不同濃度dbcAMP對(duì)SH-SY5Y細(xì)胞向γ-氨基丁酸能神經(jīng)元分化潛能的影響

王登莉,周 莉,于 升,吳 江,趙 慧

(1.吉林大學(xué)基礎(chǔ)醫(yī)學(xué)院組織學(xué)與胚胎學(xué)系,吉林長(zhǎng)春 130021;2.吉林大學(xué)第一醫(yī)院神經(jīng)內(nèi)科,吉林長(zhǎng)春 130021)

不同濃度dbcAMP對(duì)SH-SY5Y細(xì)胞向γ-氨基丁酸能神經(jīng)元分化潛能的影響

王登莉1,周 莉1,于 升1,吳 江2,趙 慧1

(1.吉林大學(xué)基礎(chǔ)醫(yī)學(xué)院組織學(xué)與胚胎學(xué)系,吉林長(zhǎng)春 130021;2.吉林大學(xué)第一醫(yī)院神經(jīng)內(nèi)科,吉林長(zhǎng)春 130021)

目的:建立具有分化成γ-氨基丁酸能神經(jīng)元潛能的神經(jīng)干細(xì)胞模型,為γ-氨基丁酸能神經(jīng)元退行性疾病的研究提供適宜的研究載體。方法:應(yīng)用雙丁酰環(huán)腺苷酸(dbc AMP)誘導(dǎo)人神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y細(xì)胞),分為0 mmol·L－1dbc AMP組(對(duì)照組)和0.3、0.6、1.0及2.0 dbc AMP組,觀察誘導(dǎo)后各組SH-SY5Y細(xì)胞的形態(tài)變化;采用Imge-Pro Plus 5.0軟件測(cè)量神經(jīng)元樣細(xì)胞神經(jīng)突起長(zhǎng)度,計(jì)算神經(jīng)突起＞30μm細(xì)胞所占細(xì)胞總數(shù)的百分率;采用免疫熒光細(xì)胞化學(xué)技術(shù)檢測(cè)γ-氨基丁酸能神經(jīng)元標(biāo)志性蛋白——谷氨酸脫羧酶65 (GAD65)的表達(dá),并計(jì)算其免疫反應(yīng)陽(yáng)性率。結(jié)果:形態(tài)學(xué)觀察,對(duì)照組SH-SY5Y細(xì)胞呈多邊形、圓形或梭型,胞膜光滑,邊界清晰;各濃度dbc AMP組隨著dbc AMP濃度的增加和誘導(dǎo)時(shí)間的延長(zhǎng),SH-SY5Y細(xì)胞胞體變小、突起變長(zhǎng);1.0 mmol·L－1dbc AMP組細(xì)胞互相交織,表現(xiàn)出成熟神經(jīng)元的表型。SH-SY5Y細(xì)胞誘導(dǎo)培養(yǎng)72 h后,與對(duì)照組(31.4%±4.2%)比較,0.3、0.6、1.0和2.0 dbc AMP組神經(jīng)突起＞30μm細(xì)胞所占細(xì)胞總數(shù)的百分率(40.1%±5.7%、47.5%±6.2%、73.1%±3.2%和74.3%±6.1%)明顯升高(P＜0.05或P＜0.01)。SH-SY5Y細(xì)胞誘導(dǎo)培養(yǎng)72 h后,與對(duì)照組(10.2%±2.1%)比較,0.3、0.6、1.0和2.0 dbc AMP組GAD65陽(yáng)性細(xì)胞表達(dá)率(22.1%±2.4%、46.9%±3.2%、70.7%±3.4%和72.3%±3.7%)明顯升高(P＜0.05 或P＜0.01)。結(jié)論:SH-SY5Y細(xì)胞具有分化為γ-氨基丁酸能神經(jīng)元樣細(xì)胞的潛能,1.0 mmol·L－1是dbc AMP的最佳誘導(dǎo)濃度。

