(xiàn)純化后的OL胞體呈圓形或橢圓形，折光性強(qiáng)，突起少，陽性細(xì)胞胞漿和突起呈棕黃色，發(fā)現(xiàn)采用此種方法培養(yǎng)出的OL純化度達(dá)95%。該方法耗材少，且方便簡易。無血清化學(xué)條件培養(yǎng)基加入后3 d，相差顯微鏡下觀察可見OL胞體增大，周圍折光強(qiáng)，突起明顯增多，相互之間連接成網(wǎng)；掃描電鏡觀察可見胞體邊緣不規(guī)則，突起分支更加清晰，分支彼此呈三維立體交錯(cuò)，部分細(xì)小突起間可見膜片狀結(jié)

構(gòu)[66]。

4 小結(jié)

OL異常在脫髓鞘疾病、神經(jīng)元損傷、精神疾病等發(fā)病機(jī)制中的作用越來越受到重視。OL原代培養(yǎng)增殖、純化和各分化階段的鑒定仍是難點(diǎn)，需要不斷優(yōu)化培養(yǎng)條件，簡化實(shí)驗(yàn)步驟，降低成本，尋求一種簡便易行、經(jīng)濟(jì)有效的辦法。在進(jìn)行體外培養(yǎng)時(shí)，可根據(jù)研究目的和需要，摸索實(shí)驗(yàn)條件，添加多種不同濃度的營養(yǎng)因子，應(yīng)用增殖、純化、定向誘導(dǎo)等培養(yǎng)基，得到高產(chǎn)出率、高純度的OL。

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Progress in Methodology for Oligodendrocyte Culture in Vitro(review)

SUN Zheng-yu,GU Li-hong,LI Lin
Department of Pharmacology,Xuanwu Hospital of Capital Medical University;Beijing Engineering Research Center for Nerve System Drugs;Key Laboratory for Neurodegenerative Diseases of Ministry of Education,Beijing 100053, China

LI Lin.E-mail:linlixw@126.com

It is important to establish a simple method of culture in vitro to obtain purity oligodendrocyte.This paper introduced the researches about co-culture in vitro with rat cortical gliocyte,proliferation and differentiation of oligodendrocyte progenitor,differentiation of oligodendrocyte and identification of cell surface antigens at different stages of development.

oligodendrocyte;oligodendrocyte precursor cell;cell culture;review

R741

A

1006-9771(2017)05-0543-05

2017-01-17

2017-02-09)

10.3969/j.issn.1006-9771.2017.05.011

[本文著錄格式]孫爭宇，顧麗紅，李林.少突膠質(zhì)細(xì)胞體外培養(yǎng)方法的研究進(jìn)展[J].中國康復(fù)理論與實(shí)踐,2017,23(5): 543-547.

CITED AS:Sun ZY,Gu LH,Li L.Progress in methodology for oligodendrocyte culture in vitro(review)[J].Zhongguo Kangfu Lilun Yu Shijian,2017,23(5):543-547.

1.國家自然科學(xué)基金項(xiàng)目(No.81673406;No.81273498)；2.北京市科技專項(xiàng)項(xiàng)目(No.Z131102002813066)。

首都醫(yī)科大學(xué)宣武醫(yī)院藥物研究室，北京市神經(jīng)藥物工程技術(shù)研究中心，神經(jīng)變性病教育部重點(diǎn)實(shí)驗(yàn)室，北京市100053。作者簡介：孫爭宇(1983-)，女，漢族，河南鄭州市人，博士研究生，主治醫(yī)師，主要研究方向：神經(jīng)藥理學(xué)。通訊作者：李林，女，漢族，北京市人，教授，博士生研究生導(dǎo)師，主要研究方向：神經(jīng)藥理學(xué)。E-mail:linlixw@126.com。