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

劉彩玉 王春芳＊ 李鵬飛 蔚洪恩 龍志晶 鄧春圣

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

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)科大學生物技術教研室,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)干細胞在誘導分化為膽堿能神經(jīng)元過程中Aldoc和Stmn1的表達變化情況。方法 從孕17天Wistar胚胎大鼠取出脊髓組織,制成細胞懸液,采用含EGF和bFGF的無血清限定性培養(yǎng)基培養(yǎng),然后進行誘導分化,觀察脊髓源神經(jīng)干細胞向膽堿能神經(jīng)元分化的情況,應用熒光定量PCR方法分析Aldoc和Stmn1基因在脊髓源神經(jīng)干細胞誘導分化為膽堿能神經(jīng)元的過程中的表達變化情況。結果 神經(jīng)干細胞在誘導分化為膽堿能神經(jīng)元后,經(jīng)免疫熒光檢測有ChAT陽性細胞表達;Aldoc基因的表達量在膽堿能神經(jīng)元較神經(jīng)干細胞低(P<0.05);Stmn1基因的表達量則在誘導分化后較神經(jīng)干細胞升高(P<0.05)。結論 Aldoc對神經(jīng)干細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)干細胞; 膽堿能神經(jīng)元; Aldoc基因; Stmn1基因; 基因表達

神經(jīng)干細胞(neural stem cell,NSCs)是一類具有分裂潛能和自我更新能力的母細胞,它可以通過不對等的分裂方式產(chǎn)生神經(jīng)組織的各類細胞,且在體外自然分化的條件下神經(jīng)干細胞也具有多向分化潛能,可分化為神經(jīng)元、星形膠質(zhì)細胞和少突膠質(zhì)細胞。近年來隨著神經(jīng)干細胞的發(fā)現(xiàn)和逐步了解,人們開始希望能通過神經(jīng)干細胞來治療脊髓損傷,其定向分化性,為修復和替代死亡的神經(jīng)細胞提供了可能。報道稱,在脊髓多層面NSC的移植可作為運動神經(jīng)元疾病將來的細胞治療策略[1],Wichterle等模擬體內(nèi)運動神經(jīng)元分化的途徑,在體外用 RA、SHH誘導鼠胚胎干細胞先向脊髓祖細胞分化,進而誘導脊髓祖細胞向運動神經(jīng)元分化,最終有20%-30%的細胞表達運動神經(jīng)元的標記物 Hb9[2]。運動神經(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,誘導脊髓源性神經(jīng)干細胞分化,所得細胞可表達ChA T[3](ChA T是膽堿能神經(jīng)元的重要標記之一),雖然已經(jīng)證實,神經(jīng)干細胞在體內(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],本實驗就NSCs誘導分化為膽堿能神經(jīng)元的過程中Aldoc與stmn1的變化情況進行了研究。

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標記的羊抗兔二抗、Cy3標記的兔抗小鼠二抗(GE);RT-PCR試劑(Ta KaRa);實時定量 PCR試劑盒 TaqMan Micro RNA Assays(Applied Biosystems);CO2培養(yǎng)箱(Forma);低溫高速離心機(Heraeus);Master-cycler型擴增儀(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剝?nèi)ゼ顾柰饽?在4℃預冷的D-Hank’s液中用吸管緩慢吹打數(shù)次,收集細胞濾液于離心管中,337 g離心5 min,棄去上清液,制備成細胞懸液,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)干細胞克隆球之后進行神經(jīng)上皮干細胞巢蛋白(Nestin)免疫熒光檢測。

3.脊髓源神經(jīng)干細胞定向誘導分化為膽堿能神經(jīng)元

取新鮮傳代的第3代NSCs,輕柔吹打分散細胞球,以5×105個細胞/ml的密度接種于預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方案,加入誘導培養(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,對照組不加任何誘導劑,每2 d換液一次,在倒置顯微鏡觀察細胞的分化情況,并拍照記錄細胞的分化形態(tài),在誘導分化2w后,用免疫熒光檢測膽堿能神經(jīng)元特異性標志物膽堿乙酰轉(zhuǎn)移酶ChA T的表達。

4.細胞免疫熒光染色

分別將貼壁4 h后的神經(jīng)干細胞和誘導分化2w后的神經(jīng)細胞用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

分別收集對照組(神經(jīng)干細胞克隆球),誘導1w和誘導2w的細胞,吸去培養(yǎng)液,用 PBS液清洗一次,每孔細胞用 1 ml的 RNAiso Reagent試劑(Ta KaRa)按說明書進行操作提取總RNA,并以紫外分光光度計定量(OD260/OD280=1.8-2.0)。逆轉(zhuǎn)錄反應體系中含: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 1h,75℃冰上冷卻15min。將所得cDNA于-20℃保存?zhèn)溆?。PCR擴增引物均參照 GenBank mRNA序列,用ABI Primer express 3.0軟件設計引物,由北京三博遠志生物技術有限責任公司合成(序列見附表)。

PCR擴增體系 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儀以如下條件進行擴增:95℃10 min,95℃15 s,60℃1 min,循環(huán)40次。反應設置4個復孔,以18S r RNA作為內(nèi)參照。采用比較CT值法,應用 RQ Study軟件(Applied Biosystems)進行miRNAs的相對定量分析。

