電磁刺激對(duì)脊髓神經(jīng)干細(xì)胞增殖與分化作用研究

馮楓 牟翔 袁華 王冰水 劉衛(wèi)

(第四軍醫(yī)大學(xué)西京醫(yī)院康復(fù)理療科，陜西 西安 710032)

·論著·

電磁刺激對(duì)脊髓神經(jīng)干細(xì)胞增殖與分化作用研究

馮楓 牟翔*袁華 王冰水 劉衛(wèi)

(第四軍醫(yī)大學(xué)西京醫(yī)院康復(fù)理療科，陜西 西安 710032)

目的探討電磁刺激對(duì)大鼠脊髓神經(jīng)干細(xì)胞(NSCs)向功能性神經(jīng)元增殖分化的作用及可能的機(jī)制。方法體外分離培養(yǎng)妊娠第14天(E14 d)的Wistar大鼠脊髓NSCs，無血清培養(yǎng)14 d后，分為對(duì)照組和電磁刺激組。電磁刺激組置于脈沖強(qiáng)磁場(chǎng)條件下用促增殖參數(shù)(0.1 Hz、4T、8次)進(jìn)行干預(yù)，每次脈沖放電20 ms。對(duì)照組不做干預(yù)處理。干預(yù)后第1天，10%胎牛血清誘導(dǎo)貼壁分化；誘導(dǎo)貼壁后第7天，蛋白免疫印跡法(Western Blot)檢測(cè)β-catenin蛋白表達(dá)情況；NCSs分化后第14天，免疫熒光染色檢測(cè)神經(jīng)元MAP2表達(dá)情況，紅外可視膜片鉗技術(shù)記錄神經(jīng)元自發(fā)放電情況。結(jié)果干預(yù)后，免疫熒光染色顯示電磁刺激組MAP2陽性細(xì)胞數(shù)45.4%±3.1%，較對(duì)照組38.3%±6.0%明顯增多(Plt;0.05)。Western Blot檢測(cè)結(jié)果顯示干預(yù)后第7天，電磁刺激組神經(jīng)干細(xì)胞β-catenin 蛋白量較對(duì)照組增高。膜片鉗實(shí)驗(yàn)結(jié)果顯示，誘導(dǎo)后第14天，可以記錄到神經(jīng)元自發(fā)放電，是有生理功能的。結(jié)論強(qiáng)電磁刺激能夠促進(jìn)大鼠脊髓神經(jīng)干細(xì)胞增殖分化為功能性神經(jīng)元。

脊髓; 神經(jīng)干細(xì)胞; 電磁刺激; 細(xì)胞分化

神經(jīng)干細(xì)胞(neural stem cells, NSCs)具有分化為神經(jīng)元、星形膠質(zhì)細(xì)胞及少突膠質(zhì)細(xì)胞的能力，它的發(fā)現(xiàn)和研究是神經(jīng)生物學(xué)領(lǐng)域重要進(jìn)展之一[1]。電磁刺激技術(shù)在中樞神經(jīng)系統(tǒng)疾病診治中的應(yīng)用受到越來越多的關(guān)注[2]。電磁刺激是通過脈沖磁場(chǎng)誘導(dǎo)產(chǎn)生的感應(yīng)電流促進(jìn)神經(jīng)干細(xì)胞增殖[3]，目前其促進(jìn)增殖的機(jī)制尚不清楚。位于中樞神經(jīng)系統(tǒng)內(nèi)的NSCs具有子代細(xì)胞可興奮性和穩(wěn)定性的雙重電生理特性[4]。本文旨在了解電磁刺激對(duì)Wistar大鼠脊髓神經(jīng)干細(xì)胞(NSCs)向功能性神經(jīng)元增殖分化的作用及可能的機(jī)制。

材料與方法

一、材料

1.主要試劑：杜爾貝克改良的含有F-12營養(yǎng)混合物的伊戈?duì)柵囵B(yǎng)基(DMEM/F12)、B27細(xì)胞培養(yǎng)添加劑(Gibco)，堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)、表皮生長(zhǎng)因子(EGF)(Sigma)，1%青鏈霉素、胎牛血清(FBS)(Hyclon)，小鼠抗大鼠微管相關(guān)蛋白2(MAP2)抗體(Sigma)，羊抗小鼠Alexa Fluor 綠色熒光二抗(Invitrogen)，十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)制備試劑盒(上海鼎杰)，肌動(dòng)蛋白(Actin)(SANTA)。人工腦脊液(artificial cerebrospinal fluid, ACSF)(mmol/L): NaCl 126，KCl 5.4，MgCl21，CaCl21.8，NaH2PO40.33，Glucose 10，羥乙基哌嗪乙磺酸(HEPES) 10，氫氧化鈉(sodium hydroxide, NaOH)調(diào)pH至7.4；電極內(nèi)液(pipette solution)(mmol/L)：KCl 20，天冬氨酸鉀potassium asparate 110，MgCl21，HEPES 5，乙二醇雙(2-氨基乙基醚)四乙酸 (EGTA) 10，三磷酸腺苷二鈉(Na2-ATP) 5，氫氧化鉀(potassium hydroxide, KOH)調(diào)pH至7.2；河豚毒素(tetrodotoxin, TTX)(Sigma)。

