董錦潤(rùn), 郭立云, 瞿家桂, 戚仁莉, 王文超, 肖春杰, 王正波,*

(1. 中國(guó)科學(xué)院生物物理研究所 腦與認(rèn)知國(guó)家重點(diǎn)實(shí)驗(yàn)室, 北京 100101; 2. 中國(guó)科學(xué)院昆明動(dòng)物研究所, 云南 昆明 650223; 3. 云南大學(xué) 生命科學(xué)院, 云南 昆明 650223; 4. 昆明醫(yī)學(xué)院第四附屬醫(yī)院 眼科, 云南 昆明 650021; 5. 中國(guó)科學(xué)技術(shù)大學(xué) 生命科學(xué)學(xué)院, 安徽 合肥230026)

獼猴神經(jīng)干細(xì)胞的誘導(dǎo)分化、干性維持及同種腦內(nèi)移植

董錦潤(rùn)1,2,3,＋, 郭立云4,＋, 瞿家桂1,2,5, 戚仁莉1,2,5, 王文超1,2, 肖春杰3,*, 王正波1,2,*

(1. 中國(guó)科學(xué)院生物物理研究所 腦與認(rèn)知國(guó)家重點(diǎn)實(shí)驗(yàn)室, 北京 100101; 2. 中國(guó)科學(xué)院昆明動(dòng)物研究所, 云南 昆明 650223; 3. 云南大學(xué) 生命科學(xué)院, 云南 昆明 650223; 4. 昆明醫(yī)學(xué)院第四附屬醫(yī)院 眼科, 云南 昆明 650021; 5. 中國(guó)科學(xué)技術(shù)大學(xué) 生命科學(xué)學(xué)院, 安徽 合肥230026)

為探索獼猴神經(jīng)干細(xì)胞分化及特性維持, 推進(jìn)神經(jīng)干細(xì)胞臨床應(yīng)用研究, 該實(shí)驗(yàn)以綠色熒光蛋白(green fluorescence protein, GFP)為標(biāo)記探討獼猴胚胎干細(xì)胞向玫瑰花環(huán)(rosettes)結(jié)構(gòu)神經(jīng)干細(xì)胞的分化及其堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor, bFGF)和表皮生長(zhǎng)因子(epidermal growth factor, EGF)的擴(kuò)增培養(yǎng)。結(jié)果表明：1)建立了穩(wěn)定高效的獼猴神經(jīng)干細(xì)胞分化體系, 在該分化體系下, GFP標(biāo)記獼猴胚胎干細(xì)胞在分化的第12天時(shí), 95%以上的細(xì)胞分化為神經(jīng)干細(xì)胞; 2)分化得到的Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞經(jīng)bFGF/EGF擴(kuò)增后, 能夠較好地維持其Rosettes結(jié)構(gòu); 3)經(jīng)bFGF/EGF擴(kuò)增后的rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞移植到獼猴腦內(nèi)后能夠較好的存活并向神經(jīng)元分化，即bFGF/EGF擴(kuò)增培養(yǎng)能較好地維持Rosettes結(jié)構(gòu)的神經(jīng)干細(xì)胞, 且移植到獼猴腦內(nèi)的該細(xì)胞亦能夠較好地存活并向神經(jīng)元分化，該結(jié)果為神經(jīng)干細(xì)胞應(yīng)用于臨床提供了基礎(chǔ)理論依據(jù)。

獼猴；神經(jīng)干細(xì)胞；玫瑰花環(huán)結(jié)構(gòu)；堿性成纖維細(xì)胞生長(zhǎng)因子；表皮生長(zhǎng)因子

由于神經(jīng)干細(xì)胞具有自我更新、多潛能分化(Gage, 2000)、遷移功能、良好的組織融合性(Amar et al, 2003)以及低免疫源性(Modo et al, 2003), 為許多難以治愈的神經(jīng)系統(tǒng)疾病提供了新的治療途徑。神經(jīng)干細(xì)胞分化技術(shù)的完善和人工誘導(dǎo)多潛能干細(xì)胞(induced pluripotent stem cells, IPS)方法的出現(xiàn)(Okita et al, 2007), 使細(xì)胞替代治療神經(jīng)系統(tǒng)疾病成為了研究熱點(diǎn)。

