劉彩玉 王春芳＊ 李鵬飛 蔚洪恩 龍志晶 鄧春圣

脊髓源神經(jīng)干細(xì)胞誘導(dǎo)分化為膽堿能神經(jīng)元過程中Aldoc和Stmn1表達(dá)的研究

劉彩玉1王春芳1＊李鵬飛1蔚洪恩1龍志晶1鄧春圣2＊

(1山西醫(yī)科大學(xué)生物技術(shù)教研室,太原030001;2中國人民解放軍第322醫(yī)院,山西大同037006)

目的 探討脊髓源神經(jīng)干細(xì)胞在誘導(dǎo)分化為膽堿能神經(jīng)元過程中Aldoc和Stmn1的表達(dá)變化情況。方法 從孕17天Wistar胚胎大鼠取出脊髓組織,制成細(xì)胞懸液,采用含EGF和bFGF的無血清限定性培養(yǎng)基培養(yǎng),然后進(jìn)行誘導(dǎo)分化,觀察脊髓源神經(jīng)干細(xì)胞向膽堿能神經(jīng)元分化的情況,應(yīng)用熒光定量PCR方法分析Aldoc和Stmn1基因在脊髓源神經(jīng)干細(xì)胞誘導(dǎo)分化為膽堿能神經(jīng)元的過程中的表達(dá)變化情況。結(jié)果 神經(jīng)干細(xì)胞在誘導(dǎo)分化為膽堿能神經(jīng)元后,經(jīng)免疫熒光檢測有ChAT陽性細(xì)胞表達(dá);Aldoc基因的表達(dá)量在膽堿能神經(jīng)元較神經(jīng)干細(xì)胞低(P<0.05);Stmn1基因的表達(dá)量則在誘導(dǎo)分化后較神經(jīng)干細(xì)胞升高(P<0.05)。結(jié)論 Aldoc對(duì)神經(jīng)干細(xì)胞的干性維持有重要作用,Stmn1在膽堿能神經(jīng)元的成熟過程中起作用。

神經(jīng)干細(xì)胞; 膽堿能神經(jīng)元; Aldoc基因; Stmn1基因; 基因表達(dá)

神經(jīng)干細(xì)胞(neural stem cell,NSCs)是一類具有分裂潛能和自我更新能力的母細(xì)胞,它可以通過不對(duì)等的分裂方式產(chǎn)生神經(jīng)組織的各類細(xì)胞,且在體外自然分化的條件下神經(jīng)干細(xì)胞也具有多向分化潛能,可分化為神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞。近年來隨著神經(jīng)干細(xì)胞的發(fā)現(xiàn)和逐步了解,人們開始希望能通過神經(jīng)干細(xì)胞來治療脊髓損傷,其定向分化性,為修復(fù)和替代死亡的神經(jīng)細(xì)胞提供了可能。報(bào)道稱,在脊髓多層面NSC的移植可作為運(yùn)動(dòng)神經(jīng)元疾病將來的細(xì)胞治療策略[1],Wichterle等模擬體內(nèi)運(yùn)動(dòng)神經(jīng)元分化的途徑,在體外用 RA、SHH誘導(dǎo)鼠胚胎干細(xì)胞先向脊髓祖細(xì)胞分化,進(jìn)而誘導(dǎo)脊髓祖細(xì)胞向運(yùn)動(dòng)神經(jīng)元分化,最終有20%-30%的細(xì)胞表達(dá)運(yùn)動(dòng)神經(jīng)元的標(biāo)記物 Hb9[2]。運(yùn)動(dòng)神經(jīng)元是膽堿能神經(jīng)元的一種,膽堿能神經(jīng)元是神經(jīng)系統(tǒng)中分泌乙酰膽堿的神經(jīng)元的通稱,本課題組在近來研究中發(fā)現(xiàn),通過RA和SHH的聯(lián)合使用,誘導(dǎo)脊髓源性神經(jīng)干細(xì)胞分化,所得細(xì)胞可表達(dá)ChA T[3](ChA T是膽堿能神經(jīng)元的重要標(biāo)記之一),雖然已經(jīng)證實(shí),神經(jīng)干細(xì)胞在體內(nèi)能夠沿著既定的路線增殖發(fā)育成各種神經(jīng)細(xì)胞主要是受基因調(diào)控的[4],但其定向分化為膽堿能神經(jīng)元的分子機(jī)制仍不清楚。研究顯示,Aldoc(Aldolase C)能夠參與神經(jīng)胚的形成[5],而stmn1(stathmin1)能夠參與神經(jīng)元的增殖與分化[6],本實(shí)驗(yàn)就NSCs誘導(dǎo)分化為膽堿能神經(jīng)元的過程中Aldoc與stmn1的變化情況進(jìn)行了研究。

