董錦潤, 郭立云, 瞿家桂, 戚仁莉, 王文超, 肖春杰, 王正波,*

(1. 中國科學(xué)院生物物理研究所 腦與認(rèn)知國家重點實驗室, 北京 100101; 2. 中國科學(xué)院昆明動物研究所, 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)干細胞的誘導(dǎo)分化、干性維持及同種腦內(nèi)移植

董錦潤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. 中國科學(xué)院生物物理研究所 腦與認(rèn)知國家重點實驗室, 北京 100101; 2. 中國科學(xué)院昆明動物研究所, 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)干細胞分化及特性維持, 推進神經(jīng)干細胞臨床應(yīng)用研究, 該實驗以綠色熒光蛋白(green fluorescence protein, GFP)為標(biāo)記探討獼猴胚胎干細胞向玫瑰花環(huán)(rosettes)結(jié)構(gòu)神經(jīng)干細胞的分化及其堿性成纖維細胞生長因子(basic fibroblast growth factor, bFGF)和表皮生長因子(epidermal growth factor, EGF)的擴增培養(yǎng)。結(jié)果表明：1)建立了穩(wěn)定高效的獼猴神經(jīng)干細胞分化體系, 在該分化體系下, GFP標(biāo)記獼猴胚胎干細胞在分化的第12天時, 95%以上的細胞分化為神經(jīng)干細胞; 2)分化得到的Rosettes結(jié)構(gòu)神經(jīng)干細胞經(jīng)bFGF/EGF擴增后, 能夠較好地維持其Rosettes結(jié)構(gòu); 3)經(jīng)bFGF/EGF擴增后的rosettes結(jié)構(gòu)神經(jīng)干細胞移植到獼猴腦內(nèi)后能夠較好的存活并向神經(jīng)元分化，即bFGF/EGF擴增培養(yǎng)能較好地維持Rosettes結(jié)構(gòu)的神經(jīng)干細胞, 且移植到獼猴腦內(nèi)的該細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)干細胞；玫瑰花環(huán)結(jié)構(gòu)；堿性成纖維細胞生長因子；表皮生長因子

由于神經(jīng)干細胞具有自我更新、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)干細胞分化技術(shù)的完善和人工誘導(dǎo)多潛能干細胞(induced pluripotent stem cells, IPS)方法的出現(xiàn)(Okita et al, 2007), 使細胞替代治療神經(jīng)系統(tǒng)疾病成為了研究熱點。

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

堿性成纖維細胞生長因子(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, 它能通過作用于細胞表面相應(yīng)的受體促進神經(jīng)干細胞的增殖和分化。表皮生長因子(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進行擴增培養(yǎng)神經(jīng)干細胞(Murphy et al, 1990; Studer et al, 1998), 而bFGF/EGF擴增是否影響干細胞特性存在爭議(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)于嚙齒類且與人類更為接近(Wolf et al, 2004), 所以，獼猴胚胎干細胞的神經(jīng)分化可為探索人類胚胎發(fā)育過程中的神經(jīng)發(fā)生提供參考, 同時可用于神經(jīng)疾病藥物篩選以及開展神經(jīng)干細胞臨床治療的相關(guān)研究。Li et al (2005)將由獼猴胚胎干細胞分化得到的神經(jīng)干細胞移植至大鼠腦內(nèi)的研究，由于異種移植而存在種屬差異性。因此, 本研究擬通過建立獼猴神經(jīng)干細胞誘導(dǎo)分化體系, 探討bFGF/EGF擴增能否維持神經(jīng)干細胞Rosettes結(jié)構(gòu)特性, 并在同物種獼猴上進行移植實驗, 為神經(jīng)干細胞移植更好的應(yīng)用于臨床提供基礎(chǔ)理論依據(jù)。

1 材料及方法

1.1 獼猴胚胎干細胞培養(yǎng)

tauGFP-rESCs細胞系LYON-ES野生型(LYONES-WT)(Wianny et al, 2008)(法國里昂干細胞與腦科學(xué)研究所饋贈, 試驗所用細胞傳代至第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, 購自ATCC)上, 其培養(yǎng)方法參照文獻 Wianny et al (2008)的報道進行。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)記獼猴胚胎干細胞向神經(jīng)干細胞分化

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。將胚胎干細胞克隆通過手工挑選, 切割成1 000個細胞左右的細胞團塊, 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)干細胞擴增

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

1.4 神經(jīng)干細胞的獼猴移植

兩只成年(5 a)肢體殘疾獼猴(由斗毆引起)由中國科學(xué)院昆明動物研究所實驗動物中心提供, 用于神經(jīng)干細胞移植實驗。

實驗獼猴術(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bFGF/EGF擴增后維持培養(yǎng)3代的5 μL(1×105個/ μL)神經(jīng)干細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)。

細胞移植2個月后, 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冰凍切片機冰凍切片;免疫組織化學(xué)染色, 方法同細胞免疫組化染色。一抗為βⅢ-tubulin 鼠抗單克隆抗體(1:50; Millipore)

細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。

2 結(jié) 果

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

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

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

3 討 論

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

圖2 Rosettes結(jié)構(gòu)神經(jīng)干細胞經(jīng)(RNSCs)bFGF/EGF擴增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)干細胞經(jīng)bFGF/EGF擴增傳代培養(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)干細胞經(jīng)擴增傳代培養(yǎng)(無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型細胞的能力。長期以來, ESCs向神經(jīng)細胞的分化一直是研究熱點。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, 是目前胚胎干細胞向神經(jīng)細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, 廣泛應(yīng)用在靈長類神經(jīng)分化研究中(Reubinoff Et al, 2001; Kuo et al, 2003; Itsykson et al, 2005)。本實驗參照Pankratz et al ( 2007)建立的綜合EB法和單層法優(yōu)點的分化體系, 建立了穩(wěn)定高效的神經(jīng)干細胞分化體系, 該體系分化采用無血清培養(yǎng)基, 得到了Nestin和Pax6高表達的神經(jīng)干細胞。

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

圖6 經(jīng)bFGF/EGF擴增后的Rosettes神經(jīng)干細胞獼猴腦內(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, 如何擴增和維持R-NSCs是開展神經(jīng)疾病細胞替代性治療研究的關(guān)鍵。Li et al(2008)發(fā)現(xiàn)肝細胞生長因子(hepatocyte growth factor, HGF)能協(xié)同bFGF促進獼猴胚胎干細胞來源的神經(jīng)前體細胞的增殖。雖然bFGF和EGF是常用的擴增和維持NSCs的關(guān)鍵生長因子(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, 會引起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)軸廣泛類型細胞分化的能力, 經(jīng)過bFGF長期擴增的細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bFGF/EGF擴增后依然能夠長期維持其干細胞特性(Hong et al, 2008; Koch et al, 2009)。本實驗中, 手工挑選的R-NSCs經(jīng)過bFGF/EGF擴增傳代2個月后,仍然能夠較好的維持Rosettes結(jié)構(gòu)特性, 表明bFGF/EGF擴增能夠較好的維持干細胞的分化特性。

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

總之, 通過bFGF/EGF擴增的神經(jīng)干細胞能夠較好地維持其干細胞特性, 移植后能夠在宿主腦內(nèi)存活并向神經(jīng)細胞分化, 這為將來人類神經(jīng)干細胞移植提供了更具參考價值的數(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, 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”項目(947703)；國家自然科學(xué)基金項目(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)