體外培養(yǎng)條件下, 獼猴胚胎干細(xì)胞(embryonic stem cell, ESC)能夠分化為神經(jīng)前體細(xì)胞、神經(jīng)元(Calhoun et al, 2003; Chen, 2003; Kuai, 2009)及膠質(zhì)細(xì)胞(Chen, 2008)；但是，隨著對(duì)神經(jīng)干細(xì)胞研究的深入, Pankratz et al (2007)發(fā)現(xiàn)在胚胎干細(xì)胞向神經(jīng)干細(xì)胞分化時(shí), 形成的玫瑰花環(huán)(rosettes)結(jié)構(gòu)細(xì)胞,才是真正意義上的早期全能神經(jīng)前體細(xì)胞，而胚胎干細(xì)胞來(lái)源的神經(jīng)前體細(xì)胞的特性維持是該領(lǐng)域的研究熱點(diǎn)和難點(diǎn)。

堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor, bFGF)是Gospodarowicz (1975)在1974年從牛腦中分離純化出的一種多肽因子。bFGF在胚腦及成年腦中均有分泌, 是一種重要的有絲分裂原, 它能通過(guò)作用于細(xì)胞表面相應(yīng)的受體促進(jìn)神經(jīng)干細(xì)胞的增殖和分化。表皮生長(zhǎng)因子(epid-ermal growth factor, EGF)是Cohen(1962)首次在小鼠的頜下腺中發(fā)現(xiàn)的一種小分子蛋白, 也具有廣泛的促絲裂增殖的作用。當(dāng)前普遍采用bFGF和EGF進(jìn)行擴(kuò)增培養(yǎng)神經(jīng)干細(xì)胞(Murphy et al, 1990; Studer et al, 1998), 而bFGF/EGF擴(kuò)增是否影響干細(xì)胞特性存在爭(zhēng)議(Du＆Zhang, 2004; Elkabetz ＆Studer 2008; Hong et al, 2008; Koch et al, 2009)。

獼猴在遺傳和生理上都優(yōu)于嚙齒類(lèi)且與人類(lèi)更為接近(Wolf et al, 2004), 所以，獼猴胚胎干細(xì)胞的神經(jīng)分化可為探索人類(lèi)胚胎發(fā)育過(guò)程中的神經(jīng)發(fā)生提供參考, 同時(shí)可用于神經(jīng)疾病藥物篩選以及開(kāi)展神經(jīng)干細(xì)胞臨床治療的相關(guān)研究。Li et al (2005)將由獼猴胚胎干細(xì)胞分化得到的神經(jīng)干細(xì)胞移植至大鼠腦內(nèi)的研究，由于異種移植而存在種屬差異性。因此, 本研究擬通過(guò)建立獼猴神經(jīng)干細(xì)胞誘導(dǎo)分化體系, 探討bFGF/EGF擴(kuò)增能否維持神經(jīng)干細(xì)胞Rosettes結(jié)構(gòu)特性, 并在同物種獼猴上進(jìn)行移植實(shí)驗(yàn), 為神經(jīng)干細(xì)胞移植更好的應(yīng)用于臨床提供基礎(chǔ)理論依據(jù)。