材料和方法

1.試劑

DMEM/F12(Gibco);B27(Sigma);視黃酸(RA,Sigma);堿性成纖維細(xì)胞生長因子(bFGF)、表皮生長因子(EGF)、腦源性神經(jīng)營養(yǎng)因子(BDNF)、神經(jīng)營養(yǎng)因子-3(NT-3)、音猬因子(SHH)(均購至R&D);胎牛血清(四季青);Nestin抗體(rabbit anti-rat nestin)(Santa Cruz);ChA T抗體(mouse anti-rat ChA T,Chemicon);Cy3標(biāo)記的羊抗兔二抗、Cy3標(biāo)記的兔抗小鼠二抗(GE);RT-PCR試劑(Ta KaRa);實(shí)時(shí)定量 PCR試劑盒 TaqMan Micro RNA Assays(Applied Biosystems);CO2培養(yǎng)箱(Forma);低溫高速離心機(jī)(Heraeus);Master-cycler型擴(kuò)增儀(Eppendorf);ABI 7300型定量 PCR儀(Applied Biosystems);倒置顯微鏡(Olympus)。

2.NSCs的培養(yǎng)與鑒定

將孕齡17 d的Wistar大鼠用10%水合氯醛麻醉(按0.4 ml/100g的用量),用75%是酒精常規(guī)消毒孕鼠腹部,于無菌條件下取出胎鼠,放入預(yù)置DHank’s液的平皿中,去除胎膜及胎盤等組織,用顯微器械取出胎鼠的脊髓組織,于解剖顯微鏡下仔細(xì)剝?nèi)ゼ顾柰饽?在4℃預(yù)冷的D-Hank’s液中用吸管緩慢吹打數(shù)次,收集細(xì)胞濾液于離心管中,337 g離心5 min,棄去上清液,制備成細(xì)胞懸液,接種到限定性無血清培養(yǎng)液中,放37℃,5%CO2培養(yǎng)箱中原代培養(yǎng),3d后更換培養(yǎng)液,以后每3d換液一次,待形成神經(jīng)干細(xì)胞克隆球之后進(jìn)行神經(jīng)上皮干細(xì)胞巢蛋白(Nestin)免疫熒光檢測。

3.脊髓源神經(jīng)干細(xì)胞定向誘導(dǎo)分化為膽堿能神經(jīng)元

取新鮮傳代的第3代NSCs,輕柔吹打分散細(xì)胞球,以5×105個(gè)細(xì)胞/ml的密度接種于預(yù)先放置有多聚賴氨酸包被蓋玻片的6孔板中,將培養(yǎng)板放入37℃、5%CO2飽和濕度的培養(yǎng)箱中讓細(xì)胞球貼壁,4h后,輕輕吸去培養(yǎng)基,參照本課題組其他人的誘導(dǎo)方案,加入誘導(dǎo)培養(yǎng)基,第1 w:DMEM/F12+2%B27+20 ng/ml bFGF+2μM RA+50 ng/ml SHH,第2 w:DMEM/F12+2%B27+20 ng/ml b FGF+2μM RA+50 ng/ml SHH+20 ng/ml NT-3+20 ng/ml BDNF,對(duì)照組不加任何誘導(dǎo)劑,每2 d換液一次,在倒置顯微鏡觀察細(xì)胞的分化情況,并拍照記錄細(xì)胞的分化形態(tài),在誘導(dǎo)分化2w后,用免疫熒光檢測膽堿能神經(jīng)元特異性標(biāo)志物膽堿乙酰轉(zhuǎn)移酶ChA T的表達(dá)。