雙丁酰環(huán)磷腺苷;人神經(jīng)母細(xì)胞瘤細(xì)胞;誘導(dǎo);分化

1 材料與方法

1.1 細(xì)胞、主要試劑和儀器SH-SY5Y細(xì)胞購(gòu)自美國(guó)典型培養(yǎng)物細(xì)胞庫(kù)(American Type Culture Collection,ATCC)。小鼠抗人谷氨酸脫羧酶65 (GAD65)單克隆抗體購(gòu)自Santa Cruz公司,驢抗小鼠Alexa Fluor 488和DAPI購(gòu)自美國(guó)Invitrogen公司,dbc AMP購(gòu)自美國(guó)Sigma公司,低糖DMEM培養(yǎng)基購(gòu)自美國(guó)Gibco公司,胎牛血清(fetal bovine serum,FBS)購(gòu)自杭州四季青生物工程公司,胰蛋白酶購(gòu)自北京鼎國(guó)生物技術(shù)有限責(zé)任公司。熒光倒置相差顯微鏡為日本Olympus公司產(chǎn)品(IX71),CO2培養(yǎng)箱為日本Sanyo公司產(chǎn)品(MCO-175),超凈工作臺(tái)為蘇凈集團(tuán)安泰公司產(chǎn)品(SW-CJ-1F)。

1.2 細(xì)胞傳代培養(yǎng)SH-SY5Y細(xì)胞采用含10% FBS、60 mg·L－1青霉素及100 mg·L－1鏈霉素的低糖DMEM培養(yǎng)液,在5%CO2、37℃飽和濕度孵箱中培養(yǎng)。待細(xì)胞長(zhǎng)到90%融合時(shí),用0.25%的胰蛋白酶消化,以1∶3或1∶4傳代,取對(duì)數(shù)生長(zhǎng)期細(xì)胞用于實(shí)驗(yàn)。

1.3 細(xì)胞分組和誘導(dǎo)將SH-SY5Y細(xì)胞按1× 104/孔的密度接種于放置了蓋玻片的24孔培養(yǎng)板中。為了減少FBS對(duì)dbc AMP誘導(dǎo)分化作用的干擾,在誘導(dǎo)實(shí)驗(yàn)中將培養(yǎng)基中FBS的濃度降低為0.5%。細(xì)胞共分為5組,每組設(shè)3個(gè)復(fù)孔,均用含0.5%FBS的低糖DMEM培養(yǎng)基培養(yǎng),包括0 mmol·L－1dbc AMP組(對(duì)照組)和0.3、0.6、1.0及2.0 mmol·L－1dbc AMP組,將24孔培養(yǎng)板置于37℃、體積分?jǐn)?shù)為5%CO2培養(yǎng)箱中培養(yǎng),每天于倒置相差顯微鏡下觀察細(xì)胞的生長(zhǎng)情況并拍照。dbc AMP誘導(dǎo)72 h后,取出細(xì)胞爬片進(jìn)行免疫熒光細(xì)胞化學(xué)染色。

1.4 神經(jīng)細(xì)胞突起長(zhǎng)度測(cè)量神經(jīng)元樣細(xì)胞神經(jīng)突起長(zhǎng)度的測(cè)量方法參照文獻(xiàn)[7-8]。以細(xì)胞核中心為測(cè)量起點(diǎn),突起末端為終點(diǎn),使用Image-Pro Plus 6.0軟件測(cè)量神經(jīng)突長(zhǎng)度,長(zhǎng)度＜30μm的突起忽略不計(jì)。根據(jù)各視野下標(biāo)尺的測(cè)量長(zhǎng)度,計(jì)算各神經(jīng)突起的實(shí)際長(zhǎng)度,并計(jì)算出各組細(xì)胞神經(jīng)突起的平均長(zhǎng)度。