1 材料及方法

1.1 獼猴胚胎干細(xì)胞培養(yǎng)

tauGFP-rESCs細(xì)胞系LYON-ES野生型(LYONES-WT)(Wianny et al, 2008)(法國(guó)里昂干細(xì)胞與腦科學(xué)研究所饋贈(zèng), 試驗(yàn)所用細(xì)胞傳代至第8～20代)培養(yǎng)在絲裂霉素C(Sigma, 5 μg/mL, 2 h)處理的CF1小鼠飼養(yǎng)層(CF1-MEF, 購(gòu)自ATCC)上, 其培養(yǎng)方法參照文獻(xiàn) Wianny et al (2008)的報(bào)道進(jìn)行。培養(yǎng)基為80% Knockout DMEM(invitrogen)+20% Knockout serum replacement(KO-SR, invitrogen)+1% 非必需氨基酸(Gibco)+1% PSG(invitrogen)+0.1 mM β-巰基乙醇(sigma)+5 ng/mL bFGF(chemicon)。

1.2 GFP標(biāo)記獼猴胚胎干細(xì)胞向神經(jīng)干細(xì)胞分化

參照Pankratz et al(2007)的神經(jīng)分化方法, 建立綜合擬胚體(embryoid Body, EB)法和單層法的分化體系。將胚胎干細(xì)胞克隆通過(guò)手工挑選, 切割成1 000個(gè)細(xì)胞左右的細(xì)胞團(tuán)塊, 懸浮培養(yǎng), 形成EB。懸浮培養(yǎng)基為：DMEM/F12(1:1, Gibco)、Neural basal medium(Gibco)、1×N2 supplement (Gibco)、2 mmol/L谷氨酰胺(Sigma)和50 U/mL 青?鏈霉素(Sigma)。將形成的EBs轉(zhuǎn)移到經(jīng)ECM(Sigma)處理成分限定的神經(jīng)分化培養(yǎng)基(neural differentiation culture medium, NDCM)中繼續(xù)培養(yǎng)。NDCM成分為：F12+ITS-x+2 μg/mL heparin+2 mmol/L glutamine (Sigma) +50 U/ mL 青?鏈霉素(Sigma)。

1.3 GFP標(biāo)記獼猴神經(jīng)干細(xì)胞擴(kuò)增

手工挑選培養(yǎng)皿中Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞,按1:3經(jīng)NDCM培養(yǎng)基添加20 ng/mL的bFGF/EGF擴(kuò)增后維持培養(yǎng)。分化培養(yǎng)實(shí)驗(yàn)的傳代方法同干細(xì)胞培養(yǎng)傳代方法, 只是不添加bFGF/EGF。

1.4 神經(jīng)干細(xì)胞的獼猴移植

兩只成年(5 a)肢體殘疾獼猴(由斗毆引起)由中國(guó)科學(xué)院昆明動(dòng)物研究所實(shí)驗(yàn)動(dòng)物中心提供, 用于神經(jīng)干細(xì)胞移植實(shí)驗(yàn)。

實(shí)驗(yàn)獼猴術(shù)前24 h禁食, 適量飲水, 常規(guī)麻醉(10 mg/kg體重鹽酸氯胺酮+20～30 mg/kg體重戊巴比妥鈉+0.3 mg/kg體重阿托品)。待動(dòng)物充分麻醉后行相關(guān)術(shù)前準(zhǔn)備, 剃毛并常規(guī)消毒頭皮后，由猴立體定位儀定位。采用30#漢密爾頓微量注射器, 將經(jīng)過(guò)bFGF/EGF擴(kuò)增后維持培養(yǎng)3代的5 μL(1×105個(gè)/ μL)神經(jīng)干細(xì)胞移植到獼猴海馬區(qū)。注射速度為0.5 μL/min, 停針1 min后, 緩慢退針。移植后每天肌注免疫抑制劑環(huán)孢霉素A(CSA)10 mg/kg。