4.細(xì)胞免疫熒光染色

分別將貼壁4 h后的神經(jīng)干細(xì)胞和誘導(dǎo)分化2w后的神經(jīng)細(xì)胞用4%的多聚甲醛室溫固定20 min后,用含0.3%Triton 100.10%羊血清室溫孵育30 min加一抗,4℃過夜,加二抗室溫孵育2 h即可在熒光顯微鏡下觀測。

5.RT-PCR檢測基因表達(dá)

分別收集對(duì)照組(神經(jīng)干細(xì)胞克隆球),誘導(dǎo)1w和誘導(dǎo)2w的細(xì)胞,吸去培養(yǎng)液,用 PBS液清洗一次,每孔細(xì)胞用 1 ml的 RNAiso Reagent試劑(Ta KaRa)按說明書進(jìn)行操作提取總RNA,并以紫外分光光度計(jì)定量(OD260/OD280=1.8-2.0)。逆轉(zhuǎn)錄反應(yīng)體系中含:2 g總 RNA;Oligo(d T)18(50 μM);5×M-MLV buffer;dNTP混合物(10 mM);RNase Inhibitor(40 U/l);RTaseM-MLV(RNase H-)(200 U/l)。42℃反應(yīng) 1h,75℃冰上冷卻15min。將所得cDNA于-20℃保存?zhèn)溆?。PCR擴(kuò)增引物均參照 GenBank mRNA序列,用ABI Primer express 3.0軟件設(shè)計(jì)引物,由北京三博遠(yuǎn)志生物技術(shù)有限責(zé)任公司合成(序列見附表)。

PCR擴(kuò)增體系 20 l中含:10 l 2×SYBR Green PCR Master Mix(Applied Biosystems),Real-time PCR引物1 l,cDNA產(chǎn)物1 l,dd H2O 8 l。在ABI7300定量PCR儀以如下條件進(jìn)行擴(kuò)增:95℃10 min,95℃15 s,60℃1 min,循環(huán)40次。反應(yīng)設(shè)置4個(gè)復(fù)孔,以18S r RNA作為內(nèi)參照。采用比較CT值法,應(yīng)用 RQ Study軟件(Applied Biosystems)進(jìn)行miRNAs的相對(duì)定量分析。

6.統(tǒng)計(jì)學(xué)處理

實(shí)驗(yàn)數(shù)據(jù)實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(ˉx±s)表示,采用SPSS13.0統(tǒng)計(jì)分析軟件進(jìn)行t檢驗(yàn)分析。

表1 18Sr RNA,Aldoc,Stmn1,ChA T基因的引物序列Table 1 Primer sequences of 18Sr RNA,Aldoc,Stmn1,ChA T for Real-time PCR

結(jié) 果

1.分離培養(yǎng)的NSCs的形態(tài)學(xué)特點(diǎn)

經(jīng)分離培養(yǎng)的NSCs細(xì)胞,培養(yǎng)5-6 d后,光鏡下可見幾個(gè)或幾十個(gè)細(xì)胞組成的懸浮微球,細(xì)胞呈群聚生長的狀態(tài),大小不一,折光性強(qiáng),隨著時(shí)間的推移,細(xì)胞球內(nèi)的細(xì)胞數(shù)目在不斷增長,細(xì)胞球的體積也隨之增大,形態(tài)規(guī)則,呈球型或橢圓型,細(xì)胞排列緊密,無明顯的突起。隨著培養(yǎng)時(shí)間的增加,細(xì)胞球的體積就會(huì)保持穩(wěn)定,不再增大(圖1)。神經(jīng)球Nestin免疫熒光標(biāo)記結(jié)果顯示神經(jīng)球內(nèi)的細(xì)胞胞漿都呈現(xiàn)強(qiáng)的紅色熒光,表明所獲得的神經(jīng)干細(xì)胞呈Nestin抗原陽性(圖2)。

2.NSCs誘導(dǎo)分化后形態(tài)學(xué)特點(diǎn)

