王鳳軍， 王洪朋， 單晶麗， 邵正凱， 姜楊薇， 陳元元， 宋婷婷， 邱麗婷

腦源性神經(jīng)生長(zhǎng)因子對(duì)mir124表達(dá)及興奮性電流的影響

王鳳軍1， 王洪朋2， 單晶麗3， 邵正凱1， 姜楊薇1， 陳元元1， 宋婷婷1， 邱麗婷1

目的 探討B(tài)DNF對(duì)海馬miRNA-124表達(dá)及齒狀回顆粒細(xì)胞興奮性突觸后電流(sEPSCs)的影響，以明確BDNF對(duì)顳葉內(nèi)側(cè)癲癇(MTLE)發(fā)病機(jī)制的影響。方法 選取哈爾濱醫(yī)科大學(xué)附屬第一臨床醫(yī)院神經(jīng)外科2008年4月-2010年10月手術(shù)治療的MTLE患者12例。RT-pcr技術(shù)檢測(cè)BDNF孵育后MTLE患者海馬組織mir-124表達(dá)、膜片鉗技術(shù)檢測(cè)BDNF對(duì)海馬顆粒細(xì)胞sEPSCs的影響。結(jié)果 (1)BDNF降低了顳葉癲癇海馬miRNA-124的表達(dá)(P<0.01)；(2)BDNF增加了顆粒細(xì)胞sEPSCs的頻率和幅度(P<0.01)。結(jié)論 BDNF降低了顳葉癲癇海馬mir-124的表達(dá)，提高顆粒細(xì)胞sEPSCs的頻率和幅度，從而可能對(duì)人類MTLE的發(fā)病起到促進(jìn)作用。

腦源性神經(jīng)生長(zhǎng)因子； 顳葉內(nèi)側(cè)癲癇； miRNA-124； 自發(fā)性興奮性突觸后電流

癲癇是中樞神經(jīng)系統(tǒng)常見(jiàn)疾病，以反復(fù)癇樣發(fā)作為特征，全世界大約有5000萬(wàn)患者，癲癇的發(fā)作類型超過(guò)40種，MTLE為最常見(jiàn)的一種難治性癲癇。近年來(lái)， 腦源性神經(jīng)生長(zhǎng)因子(BDNF)成為癲癇的研究熱點(diǎn)。BDNF于1982年由德國(guó)科學(xué)家Brade從豬腦里提取的一類神經(jīng)生長(zhǎng)因子[1]，其對(duì)維持神經(jīng)細(xì)胞的生理功能有重要的作用，分布廣泛，海馬和皮質(zhì)分布最多，其它腦區(qū)也有表達(dá)[2]。BDNF能夠增強(qiáng)大鼠海馬齒狀回的突觸信號(hào)傳導(dǎo)[3，4]及通過(guò)調(diào)節(jié)NMDA受體的活性來(lái)增強(qiáng)突觸傳遞功能[5]和影響TrkB信號(hào)而促進(jìn)了癲癇發(fā)病[6]。然而也有報(bào)道BDNF對(duì)癲癇的發(fā)生起到明顯抑制作用[7]。BDNF在多種癲癇模型鼠腦內(nèi)表達(dá)明顯上調(diào)[8～10]。Hideo等報(bào)道癲癇動(dòng)物模型驚厥發(fā)作4 h后海馬區(qū)BDNF miRNA表達(dá)明顯增高[11]。BDNF的多態(tài)性與不同種族之間的聯(lián)系可能不同[12]及通過(guò)改變氯離子協(xié)同轉(zhuǎn)運(yùn)蛋白對(duì)顳葉癲癇的發(fā)生起到調(diào)節(jié)作用[13]。眾多研究表明BDNF與癲癇二者關(guān)系密切，但BDNF對(duì)癲癇的發(fā)病究竟是促進(jìn)還是抑制作用不得而知。miRNA對(duì)細(xì)胞基本生理過(guò)程有著重要的調(diào)控作用，miRNA的表達(dá)異常必然會(huì)導(dǎo)致細(xì)胞功能紊亂，引起疾病的發(fā)生。現(xiàn)今已發(fā)現(xiàn)的miRNA中，約70%在哺乳類動(dòng)物的腦組織中有表達(dá)，如腦組織所特有的miRNA(miRNA-124a、miRNA-128、miRNA-101等)，腦組織富含的miRNA(如miRNA-125b)，在神經(jīng)系統(tǒng)的不同生理過(guò)程、不同信號(hào)傳導(dǎo)通路的基因表達(dá)調(diào)控，如神經(jīng)系統(tǒng)發(fā)生和發(fā)育、神經(jīng)干細(xì)胞分化、細(xì)胞凋亡等過(guò)程中發(fā)揮重要作用[14]。多項(xiàng)研究表明miRNA與癲癇發(fā)病密切相關(guān)如miRNA-124、miRNA-203能夠抑制癲癇的發(fā)作[15～17]。部分miRNA在人類和癲癇的模型鼠都會(huì)不同程度表達(dá)上調(diào)[18]。BDNF與miRNA在癲癇的發(fā)病機(jī)制中可能有交互作用[19]。以上研究表明BDNF與miRNA對(duì)顳葉癲癇的發(fā)生可能存在一定的作用，探索此種機(jī)制或許會(huì)成為治療顳葉內(nèi)側(cè)癲癇的一個(gè)新手段。