1.5 免疫熒光細(xì)胞化學(xué)染色和免疫陽(yáng)性細(xì)胞計(jì)數(shù)將各組細(xì)胞用冷丙酮固定后,0.01 mol·L－1PBS(p H 7.2)沖洗細(xì)胞;5%驢血清室溫封閉30 min,滴加小鼠抗人GAD65單克隆抗體(效價(jià)1∶25)于4℃孵育過(guò)夜(陰性對(duì)照組用PBS代替GAD65一抗);0.01 mol·L－1PBS沖洗后,避光滴加熒光標(biāo)記的驢抗小鼠Alexa Fluor 488抗體(效價(jià)1∶500),于37℃孵育50 min;0.01 mol·L－1PBS沖洗,DAPI(工作濃度為1 mg·L－1)滴染細(xì)胞核5 min,0.01 mol·L－1PBS沖洗;甘油磷酸緩沖液封片后熒光顯微鏡觀察、拍照并計(jì)數(shù)。20倍物鏡下,每個(gè)樣品隨機(jī)選取10個(gè)視野,分別計(jì)算每個(gè)視野下的GAD65免疫反應(yīng)陽(yáng)性細(xì)胞數(shù)。

1.6 統(tǒng)計(jì)學(xué)分析應(yīng)用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)處理。各組細(xì)胞神經(jīng)突起的長(zhǎng)度和神經(jīng)突起＞30μm細(xì)胞數(shù)的百分率以±s表示,組間比較采用t檢驗(yàn)。

2 結(jié)果

2.1 dbc AMP誘導(dǎo)后SH-SY5Y細(xì)胞形態(tài)學(xué)對(duì)照組SH-SY5Y細(xì)胞培養(yǎng)48或72 h時(shí)細(xì)胞呈多邊形、圓形或梭型,胞膜光滑,邊界清晰。各濃度dbc AMP組細(xì)胞培養(yǎng)24 h時(shí)可見細(xì)胞形態(tài)發(fā)生改變,細(xì)胞兩端開始伸出短小的樹突樣突起。48 h 時(shí)0.3 mmol·L－1dbc AMP組只有少數(shù)短突起; 0.6 mmol·L－1dbc AMP組突起增多但突起較短,突起交織現(xiàn)象少;1.0 mmol·L－1dbc AMP組細(xì)胞兩端出現(xiàn)長(zhǎng)而細(xì)的軸突樣突起,并相互交織連接。隨著dbc AMP誘導(dǎo)時(shí)間的延長(zhǎng),各濃度dbc AMP組細(xì)胞形態(tài)變化均很明顯,誘導(dǎo)72 h時(shí)1.0 mmol·L－1dbc AMP組越來(lái)越多的細(xì)胞突起變長(zhǎng),細(xì)胞相互交織現(xiàn)象更明顯,表現(xiàn)出成熟神經(jīng)元的表型,且細(xì)胞的平均神經(jīng)突起長(zhǎng)度也隨著dbc AMP濃度的增加而變長(zhǎng)。見圖1。

在誘導(dǎo)72 h時(shí),1.0 mmol·L－1dbc AMP組突起＞30μm的細(xì)胞數(shù)占細(xì)胞總數(shù)的(73.1± 3.2)%,與對(duì)照組[(31.4±4.2)%]比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.01);神經(jīng)突起平均長(zhǎng)度為(98.37±4.57)μm,與對(duì)照組[(41.77±4.58)μm]比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)(表1)。2.0 mmol·L－1dbc AMP組神經(jīng)突起平均長(zhǎng)度增加,達(dá)到(112.86±5.25)μm,突起＞30μm的細(xì)胞所占比例為(74.3±6.1)%,與1.0 mmol·L－1dbc AMP組比較無(wú)明顯變化(P＞0.05)(表2),并且同一放大倍數(shù)視野下細(xì)胞總數(shù)減少。group;E,F:0.6 mmol·L－1dbc AMP group;G,H:1.0 mmol·L－1dbc AMP group;I,J:2.0 mmol·L－1dbc AMP group.“→”:Long neurites after dbc AMP induction;Bar＝50μm.