2.實(shí)驗(yàn)動(dòng)物：孕14 d Wistar大鼠，由第四軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。

二、方法

1.神經(jīng)干細(xì)胞原代培養(yǎng)：取妊娠第14天(embryonic，E14 d)孕鼠行6%水合氯醛0.6 mL/100 g腹腔麻醉處死，于75%乙醇中消毒3 min，無菌條件下取出子宮，置于預(yù)冷的無二價(jià)陽離子的Hank's平衡鹽緩沖液(D-Hank's)中。解剖顯微鏡下取出胚胎(n=6)，剝離脊髓組織并去除硬脊膜，眼科剪充分剪碎(1 mm3)，1 mL加樣槍反復(fù)吹打組織塊約50次，過濾制成細(xì)胞懸液，1000 r/min離心5 min，后小心去除上清液，進(jìn)行無血清培養(yǎng)。

2.實(shí)驗(yàn)分組：將培養(yǎng)的第3代NSCs制成單細(xì)胞懸液，隨機(jī)分裝在2個(gè)1 mL注射器內(nèi)，A組標(biāo)記為對(duì)照組不行電磁干預(yù)，B組為實(shí)驗(yàn)組，置于脈沖強(qiáng)磁場(chǎng)內(nèi)，給予0.1 Hz、4T，持續(xù)20 ms脈沖強(qiáng)磁場(chǎng)干預(yù)8次[3]。

3.形態(tài)學(xué)實(shí)驗(yàn)：干預(yù)后第1 天，10%胎牛血清誘導(dǎo)貼壁分化，分化后第7天，在相差顯微鏡下觀察兩組NSCs生長(zhǎng)形態(tài)；分化后第14天，進(jìn)行細(xì)胞免疫熒光染色鑒定NSCs的子代細(xì)胞。

4.蛋白免疫印跡實(shí)驗(yàn)(Western Blot)：電磁刺激干預(yù)、并貼壁分化后第7天，分別提取實(shí)驗(yàn)組及對(duì)照組細(xì)胞胞質(zhì)蛋白，行SDS-PAGE凝膠電泳，內(nèi)參為Actin。采用 Alpha 圖像軟件處理系統(tǒng)，分析兩組目標(biāo)條帶的光密度值和灰度值，計(jì)算兩組目的/內(nèi)參的比值，即 β-連環(huán)蛋白(β-catenin)的相對(duì)表達(dá)量。

5.膜片鉗實(shí)驗(yàn)：脈沖強(qiáng)磁場(chǎng)干預(yù)后第14天，應(yīng)用全細(xì)胞膜片鉗技術(shù)記錄神經(jīng)元自發(fā)放電。所用微電極用兩步法拉制而成，充灌電極內(nèi)液后電阻在3 MΩ左右。用ACSF以3 mL/min恒速灌流沖洗細(xì)胞表面，形成高阻封接(gt;5 GΩ)后，脈沖式抽吸打破細(xì)胞膜，形成全細(xì)胞記錄，電流鉗模式下進(jìn)行記錄。實(shí) 驗(yàn) 過 程 由 計(jì) 算 機(jī) 軟 件pCLAMP9(Axon Instrument)控制，數(shù)-模轉(zhuǎn)換器完成刺激信號(hào)的產(chǎn)生、反饋信號(hào)的采集以及數(shù)據(jù)分析。實(shí)驗(yàn)在室溫(20～24 ℃)下進(jìn)行。

結(jié) 果

一、細(xì)胞形態(tài)觀察和鑒定

神經(jīng)干細(xì)胞培養(yǎng)至第7天，在相差顯微鏡下可見培養(yǎng)瓶中體積較大的神經(jīng)球懸浮生長(zhǎng)，形態(tài)呈桑葚狀，胞質(zhì)透亮，折光性強(qiáng)(圖1)；至第14天，對(duì)成熟神經(jīng)元標(biāo)記物微管相關(guān)蛋白2(MAP2)進(jìn)行免疫熒光標(biāo)記，鏡下可見綠色熒光標(biāo)記的MAP2陽性的神經(jīng)元，并且電磁刺激組MAP2陽性細(xì)胞數(shù)量大于對(duì)照組(圖2)。

圖1 相差顯微鏡下對(duì)照組和電磁刺激組神經(jīng)球形態(tài) (×40)

Fig 1 Morphology of neurosphere under phase contrast microscope in control and electromagnetic stimulation group (×40)

A: Control group; B: In electromagnetic stimulation group, the size of neurosphere was larger than that of the control group.