神經(jīng)干細(xì)胞接種于蓋玻片上4 h后,神經(jīng)干細(xì)胞球邊緣有突起伸出,呈放射狀。誘導(dǎo)3 d時(shí),發(fā)現(xiàn)細(xì)胞突起明顯增粗伸長。培養(yǎng)至第7 d,從神經(jīng)干細(xì)胞球周圍遷出許多細(xì)胞(圖3),可見許多分化的細(xì)胞呈典型的神經(jīng)元樣細(xì)胞形態(tài):胞體飽滿而不規(guī)則,有多個(gè)細(xì)長突起。培養(yǎng)14 d時(shí),大多數(shù)神經(jīng)細(xì)胞已經(jīng)從神經(jīng)干細(xì)胞球中央遷出(圖4),且神經(jīng)細(xì)胞之間的突起相互交錯(cuò)呈網(wǎng)絡(luò)結(jié)構(gòu)(圖5),免疫熒光檢測可見細(xì)胞胞漿呈紅色熒光,即有膽堿能神經(jīng)元特異標(biāo)志ChA T的陽性表達(dá)(圖6)。

圖1 培養(yǎng)14 d的神經(jīng)干細(xì)胞球,形態(tài)規(guī)則,輪廓清楚,折光性強(qiáng)。標(biāo)尺示50μm圖2 免疫熒光染色顯示,神經(jīng)干細(xì)胞球內(nèi)細(xì)胞胞漿呈Nestin陽性(紅色)。標(biāo)尺示40μm圖3 誘導(dǎo)分化1 w后,一些細(xì)胞從神經(jīng)干細(xì)胞球周圍遷出,呈放射狀。標(biāo)尺示200μm圖4 誘導(dǎo)分化2 w后,大多數(shù)神經(jīng)細(xì)胞已經(jīng)從神經(jīng)干細(xì)胞球中央中遷出。標(biāo)尺示100μm圖5 誘導(dǎo)分化2w后,部分神經(jīng)細(xì)胞之間的突起相互交錯(cuò)呈網(wǎng)絡(luò)結(jié)構(gòu)。標(biāo)尺示100μm圖6 誘導(dǎo)分化2w后,免疫熒光檢測顯示ChAT免疫反應(yīng)陽性的膽堿能神經(jīng)元之間的突起相互連接。標(biāo)尺示80μmFig.1 Neural stem cell sphere was showed regular morphology with clear boundary and strong light ref ractivity after 14 days.bar=50μmFig.2 Fluorescent images of NSCs was showed the cytoplasm immunopositive for nestin(red).bar=40μmFig.3 After neural stem cells were differeted in 1w,some of them were migrated f rom neural stem cells sp here in shape of radial.bar=200μmFig.4 After neural stem cells were differeted in 2w,most of neural cells migrated from the centre of neual stem cell sphere.bar=100μmFig.5 Some neutites formed a net after 2w in differetiation.bar=100μmFig.6 Fluorescent imagesof ChAT-posibive cholinergic neurons complete neutites werefound after 2w in differetiation.bar=80μm

3.RT-PCR檢測基因表達(dá)

實(shí)時(shí)定量PCR檢測結(jié)果顯示:Aldoc在誘導(dǎo)前后有變化,在誘導(dǎo)1w時(shí)其表達(dá)量比對(duì)照組低,誘導(dǎo)2 w時(shí)表達(dá)量仍較誘導(dǎo)1w時(shí)低(圖7);Stmn1在誘導(dǎo)1w時(shí)表達(dá)量較對(duì)照組低,誘導(dǎo)2 w時(shí)表達(dá)較誘導(dǎo)1w組高,也顯著高于對(duì)照組(圖8);ChA T誘導(dǎo)2w后表達(dá)量比未誘導(dǎo)組高(圖9);均存在統(tǒng)計(jì)學(xué)意義(P<0.05)。

圖7 Aldoc相對(duì)定量結(jié)果:誘導(dǎo)1w時(shí)較對(duì)照組低,誘導(dǎo)2w時(shí)仍較1w時(shí)低。(A:1w;B:2w;C:對(duì)照)Fig.7 The relative quantitative resultsof target gene Aldoc:in comparison to control group,Aldoc was low-expressed after 1w,and still was low-expressed in comparison to 1w after 2w.(A:1w;B:2w;C:control)