圖1 各組SH-SY5Y細(xì)胞形態(tài)學(xué)Fig.1 Morphology of SH-SY5Y cells in various groupsA,C,E,G,I:48 h after dbc AMP treatment;B,D,F,H,J:72 h after dbc AMP treatment;A,B:Control group;C,D:0.3 mmol·L－1dbc AMP

表1 各組SH-SY5Y細(xì)胞神經(jīng)突起的平均長(zhǎng)度Tab.1 Average lengths of neurites of SH-SY5Y cells in various groups(n＝3,±s,l/μm)

表1 各組SH-SY5Y細(xì)胞神經(jīng)突起的平均長(zhǎng)度Tab.1 Average lengths of neurites of SH-SY5Y cells in various groups(n＝3,±s,l/μm)

?P＜0.05,??P＜0.01 vs control group.

Group Average length of neurite (t/h) 48 72 Control 41.43±4.66 41.77±4.58 dbc AMP(mmol·L－1) 0.3 53.61±6.91?70.11±6.26?0.6 70.17±7.25?80.61±7.10?1.0 84.02±7.83??98.37±7.83??2.0 104.53±8.22??112.86±8.50??

表2 各組神經(jīng)突起＞30μm細(xì)胞占總細(xì)胞數(shù)的百分率Tab.2 Percentages of cells with neurites longer than 30μm in various groups(n＝3,±s,η/%)

表2 各組神經(jīng)突起＞30μm細(xì)胞占總細(xì)胞數(shù)的百分率Tab.2 Percentages of cells with neurites longer than 30μm in various groups(n＝3,±s,η/%)

?P＜0.05,??P＜0.01 vs control group.

Group Percentage of cells with neurites＞30μm (t/h) 48 72 Control 10.32±3.56 31.46±4.22 dbc AMP(mmol·L－1) 0.3 37.23±4.79?40.17±5.77?0.6 42.72±4.44?45.53±6.23?1.0 62.46±3.44??73.16±3.22??2.0 72.64±5.77??74.33±6.11??

2.2 免疫熒光細(xì)胞化學(xué)染色及免疫陽(yáng)性細(xì)胞計(jì)數(shù)

通過(guò)免疫熒光細(xì)胞化學(xué)染色鑒定γ-氨基丁酸能神經(jīng)元的標(biāo)志物GAD65的表達(dá),陽(yáng)性產(chǎn)物被熒光標(biāo)記的抗體染成綠色,分布在細(xì)胞膜上,而陰性對(duì)照(即2.0 mmol·L－1dbc AMP組染色過(guò)程中未加GAD65一抗)僅有細(xì)胞核被DAPI染成藍(lán)色,細(xì)胞膜上無(wú)特異性染色。0.3 mmol·L－1dbc AMP 組GAD65陽(yáng)性細(xì)胞數(shù)量少,陽(yáng)性率為(22.1± 2.40)%;0.6 mmol·L－1dbc AMP組GAD65陽(yáng)性細(xì)胞數(shù)增多,陽(yáng)性率達(dá)到(46.9±3.2)%; 1.0 mmol·L－1dbc AMP組陽(yáng)性細(xì)胞率高達(dá)(70.7±3.4)%,與對(duì)照組[(10.2±2.1)%]比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.01);2.0 mmol·L－1dbc AMP組GAD65陽(yáng)性細(xì)胞率為(72.3± 3.7)%,與1.0 mmol·L－1dbc AMP組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。見圖2(插頁(yè)一)。