6.統(tǒng)計學處理

實驗數(shù)據(jù)實驗數(shù)據(jù)用均數(shù)±標準差(ˉx±s)表示,采用SPSS13.0統(tǒng)計分析軟件進行t檢驗分析。

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 果

1.分離培養(yǎng)的NSCs的形態(tài)學特點

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

2.NSCs誘導分化后形態(tài)學特點

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

圖1 培養(yǎng)14 d的神經(jīng)干細胞球,形態(tài)規(guī)則,輪廓清楚,折光性強。標尺示50μm圖2 免疫熒光染色顯示,神經(jīng)干細胞球內(nèi)細胞胞漿呈Nestin陽性(紅色)。標尺示40μm圖3 誘導分化1 w后,一些細胞從神經(jīng)干細胞球周圍遷出,呈放射狀。標尺示200μm圖4 誘導分化2 w后,大多數(shù)神經(jīng)細胞已經(jīng)從神經(jīng)干細胞球中央中遷出。標尺示100μm圖5 誘導分化2w后,部分神經(jīng)細胞之間的突起相互交錯呈網(wǎng)絡結構。標尺示100μm圖6 誘導分化2w后,免疫熒光檢測顯示ChAT免疫反應陽性的膽堿能神經(jīng)元之間的突起相互連接。標尺示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

實時定量PCR檢測結果顯示:Aldoc在誘導前后有變化,在誘導1w時其表達量比對照組低,誘導2 w時表達量仍較誘導1w時低(圖7);Stmn1在誘導1w時表達量較對照組低,誘導2 w時表達較誘導1w組高,也顯著高于對照組(圖8);ChA T誘導2w后表達量比未誘導組高(圖9);均存在統(tǒng)計學意義(P<0.05)。

圖7 Aldoc相對定量結果:誘導1w時較對照組低,誘導2w時仍較1w時低。(A:1w;B:2w;C:對照)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相對定量結果:誘導1w時較對照組低,誘導2w時又較1w時高。(A:1w;B:2w;C:對照)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相對定量結果:誘導2w后實驗組較對照組高。(A:實驗組;B:對照)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動神經(jīng)元損傷是當今神經(jīng)科學的重大難題,干細胞治療具有良好的應用前景。但移植入脊髓的干細胞多分化為神經(jīng)膠質(zhì)細胞[7],由此我們在應用前先根據(jù)靶組織類型對神經(jīng)干細胞進行定向誘導分化,因此神經(jīng)干細胞定向誘導分化為所需神經(jīng)元的分子機制成為關鍵問題。研究顯示,在體外用 RA、SHH誘導胚胎干細胞向運動神經(jīng)元分化,最終可表達運動神經(jīng)元的標記物Hb9[8-9];同時本課題組也發(fā)現(xiàn),通過RA和SHH的聯(lián)合使用,誘導脊髓源性神經(jīng)干細胞分化,所得細胞可表達 ChA T[3]。因此,實驗中參考Li等人的方法[9],采用 RA、SHH、NF3和BDNF為誘導劑,誘導神經(jīng)干細胞向膽堿能神經(jīng)元分化。這些研究都是在觀察誘導分化前后運動神經(jīng)元標記基因的表達變化情況,而對細胞分化過程中分子機制的研究比較少。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],且本課題組前期對神經(jīng)干細胞和運動神經(jīng)元差異表達蛋白的研究也顯示Aldoc在神經(jīng)干細胞較運動神經(jīng)元中高表達,Stmn1在運動神經(jīng)元較神經(jīng)干細胞中高表達,就此我們在本實驗中觀察了Aldoc和Stmn1在NSCs誘導分化為膽堿能神經(jīng)元過程中的變化情況,進一步探討神經(jīng)干細胞的分化機制。

本實驗研究發(fā)現(xiàn):Aldoc在脊髓源神經(jīng)干細胞誘導前后有變化,在誘導1w時其表達量比對照組低,誘導2w時表達量仍較誘導1w時低。Henriette等人發(fā)現(xiàn)Aldoc作為糖酵解酶的成員之一,能夠為轉(zhuǎn)錄因子 Pax-6合成一個靶標,而 Pax-6與神經(jīng)發(fā)育有緊密聯(lián)系[5];且課題組前期對神經(jīng)干細胞和運動神經(jīng)元的差異蛋白研究中也顯示Aldoc在運動神經(jīng)元較干細胞表達低[10],由此我們推測Aldoc在參與神經(jīng)胚形成的同時,可能對神經(jīng)干細胞的干性維持有一定作用。

我們的實驗結果顯示Stmn1在誘導1w時表達量較對照組降低,誘導2w時表達較誘導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,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,從而降低 Stmn1基因的表達,調(diào)控神經(jīng)元的增殖與分化[14]。且課題組前期對神經(jīng)干細胞和運動神經(jīng)元的差異蛋白研究也顯示Stmn1在運動神經(jīng)元較神經(jīng)干細胞表達高[10],而且有報道稱Stmn1在神經(jīng)元表達較高[15-16]。由此我們推斷Stmn1可能是在脊髓源神經(jīng)干細胞誘導分化為膽堿能神經(jīng)元的后期,即膽堿能神經(jīng)元的成熟過程中發(fā)揮作用。

通過實驗研究,我們檢測到Aldoc和Stmn1基因在NSCs分化中的表達變化情況,進一步使我們了解了神經(jīng)干細胞向膽堿能神經(jīng)元分化過程中的分子機制,可能對神經(jīng)干細胞的移植和基因治療的調(diào)控提供理論基礎,為神經(jīng)干細胞移植應用于脊髓損傷的修復奠定實驗基礎。

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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

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

劉彩玉,女(1984年),漢族,碩士研究生。

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