圖8 Stmn1相對(duì)定量結(jié)果:誘導(dǎo)1w時(shí)較對(duì)照組低,誘導(dǎo)2w時(shí)又較1w時(shí)高。(A:1w;B:2w;C:對(duì)照)Fig.8 The relative quantitative resultsof target gene Stmn1,in comparison to control group,Stmn1 was low-expressed after 1w,and was high-expressed in comparison to 1w after 2w.(A:1w;B:2w;C:control)

圖9 ChAT相對(duì)定量結(jié)果:誘導(dǎo)2w后實(shí)驗(yàn)組較對(duì)照組高。(A:實(shí)驗(yàn)組;B:對(duì)照)Fig.9 The relative quantitative results of target gene ChA T,in comparison to control group,ChA T was high-expressed in experimental group after 2w.(A:experimental group;B:control)

討 論

脊髓損傷所引起的運(yùn)動(dòng)神經(jīng)元損傷是當(dāng)今神經(jīng)科學(xué)的重大難題,干細(xì)胞治療具有良好的應(yīng)用前景。但移植入脊髓的干細(xì)胞多分化為神經(jīng)膠質(zhì)細(xì)胞[7],由此我們?cè)趹?yīng)用前先根據(jù)靶組織類型對(duì)神經(jīng)干細(xì)胞進(jìn)行定向誘導(dǎo)分化,因此神經(jīng)干細(xì)胞定向誘導(dǎo)分化為所需神經(jīng)元的分子機(jī)制成為關(guān)鍵問題。研究顯示,在體外用 RA、SHH誘導(dǎo)胚胎干細(xì)胞向運(yùn)動(dòng)神經(jīng)元分化,最終可表達(dá)運(yùn)動(dòng)神經(jīng)元的標(biāo)記物Hb9[8-9];同時(shí)本課題組也發(fā)現(xiàn),通過RA和SHH的聯(lián)合使用,誘導(dǎo)脊髓源性神經(jīng)干細(xì)胞分化,所得細(xì)胞可表達(dá) ChA T[3]。因此,實(shí)驗(yàn)中參考Li等人的方法[9],采用 RA、SHH、NF3和BDNF為誘導(dǎo)劑,誘導(dǎo)神經(jīng)干細(xì)胞向膽堿能神經(jīng)元分化。這些研究都是在觀察誘導(dǎo)分化前后運(yùn)動(dòng)神經(jīng)元標(biāo)記基因的表達(dá)變化情況,而對(duì)細(xì)胞分化過程中分子機(jī)制的研究比較少。研究發(fā)現(xiàn),Aldoc參與神經(jīng)胚的形成[5],Stmn1參與神經(jīng)元的增殖與分化[6],且本課題組前期對(duì)神經(jīng)干細(xì)胞和運(yùn)動(dòng)神經(jīng)元差異表達(dá)蛋白的研究也顯示Aldoc在神經(jīng)干細(xì)胞較運(yùn)動(dòng)神經(jīng)元中高表達(dá),Stmn1在運(yùn)動(dòng)神經(jīng)元較神經(jīng)干細(xì)胞中高表達(dá),就此我們?cè)诒緦?shí)驗(yàn)中觀察了Aldoc和Stmn1在NSCs誘導(dǎo)分化為膽堿能神經(jīng)元過程中的變化情況,進(jìn)一步探討神經(jīng)干細(xì)胞的分化機(jī)制。

本實(shí)驗(yàn)研究發(fā)現(xiàn):Aldoc在脊髓源神經(jīng)干細(xì)胞誘導(dǎo)前后有變化,在誘導(dǎo)1w時(shí)其表達(dá)量比對(duì)照組低,誘導(dǎo)2w時(shí)表達(dá)量仍較誘導(dǎo)1w時(shí)低。Henriette等人發(fā)現(xiàn)Aldoc作為糖酵解酶的成員之一,能夠?yàn)檗D(zhuǎn)錄因子 Pax-6合成一個(gè)靶標(biāo),而 Pax-6與神經(jīng)發(fā)育有緊密聯(lián)系[5];且課題組前期對(duì)神經(jīng)干細(xì)胞和運(yùn)動(dòng)神經(jīng)元的差異蛋白研究中也顯示Aldoc在運(yùn)動(dòng)神經(jīng)元較干細(xì)胞表達(dá)低[10],由此我們推測Aldoc在參與神經(jīng)胚形成的同時(shí),可能對(duì)神經(jīng)干細(xì)胞的干性維持有一定作用。