3 討論

γ-氨基丁酸能神經(jīng)元是人類大腦發(fā)生過(guò)程中基本神經(jīng)元類型,包括抑制性神經(jīng)元和興奮性神經(jīng)元,前者屬中間神經(jīng)元,釋放抑制性神經(jīng)遞質(zhì)γ-氨基丁酸,參與形成局部抑制回路[9]。在胚胎神經(jīng)干細(xì)胞和成體神經(jīng)干細(xì)胞體外培養(yǎng)時(shí),如果只加入血清不加入任何其他誘導(dǎo)因素,神經(jīng)干細(xì)胞分化的神經(jīng)元均為γ-氨基丁酸能神經(jīng)元,該現(xiàn)象提示γ-氨基丁酸能神經(jīng)元是神經(jīng)干細(xì)胞自然分化的產(chǎn)物。本實(shí)驗(yàn)選擇的分化誘導(dǎo)劑為細(xì)胞本身就存在的第二信使c AMP的衍生物dbc AMP,檢測(cè)細(xì)胞分化的標(biāo)志性蛋白為γ-氨基丁酸能神經(jīng)元產(chǎn)生γ-氨基丁酸的限速酶GAD65,以此探討SH-SY5Y細(xì)胞系是否具有神經(jīng)干細(xì)胞特性和能否成為人神經(jīng)干細(xì)胞模型。

由于SH-SY5Y細(xì)胞能夠在體外長(zhǎng)期培養(yǎng),通過(guò)誘導(dǎo)分化可使細(xì)胞形態(tài)接近成熟神經(jīng)元,在實(shí)驗(yàn)中提供穩(wěn)定的細(xì)胞數(shù),具有許多神經(jīng)元的生化和生理特征而被作為實(shí)驗(yàn)性神經(jīng)科學(xué)研究的神經(jīng)元細(xì)胞模型[10]。SH-SY5Y細(xì)胞形態(tài)學(xué)改變是其細(xì)胞分化的表性特征,形態(tài)學(xué)的成熟程度是判斷分化誘導(dǎo)成功與否的重要標(biāo)志。細(xì)胞形態(tài)分化成熟的標(biāo)準(zhǔn)為有1個(gè)或1個(gè)以上的突起長(zhǎng)度延伸至胞體直徑2倍以上[11];神經(jīng)元細(xì)胞的神經(jīng)突起長(zhǎng)度的測(cè)量方法參照Neumann等[7]和Li等[8]的報(bào)道,長(zhǎng)度＜30μm的突起忽略不計(jì)。本研究結(jié)合這2種標(biāo)準(zhǔn),較為準(zhǔn)確地對(duì)分化后的細(xì)胞進(jìn)行了系統(tǒng)的測(cè)量。本實(shí)驗(yàn)結(jié)果表明:一定濃度dbc AMP可誘導(dǎo)SH-SY5Y細(xì)胞形態(tài)發(fā)生明顯的改變,隨著誘導(dǎo)劑濃度的逐漸增加,SH-SY5Y細(xì)胞從最初的多邊形、圓形或梭型逐漸演變?yōu)橛休^長(zhǎng)細(xì)胞突起的形態(tài),這些突起逐漸伸長(zhǎng)并相互交織成網(wǎng),最長(zhǎng)的突起可達(dá)120μm,約為細(xì)胞體直徑的6倍。Dodurga等[12]發(fā)現(xiàn): SH-SY5Y細(xì)胞在10 mmol·L－1RA誘導(dǎo)第3天出現(xiàn)神經(jīng)突起,并隨著誘導(dǎo)時(shí)間的延長(zhǎng),神經(jīng)突起隨之變長(zhǎng),呈現(xiàn)出神經(jīng)元表型。上述結(jié)果表明SH-SY5Y細(xì)胞在一定誘導(dǎo)條件下具有分化為神經(jīng)元樣細(xì)胞的潛能。