1.5 免疫組織化學(xué)染色

貼壁培養(yǎng)的細(xì)胞用PBS洗滌2遍、4%多聚甲醛(Sigma)室溫下固定20 min、0.4% Triton X-100 (Sigma)透膜15 min、PBS洗滌2遍; 加入5%的BSA室溫下封閉1 h, 然后加入一抗4 ℃孵育過(guò)夜; 去掉一抗后, PBS沖洗3遍, 然后加入二抗室溫下孵育1 h; PBS洗滌3次后用hochest33342標(biāo)記細(xì)胞核。一抗包括：Nestin 鼠抗單克隆抗體(1:200; Chemicon)、βⅢ-tubulin 鼠抗單克隆抗體(1:200; Millipore)及神經(jīng)膠質(zhì)原纖維酸性蛋白(GFAP)鼠抗單克隆抗體(1:1 000; Chemicon)。

細(xì)胞移植2個(gè)月后, 麻醉獼猴(方法同前)。0.9%生理鹽水經(jīng)心臟灌流、4%多聚甲醛灌流固定、取腦; 4%多聚甲醛后固定3～4 d 經(jīng)15%、20%、30%梯度蔗糖脫水; LeicaCM1850冰凍切片機(jī)冰凍切片;免疫組織化學(xué)染色, 方法同細(xì)胞免疫組化染色。一抗為βⅢ-tubulin 鼠抗單克隆抗體(1:50; Millipore)

細(xì)胞免疫組化和腦片組化結(jié)果均在共聚焦顯微鏡下(Zeiss, LSM 510 META)檢測(cè)。

2 結(jié) 果

在該實(shí)驗(yàn)分化體系下, Lyon-ES在分化第12天時(shí), 95%以上的細(xì)胞為神經(jīng)干細(xì)胞, 細(xì)胞排列具有典型的玫瑰花型結(jié)構(gòu)(圖1A, B, E, F)。細(xì)胞免疫組化實(shí)驗(yàn)結(jié)果表明, 幾乎所有的細(xì)胞都呈現(xiàn)為神經(jīng)干細(xì)胞蛋白標(biāo)記物Nestin(圖1B)和Pax6(圖1F)陽(yáng)性。

分化得到的Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞經(jīng)手工挑選后貼壁培養(yǎng), 若同時(shí)添加bFGF/EGF, 可維持其Rosettes結(jié)構(gòu)2個(gè)月(圖2, 三角符號(hào)所示), 90%以上的細(xì)胞保持不分化特性(圖3);若不添加bFGF/EGF培養(yǎng), 部分細(xì)胞將開(kāi)始分化(圖4)。經(jīng)細(xì)胞組織化學(xué)實(shí)驗(yàn), Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞在分化第21天時(shí), 大量細(xì)胞表達(dá)神經(jīng)元蛋白標(biāo)記物β -tublin-Ⅲ(圖5B), 在分化第56天時(shí), 大多數(shù)細(xì)胞表達(dá)神經(jīng)膠質(zhì)細(xì)胞蛋白標(biāo)記物GFAP(圖5F)。

將經(jīng)bFGF/EGF擴(kuò)增后的Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞移植到兩只獼猴海馬后兩個(gè)月, 發(fā)現(xiàn)移植細(xì)胞能夠較好的在獼猴腦內(nèi)存活(圖4A), 且能夠表達(dá)神經(jīng)元蛋白標(biāo)記物β-tublin-Ⅲ(圖4B), 表明這些細(xì)胞已經(jīng)分化為神經(jīng)元。

3 討 論

圖1 LYON-ES細(xì)胞在神經(jīng)分化第12天分化為神經(jīng)干細(xì)胞Fig.1 LYON-ES cells differentiated into neural stem cells at the 12th day

圖2 Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞經(jīng)(RNSCs)bFGF/EGF擴(kuò)增Fig.2 Rosettes neural stem cells(RNSCs) proliferate with bFGF/EGF

圖3 手工挑選Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞經(jīng)bFGF/EGF擴(kuò)增傳代培養(yǎng)Fig.3 The Rosettes neural stem cells proliferation and differentiation with bFGF and EGF

圖4 手工挑選Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞經(jīng)擴(kuò)增傳代培養(yǎng)(無(wú)bFGF/EGF)Fig.4 The Rosettes neural stem cells proliferation and differentiation without bFGF and EGF