Scale bar=50 μm.

圖2 對(duì)照組和電磁刺激組微管相關(guān)蛋白2免疫熒光染色 (×20)

Fig 2 Microtubule associated protein 2 immunofluorescence staining in control and electromagnetic stimulation group (×20)

A: Control group; B: In electromagnetic stimulation group, the number of MAP2 positive cells was greater than that in the control group.

二、Western Blot結(jié)果

在電磁刺激后第7天，Western Blot 檢測(cè)實(shí)驗(yàn)組和對(duì)照組神經(jīng)干細(xì)胞β-catenin 蛋白的表達(dá)(圖3)。實(shí)驗(yàn)組神經(jīng)干細(xì)胞β-catenin蛋白量(β-catenin: 326955, Actin: 332613, 目的/內(nèi)參: 0.9830)較對(duì)照組(β-catenin: 135488, Actin: 318421, 目的/內(nèi)參: 0.4255)增高約2倍。

圖3 對(duì)照組和電磁刺激組神經(jīng)干細(xì)胞β-catenin 蛋白的表達(dá)

Fig 3 β-catenin protein expression on neural stem cells in control and electromagnetic stimulation group

A: Control group; B: In electromagnetic stimulation group, the expression of β-catenin protein of NSCs was about 2 times higher than that of the control group.

圖4 神經(jīng)元具有自發(fā)放電，且該動(dòng)作電位可被TTX阻斷抑制

Fig 4 Neurons differentiated from NSCs could discharge spontaneously and be inhibited by TTX

三、膜片鉗實(shí)驗(yàn)結(jié)果

電磁刺激后第14天，應(yīng)用全細(xì)胞膜片鉗技術(shù)可記錄到NSCs分化的神經(jīng)元具有自發(fā)放電，動(dòng)作電位可被快鈉離子通道阻斷劑TTX(0.5 μM)阻斷(圖4)。表明，電磁刺激后，脊髓NSCs分化為成熟的神經(jīng)元，具有電生理活性。

討 論

近年來研究發(fā)現(xiàn)神經(jīng)干細(xì)胞與其他干細(xì)胞相比具有其自身獨(dú)特的優(yōu)勢(shì)，因此其成為神經(jīng)領(lǐng)域研究的熱點(diǎn)之一[5]。另外，胚胎源性神經(jīng)干細(xì)胞由于其自身獨(dú)特的特點(diǎn)，如與脊髓損傷區(qū)的細(xì)胞同源、來源豐富和易于培養(yǎng)等，被認(rèn)為是一種治療脊髓損傷的理想干細(xì)胞[6-7]。研究發(fā)現(xiàn)移植的神經(jīng)干細(xì)胞在體內(nèi)能繼續(xù)存活，而且存活下來的移植細(xì)胞能從移植部位向受損神經(jīng)區(qū)域遷移，并基本保留了神經(jīng)干細(xì)胞自我更新和多向分化的能力，移植細(xì)胞在宿主體內(nèi)分泌特殊的遞質(zhì)和神經(jīng)營養(yǎng)因子，改善損傷組織的局部微環(huán)境，促進(jìn)神經(jīng)元存活和軸突生長(zhǎng)，使受損神經(jīng)得以較快修復(fù)或者再生，重新建立起來的突觸聯(lián)系在神經(jīng)細(xì)胞之間形成新的神經(jīng)環(huán)路，在結(jié)構(gòu)和功能上替代受損的神經(jīng)元[8-10]。

脈沖強(qiáng)磁場(chǎng)與超高壓和極低溫一樣，屬于極端的物理實(shí)驗(yàn)條件，已有文獻(xiàn)報(bào)導(dǎo)， 脈沖強(qiáng)磁場(chǎng)(0.1 Hz 、4T、8次)是促神經(jīng)干細(xì)胞增殖最佳參數(shù)，增殖效應(yīng)在干預(yù)后第七天最顯著，且增殖的神經(jīng)干細(xì)胞仍具有向神經(jīng)元和膠質(zhì)細(xì)胞分化的能力[3]，該參數(shù)下脈沖強(qiáng)磁場(chǎng)可能是通過 Wnt/β-catenin通路發(fā)揮作用。