1 資料與方法

1.1 試劑與材料 腦源性神經(jīng)生長(zhǎng)因子(BDNF),甲碘荷包牡丹堿(BMI),生物胞素(Biocytin)異去甲檳榔次堿(Isoguvacine hydrochloride),木防己苦毒素(picrotoxin,PTX)多聚甲醛(paraformaldehyde)， NaCl，NaH2PO4，KCl，CaCl2，MgSO4·7H2O，NaHCO3， KOH，EGTA，MgCl2，HEPES， dextrose，K2-ATP，Sucrose以上試劑均購(gòu)自Sigma公司(St Louis,MO,USA)。超純水、蒸餾水、組織膠水、PCR 引物(上海生物工程技術(shù)有限公司)。

選取哈爾濱醫(yī)科大學(xué)附屬第一臨床醫(yī)院神經(jīng)外科2008年4月-2010年10月手術(shù)治療的MTLE患者12例手術(shù)時(shí)切下的海馬組織為標(biāo)本，男性7例，女性5例，年齡15～35歲，平均年齡(25.91±5.51)歲。應(yīng)用BDNF灌流前后對(duì)照。

1.2 方法 RT-PCR術(shù)中海馬組織切下后迅速用冰冷的(4 ℃)S-ACSF保存，使用震動(dòng)切片機(jī)將海馬組織切成350-400 μm腦片在培養(yǎng)槽中孵育1 h，槽內(nèi)裝有A-CSF并持續(xù)的供給95%O2和5%CO2，溫度控制在32 ℃。部分腦片在A-CSF含有BDNF(100 ng/ml)組孵育1 h后與A-CSF組對(duì)比檢測(cè)海馬組織mir-124的表達(dá)。實(shí)驗(yàn)方法按照試劑盒說(shuō)明書(shū)，循環(huán)閾值的形式表達(dá)結(jié)果，均值加減SD，各組間的差異以△△CT=△CT(目的基因)-△CT(actin)，通過(guò)2-△△CT方法來(lái)檢測(cè)各組相對(duì)miRNA的表達(dá)[20]。

Whole-cell記錄 術(shù)中海馬組織切下后迅速用冰冷的(4 ℃)S-ACSF保存，使用震動(dòng)切片機(jī)將海馬組織切成350～400 μm腦片，顯微鏡下齒狀回顆粒細(xì)胞形成全細(xì)胞記錄將膜電位鉗制在-65 mV,灌流液為ACSF中加入PTX(100 μmol)或BMI(10 μmol)，電壓鉗下記錄sEPSCs，觀測(cè)應(yīng)用BDNF后sEPSCs頻率、幅度的變化。

2 結(jié) 果

2.1 BDNF降低了MTLE腦組織mir-124的表達(dá)(P<0.01) 術(shù)中海馬組織制備350～400 μm腦片，腦片在A-CSF含有BDNF(100 ng/ml)組孵育1 h后與腦片在僅有A-CSF組比較mir-124的表達(dá)明顯降低(P<0.01)(見(jiàn)圖1)。