ESCs具有分化為身體的所有類(lèi)型細(xì)胞的能力。長(zhǎng)期以來(lái), ESCs向神經(jīng)細(xì)胞的分化一直是研究熱點(diǎn)。目前, 體外定向誘導(dǎo)ESCs向神經(jīng)分化的方法多樣, 依據(jù)分化原理可以分為以下幾種：化學(xué)物質(zhì)誘導(dǎo)法(Bain et al, 1995)、EB形成法(Reubinoff et al, 2001; Kuo et al, 2003; Itsykson et al, 2005)、共培養(yǎng)或條件培養(yǎng)基誘導(dǎo)法(Takagi et al, 2005;Yan et al, 2005)、基因修飾法和單層培養(yǎng)誘導(dǎo)分化法Nestin (Andressen et al, 2001)、Sox2(Li et al, 1998)、Nurr1(Kim et al, 2002)、bHLH(Kanda et al, 2004)等。其中EB形成法又稱譜系選擇法, 是目前胚胎干細(xì)胞向神經(jīng)細(xì)胞分化研究中最常用的方法。該分化方法具有三維結(jié)構(gòu), 可以模擬早期胚胎發(fā)育的許多特點(diǎn), 廣泛應(yīng)用在靈長(zhǎng)類(lèi)神經(jīng)分化研究中(Reubinoff Et al, 2001; Kuo et al, 2003; Itsykson et al, 2005)。本實(shí)驗(yàn)參照Pankratz et al ( 2007)建立的綜合EB法和單層法優(yōu)點(diǎn)的分化體系, 建立了穩(wěn)定高效的神經(jīng)干細(xì)胞分化體系, 該體系分化采用無(wú)血清培養(yǎng)基, 得到了Nestin和Pax6高表達(dá)的神經(jīng)干細(xì)胞。

圖5 經(jīng)bFGF/EGF擴(kuò)增后的Rosettes神經(jīng)干細(xì)胞向神經(jīng)細(xì)胞分化Fig.5 The Rosettes neural stem cells proliferate with bFGF/EGF and differentiate into neurons and glia cells

圖6 經(jīng)bFGF/EGF擴(kuò)增后的Rosettes神經(jīng)干細(xì)胞獼猴腦內(nèi)移植Fig.6 The rosettes neural stem cells proliferated by adding bFGF/EGF were transplanted into rhesus monkey brain

Rosettes結(jié)構(gòu)的NSCs(R-NSCs)具有廣泛的發(fā)育潛能。因此, 如何擴(kuò)增和維持R-NSCs是開(kāi)展神經(jīng)疾病細(xì)胞替代性治療研究的關(guān)鍵。Li et al(2008)發(fā)現(xiàn)肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor, HGF)能協(xié)同bFGF促進(jìn)獼猴胚胎干細(xì)胞來(lái)源的神經(jīng)前體細(xì)胞的增殖。雖然bFGF和EGF是常用的擴(kuò)增和維持NSCs的關(guān)鍵生長(zhǎng)因子(Murphy et al, 1990 ; Studer et al, 1998), 可有些學(xué)者認(rèn)為, 當(dāng)R-NSCs在bFGF/ EGF存在時(shí), 會(huì)引起Rosettes結(jié)構(gòu)的改變從而導(dǎo)致其向終端分化。bFGF具有誘導(dǎo)喙尾(rostro-caudal)軸廣泛類(lèi)型細(xì)胞分化的能力, 經(jīng)過(guò)bFGF長(zhǎng)期擴(kuò)增的細(xì)胞將很難分化成特定區(qū)域的神經(jīng)元(Du＆Zhang, 2004)。也有學(xué)者認(rèn)為, 經(jīng)過(guò)bFGF/EGF擴(kuò)增后依然能夠長(zhǎng)期維持其干細(xì)胞特性(Hong et al, 2008; Koch et al, 2009)。本實(shí)驗(yàn)中, 手工挑選的R-NSCs經(jīng)過(guò)bFGF/EGF擴(kuò)增傳代2個(gè)月后,仍然能夠較好的維持Rosettes結(jié)構(gòu)特性, 表明bFGF/EGF擴(kuò)增能夠較好的維持干細(xì)胞的分化特性。