誘導(dǎo)劑的種類、濃度及作用時(shí)間是成功誘導(dǎo)分化干細(xì)胞的重要因素。c AMP是細(xì)胞內(nèi)第2信使,其不僅在調(diào)節(jié)新陳代謝過(guò)程中起到很重要的作用,而且能夠改變細(xì)胞形態(tài),影響細(xì)胞分化。體外實(shí)驗(yàn)中,c AMP衍生物dbc AMP可以滲透到細(xì)胞內(nèi),提高細(xì)胞內(nèi)c AMP水平,從而模擬體內(nèi)某種刺激,所以本實(shí)驗(yàn)選擇dbc AMP為SH-SY5Y細(xì)胞分化的誘導(dǎo)劑[13]。本研究結(jié)果顯示:經(jīng)不同濃度dbc AMP處理后,光學(xué)顯微鏡下可見SH-5Y5Y細(xì)胞形態(tài)發(fā)生顯著變化,細(xì)胞喪失了未分化的腫瘤細(xì)胞形態(tài);當(dāng)dbc AMP濃度為1.0 mmol·L－1時(shí),細(xì)胞胞體明顯變小,而且伸出長(zhǎng)突起,具有類似神經(jīng)元的形態(tài);而0.3和0.6 mmol·L－1dbc AMP作用下胞體未見明顯變化。

2.0 mmol·L－1dbc AMP誘導(dǎo)下,SH-SY5Y細(xì)胞神經(jīng)突起長(zhǎng)度及GAD65陽(yáng)性細(xì)胞率與1.0 mmol·L－1dbc AMP組比較無(wú)明顯改變,并且細(xì)胞總數(shù)減少,說(shuō)明高濃度dbc AMP對(duì)細(xì)胞生長(zhǎng)有毒副作用。因此本研究最終確定1.0 mmol·L－1是dbc AMP的最佳誘導(dǎo)濃度。

γ-氨基丁酸能神經(jīng)元標(biāo)志性蛋白GAD有2種同工酶,根據(jù)其相對(duì)分子質(zhì)量的不同分為GAD65 和GAD67。在神經(jīng)系統(tǒng)中,GAD65和GAD67通常存在于相同的γ-氨基丁酸能神經(jīng)元內(nèi),但分別由不同的基因編碼,在含量和亞細(xì)胞分布上存在差異[14]。本研究采用GAD65標(biāo)記γ-氨基丁酸能神經(jīng)元,通過(guò)免疫熒光細(xì)胞化學(xué)染色,熒光顯微鏡下可見GAD65表達(dá)部位位于細(xì)胞膜上。

綜上所述,SH-SY5Y細(xì)胞能夠成為人神經(jīng)干細(xì)胞模型,其具有神經(jīng)干細(xì)胞特性,在一定條件下可分化為γ-氨基丁酸能神經(jīng)元。本實(shí)驗(yàn)結(jié)果為探討胚胎神經(jīng)干細(xì)胞定向分化的分子機(jī)制提供了適宜的研究載體。

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Influence of different concentrations of dbc AMP in differentiation potentiality of SH-SY5Y cells to GABAergic-like cells

WANG Deng-li1,ZHOU Li1,YU Sheng1,WU Jiang2,ZHAO Hui1
(1.Department of Histology and Embryology,School of Basic Medical Sciences,Jilin University,Changchun 130021,China;2.Department of Neurology,First Hospital,Jilin University,Changchun 130021,China)