有關(guān)神經(jīng)干細(xì)胞增殖分化的調(diào)節(jié)機(jī)制，Wnt 信號(hào)通路被認(rèn)為在胚胎神經(jīng)系統(tǒng)生長(zhǎng)發(fā)育過程中起關(guān)鍵作用。Wnt 是一條十分保守的信號(hào)傳導(dǎo)通路，從低等生物體到高等生物都具有高度同源性，Wnt/β-catenin 途徑是該通路的經(jīng)典分支，在神經(jīng)系統(tǒng)的發(fā)育中起重要作用[11-12]。β-catenin又叫連環(huán)蛋白，是Wnt/β-catenin途徑中的多功能蛋白，當(dāng)Wnt通路處于關(guān)閉狀態(tài)時(shí)，胞漿內(nèi)的降解蛋白復(fù)合體卻是活化狀態(tài)，結(jié)合β-catenin并使之磷酸化，被磷酸化的β-catenin降解，無法進(jìn)入細(xì)胞核與轉(zhuǎn)錄因子作用，不能啟動(dòng)核內(nèi)靶基因。Wnt通路被激活時(shí)，胞質(zhì)內(nèi)調(diào)節(jié)β-catenin的降解復(fù)合體解離而失活，β-catenin去磷酸化，與核內(nèi)的轉(zhuǎn)錄因子結(jié)合并相互作用，激活下游靶基因，啟動(dòng)基因的轉(zhuǎn)錄及表達(dá)。因此，在Wnt通路激活時(shí)，胞質(zhì)內(nèi)調(diào)控細(xì)胞增殖的關(guān)鍵分子β-catenin的表達(dá)水平是增高的[13-15]。本次實(shí)驗(yàn)顯示脈沖強(qiáng)磁場(chǎng)作用于神經(jīng)干細(xì)胞后，細(xì)胞β-catenin表達(dá)上調(diào)，而β-catenin表達(dá)增高又是Wnt信號(hào)激活的標(biāo)志，所以，脈沖強(qiáng)磁場(chǎng)促神經(jīng)干細(xì)胞增殖的機(jī)制可能是通過Wnt/β-catenin途徑介導(dǎo)的。

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Electromagneticstimulationonproliferationanddifferentiationofspinalcordneuralstemcells

FENGFeng,MUXiang,YUANHua,WANGBingshui,LIUWei

DepartmentofPhysiotherapy,XijingHospital,FourthMilitaryMedicalUniversity,Xi'an710032, China

ObjectiveThe study aims to investigate the effects of electromagnetic stimulation on proliferation and differentiation of rat spinal cord neural stem cells (NSCs) and the possible mechanisms.MethodsNSCs were isolated from the spinal cord of embryonic 14 d (E14 d) Wistar rats, and cultured in serum-free medium for two weeks. Then they were divided into the experiment and control groups. NSCs of experimental group were exposed in 0.1 Hz, 4T high-intensity pulsed electromagnetic field for 8 times, each 20 ms pulse discharge, and NSCs of control group were given sham stimulation. On the first day after intervention, 10% fetal bovine serum was used to induce adherence and differentiation; protein immunoblotting (Western Blot) was used to detect β-catenin protein expression one week after adherence, and NCSs could differentiate into MAP2 positive neurons by immunofluorescence staining two weeks later. Neuronal spontaneous discharge case was recorded by infrared visual patch clamp after two weeks.ResultsAfter the intervention, immunofluorescence staining revealed that the MAP2 positive cells of electromagnetic stimulation group was 45.4% ± 3.1%, which was significantly increased compared with 38.3%±6.0% of the control group (Plt;0.05). Western Blot showed that 7 days after the intervention the β-catenin protein level in electromagnetic stimulation group was higher than that of the control group. Spontaneous neuronal discharge which was recorded by patch clamp at 14th day after induction showed its physiological function.ConclusionHigh-intensity pulsed electromagnetic stimulation can promote the rat spinal cord neural stem cells to proliferate and differentiate into functional neurons.

Spinal cord; Neural stem cells; Electromagnetic stimulation; Cell proliferation

1671-2897(2017)16-035-04

R 34.1

A

國家國際合作基金資助項(xiàng)目(2013DFA32610)；陜西省國際科技合作與交流計(jì)劃資助項(xiàng)目(2015KW-035)

馮楓，主治醫(yī)師，E-mail: 6762270 @qq.com

*通訊作者： 牟翔，教授、主任醫(yī)師，E-mail: pro.mu@fmmu.edu.cn

2016-09-23；

2016-12-15)