當(dāng)前進(jìn)行的神經(jīng)干細(xì)胞移植實(shí)驗(yàn)研究中, 多數(shù)都通過(guò)bFGF/EGF擴(kuò)增, 移植后細(xì)胞的存活、分化及遷移效果良好(Muotri et al, 2005)。由獼猴胚胎干細(xì)胞分化得到的神經(jīng)干細(xì)胞在大鼠上異種移植也能夠存活并分化(Li et al, 2005)。本實(shí)驗(yàn)中, 經(jīng)bFGF/ EGF擴(kuò)增的獼猴Rosettes結(jié)構(gòu)神經(jīng)干細(xì)胞經(jīng)同種移植后2個(gè)月, 仍然能夠較好地存活并向神經(jīng)元分化。

總之, 通過(guò)bFGF/EGF擴(kuò)增的神經(jīng)干細(xì)胞能夠較好地維持其干細(xì)胞特性, 移植后能夠在宿主腦內(nèi)存活并向神經(jīng)細(xì)胞分化, 這為將來(lái)人類(lèi)神經(jīng)干細(xì)胞移植提供了更具參考價(jià)值的數(shù)據(jù)。

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Rhesus monkey embryonic stem cells differentiation, proliferation and allotransplantation

DONG Jin-Run1,2,3,+, GUO Li-Yun4,+, QU Jia-Gui1,2,5, QI Ren-Li1,2,5, WANG Wen-Chao1,2, XIAO Chun-Jie3,*, WANG Zheng-Bo1,2,＊

(1. State Kay Laboratory of Brain and Cognitive Science, Institute of Biophysics, the Chinese Academy of Sciences, Beijing 100101, China; 2. Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China; 3.Life Sciences Faculty of Yunnan University, Kunming 650223, China; 4.Department of Ophthalmology, the Fourth Affiliated Hospital of Kunming Medical College, Kunming 650021,China; 5School of Life Science, University of Science and Technology of China, Hefei 230026, China)

To investigate the characteristics of rhesus monkey embryonic stem cells and to promote their clinical application, the differentiation and proliferation of rosettes neural stem cells from GFP marked rhesus monkey embryonic stem cells were studied The results showed that: 1) A stable and high-efficient neural differentiation system was established. More than 95% of the embryonic stem cells were differentiated into neural stem cells on the 12thdays of differentiation; 2) the rosettes neural stem cells differentiated from the rhesus monkey embryonic stem cells could maintain their rosettes-shape by proliferating with bFGF/EGF; 3) the neural stem cells could differentiate into neurons after transplanted into the rhesus monkey brain. In conclusion, the rosettes neural stem cells differentiated from rhesus monkey embryonic stem cells could maintain their characteristics after proliferation with bFGF/EGF and they could survive and differentiate into neurons after transplanted into the rhesus monkey brain, which strongly supports the clinical application of neural stem cells in the future.

Rhesus monkey; Neural stem cells; Rosettes; Basic fibroblast growth factor;Epidermal growth factor

董錦潤(rùn), E-mail: vivid_run@126.com;郭立云,昆明醫(yī)學(xué)院在讀博士研究生, kitteyyun@yahoo.com.cn

Q959.848；Q421; Q813.7

A

0254-5853-(2012)01-0043-06

book=48,ebook=388

10.3724/SP.J.1141.2012.01043

2011-12-01;接受日期：2011-12-22

；“973”項(xiàng)目(947703)；國(guó)家自然科學(xué)基金項(xiàng)目(31070963, 30670669)

?通信作者(Corresponding authors), E-mail: wangzb@mail.kiz.ac.cn

＋并列第一作者(Authors contributed equally to the work)