ObjectiveTo establish a nerve stem cell model possessing the potentiality of differentiating into γ-aminobutyric acid neuron-like cells(GABAergic-like cells),and provide eligible investigative vector for study on GABAergic neurons degenerative diseases.Methods Dibutyryl cyclic adenosine monophosphate(dbc AMP)was applied to induce human neuroblastoma SH-SY5Y cells,and the SH-SY5Y cells were divided into control group (0 mmol·L－1dbc AMP),0.3 mmol·L－1dbc AMP group,0.6 mmol·L－1dbc AMP group,1.0 mmol·L－1dbc AMP group and 2.0 mmol·L－1dbc AMP group;the morphological changes of SH-SY5Y cells were observed.The Image-Pro Plus 5.0 software was used to measure the length of neurites of the neuron-like cells,and the percentage of the SH-SY5Y cells with neurites longer than 30μm was calculated.The immunofluorescence cytochemistry technique was utilized to test GAD65 positive cells,and the positive rate of immune response was calculated.ResultsThe results of light microscope observation showed that the cells in control group were polygonal,circular or shuttle type with smooth membrane and clear boundaries.With the increasing of the concentrations of dbc AMP and the prolongation of time,the morphology of SH-SY5Y cells’bodies became smaller with longer processes in dbc AMP groups.The cells interwined each other and showed mature neuron phenotype in 1.0 mmol·L－1dbc AMP group.Compared with control group(31.4%±4.2%),the percentages of the cell with neurites longer than 30μm in 0.3,0.6,1.0,and 2.0 dbc AMP groups(40.1%±5.7%,47.5%±6.2%, 73.1%±3.2%,and 74.3%±6.1%)72 h after induction were significantly increased(P＜0.05 or P＜0.01).Compared with control group(10.2%±2.1%),the GAD65 positive expression rates in 0.3,0.6,1.0,and 2.0 mmol·L－1,dbc AMP groups(22.1%±2.4%,46.9%±3.2%,70.7%±3.4%,and 72.3%±3.7%)72 h after induction were significantly increased(P＜0.05 or P＜0.01).ConclusionThe SH-SY5Y cells have the potentiality of differentiating into GABAergic-like cells,and 1.0 mmol·L－1is the optimal concentration of dbc AMP.

dibutyryl cyclic adenosine monophosphate;human neuroblastoma cells;induction;differentiation神經(jīng)干細(xì)胞研究多使用小鼠神經(jīng)母細(xì)胞瘤(N2A細(xì)胞株)作為細(xì)胞模型[1],但在研究人胚胎神經(jīng)干細(xì)胞時(shí),N2A細(xì)胞株有一定的局限性,因?yàn)樾∈筮z傳學(xué)特征與人類相比有一定差異,尤其是在基因重組研究中不能互為替代,因此尋找一個(gè)有神經(jīng)干細(xì)胞特征、適合于研究人胚胎神經(jīng)干細(xì)胞定向分化的細(xì)胞模型尤為重要。人神經(jīng)母細(xì)胞瘤細(xì)胞系SH-SY5Y來(lái)源于不成熟的腫瘤性神經(jīng)嵴細(xì)胞,具有干細(xì)胞特性,是研究神經(jīng)細(xì)胞終末分化的重要細(xì)胞模型。雙丁酰環(huán)磷腺苷(dibutyryl cyclic adenosine monophosphate,dbc AMP)[2]、腦源性神經(jīng)營(yíng)養(yǎng)因子(brain-derived neurotrophic factor, BDNF)[3]、佛波醇酯(tetradecanoyl phorbolacetate,TPA)[4]和維甲酸(retinoic acid, RA)[5]均能誘導(dǎo)SH-SY5Y細(xì)胞分化。在RA和BDNF共存的情況下,SH-SY5Y細(xì)胞具有向膽堿能神經(jīng)元分化的表型[6]。但SH-SY5Y細(xì)胞能否分化為γ-氨基丁酸能神經(jīng)元類型,目前尚未見報(bào)道。本研究采用不同濃度dbc AMP誘導(dǎo)SH-SY5Y細(xì)胞,應(yīng)用免疫熒光細(xì)胞化學(xué)技術(shù)檢測(cè)SH-SY5Y細(xì)胞向γ-氨基丁酸能神經(jīng)元分化的潛能,為建立一種適合于研究人類神經(jīng)干細(xì)胞定向分化分子機(jī)制的細(xì)胞模型提供實(shí)驗(yàn)依據(jù)。

R741

A

2013-11-22

國(guó)家自然科學(xué)基金資助課題(81171219);吉林大學(xué)211工程項(xiàng)目資助課題(2010)

王登莉(1987－),女,江蘇省南通市人,在讀醫(yī)學(xué)碩士,主要從事神經(jīng)干細(xì)胞研究。

趙 慧(Tel:0431-85619477,E-mail:zhaohui＠jlu.edu.cn)

1671-587Ⅹ(2014)05-0933-05

10.13481/j.1671-587x.20140505