我們的實(shí)驗(yàn)結(jié)果顯示Stmn1在誘導(dǎo)1w時(shí)表達(dá)量較對(duì)照組降低,誘導(dǎo)2w時(shí)表達(dá)較誘導(dǎo)1w組又升高。Stmn1作為微管蛋白調(diào)控基因,在早期的中央和周圍神經(jīng)系統(tǒng)高表達(dá),可參與神經(jīng)系統(tǒng)的發(fā)育[11],并且可以編碼微管蛋白,對(duì)有絲分裂紡錘體的組裝和消失有重要作用[12];但因其缺少Stmn2的N-末端同源區(qū),故不能與CaMy1綁定,從而不能調(diào)控神經(jīng)微管的形成[13]。而Stmn1能夠通過綁定到其上的Ascl1與 b HL H家族的 Hes1基因作用,Hes1能夠抑制Ascl1的表達(dá),從而降低 Stmn1基因的表達(dá),調(diào)控神經(jīng)元的增殖與分化[14]。且課題組前期對(duì)神經(jīng)干細(xì)胞和運(yùn)動(dòng)神經(jīng)元的差異蛋白研究也顯示Stmn1在運(yùn)動(dòng)神經(jīng)元較神經(jīng)干細(xì)胞表達(dá)高[10],而且有報(bào)道稱Stmn1在神經(jīng)元表達(dá)較高[15-16]。由此我們推斷Stmn1可能是在脊髓源神經(jīng)干細(xì)胞誘導(dǎo)分化為膽堿能神經(jīng)元的后期,即膽堿能神經(jīng)元的成熟過程中發(fā)揮作用。

通過實(shí)驗(yàn)研究,我們檢測到Aldoc和Stmn1基因在NSCs分化中的表達(dá)變化情況,進(jìn)一步使我們了解了神經(jīng)干細(xì)胞向膽堿能神經(jīng)元分化過程中的分子機(jī)制,可能對(duì)神經(jīng)干細(xì)胞的移植和基因治療的調(diào)控提供理論基礎(chǔ),為神經(jīng)干細(xì)胞移植應(yīng)用于脊髓損傷的修復(fù)奠定實(shí)驗(yàn)基礎(chǔ)。

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The expression of Aldoc and Stmn1 during the differentiation of spinal cord-der ived neural stem cells into cholinergic neurons

Liu Caiyu1,Wang Chunfang1＊,Li Pengfei1,Wei Hongen1,Long Zhijing1,Deng Chunsheng2＊
(1Department of Biotechnology,Shanxi Medical University,Taiyuan 030001;2The 322nd Hospital of PLA,Datong Shanxi 037006 China)

Objective To detect the expression changes of Aldoc and Stmn1 gene during the differentiation of neural stem cells derived f rom the embryonic spinal cord into cholinergic neurons in rats.Methods The neural stem cells were harvested from the spinal cord of 17-day-old fetal rats,cultured in a serumf ree limited medium containing EGF and bFGF,and then induced by a conditioned medium.The real time quantitative PCR was used to detect the expression of Aldoc and Stmn1 mRNA during the differentiation.Results The cells were positive for ChA T by immunostaining analyses after the induction.The expression of Aldoc was decreased in cholinergic neurons(P<0.05);while the expression of Stmn1 was increased in cholinergic neurons(P<0.05).Conclusion Aldoc gene may play an important role in the maintenance of neural stem cells,whereas Stmn1 gene may play a role during the maturation process of cholinergic neurons.

Neural stem cell; Cholinergic neuron; Aldoc; Stmn1; Gene expression

R329

A

10.3870/zgzzhx.2011.03.010

2010-02-10

2011-04-01

山西省自然科學(xué)基金(2009011057-1);中國人民解放軍第322醫(yī)院自然科學(xué)基金(2011001)

劉彩玉,女(1984年),漢族,碩士研究生。

＊通訊作者(To whom correspondence should be addressed)