2.2 BDNF升高了顆粒細(xì)胞sEPSCs的頻率和幅度 術(shù)中海馬組織制備350-400 μm腦片，顯微鏡下識(shí)別齒狀回顆粒細(xì)胞，膜電位鉗制-65 mV， 成功記錄sEPSCs，灌流液內(nèi)PTX(100 μmol)或BMI(10 μmol)消除GABA影響。灌流液內(nèi)加入BDNF(100 ng/ml)。記錄6個(gè)顆粒細(xì)胞測(cè)量sEPSC的動(dòng)力學(xué)特性。常規(guī)監(jiān)測(cè)輸入阻抗(10-25 MΩ)，以輸入阻抗變化不超過(guò)15%為標(biāo)準(zhǔn)。統(tǒng)計(jì)結(jié)果表明BDNF應(yīng)用前后比較sEPSC幅度明顯升高，差異顯著(P<0.01)；BDNF灌流前后比較sEPSC頻率明顯升高，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)。洗脫組與灌流前比較，無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)(見(jiàn)圖2)。

圖1 BDNF應(yīng)用前后mir-124的表達(dá)

與灌流前比較**P<0.01；與Washout比較**P<0.01

圖2 BDNF上調(diào)了顆粒細(xì)胞sEPSCs的頻率和幅度

3 討 論

癲癇是最為常見(jiàn)的慢性腦疾病，病因復(fù)雜，患病率高為其特點(diǎn),對(duì)癲癇的發(fā)病機(jī)制研究，在神經(jīng)科學(xué)的研究中占有重要的意義。BDNF為癲癇研究的熱點(diǎn)之一，對(duì)癲癇的發(fā)病作用結(jié)果不一甚至有些相對(duì)立。多數(shù)學(xué)者認(rèn)為BDNF對(duì)癲癇的發(fā)生有促進(jìn)作用，BDNF對(duì)神經(jīng)遞質(zhì)有調(diào)節(jié)作用，可以促進(jìn)神經(jīng)元NPY的表達(dá)[21]和提高了興奮性谷氨酸的濃度[22]，及胞膜NMDA受體[23]。BDNF可調(diào)節(jié)突觸的形成，增加樹(shù)突棘突的密度及TRPC3通道[24,25]。研究表明部分miRNA與癲癇發(fā)病密切相關(guān)[26]，在人類TLE或動(dòng)物模型中mir-146a都有較高的表達(dá)[27]。有研究表明mir-134有明顯的致癲癇作用[28,29]，MiR-155的拮抗劑通過(guò)激活BDNF對(duì)癲癇持續(xù)狀態(tài)發(fā)作起到了保護(hù)作用。大多數(shù)研究都是以動(dòng)物癲癇模型為基礎(chǔ)，探討其在癲癇發(fā)生發(fā)展中的作用，本實(shí)驗(yàn)研究以顳葉內(nèi)側(cè)癲癇患者術(shù)中切下的海馬為標(biāo)本。結(jié)果表明BDNF對(duì)mir-124的表達(dá)及sEPSCs的影響表明其對(duì)癲癇的發(fā)生可能有其促進(jìn)作用，但二者是否具有相關(guān)性，我們將繼續(xù)進(jìn)行下一步研究。

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The effect of BDNF on levels of miRNA-124 and sEPSCs

WANG Fengjun,WANG Hongpeng,SHAN Jingli,et al.

(Department of Neurology,The Fourth Hospital of Harbin Medical University,Harbin 150001,China)

Objective To study the effect of brain derived neurotrophic factor (BDNF) in the pathogenesis from patients with MTLE.Methods The study was performed between April 2008 and October 2010.Surgically removed specimens were collected from the patients with MTLE.All patients gave written informed consent for research use of the biopsy materials.Surgically resected hippocampal were collected and immediately immersed in oxygenated ice-cold SACSF.The expression of Mir-124 was detected by RT-PCR and sEPSCs by patch-clamp.Results Expression of mir-124 was significantly decreased in Dentate Gyrus of MTLE compared with that of controls (P<0.01).BDNF increases frequency and amplitude of sEPSC (P<0.01).Conclusions The results indicate that BDNF significantly decreased the expression of mir-124 as in Dentate Gyrus from patients with MTLE.Our electrophysiological findings indicate that BDNF increase excitability on dentate granule cells.In conclusion,our work supports a role of BDNF in pathophisyology of MTLE and raises the possibility that interference with BDNF action may be a therapeutic method for patients of MTLE.

BDNF; mir-124; MTLE; sEPSCs

2017-04-23；

2017-05-30

黑龍江省教育廳面上項(xiàng)目(No.12541509)

(1.哈爾濱醫(yī)科大學(xué)附屬第四醫(yī)院神經(jīng)內(nèi)科，黑龍江 哈爾濱 150001；2.牡丹江市婦女兒童醫(yī)院,黑龍江 牡丹江 157000;3.哈爾濱市第二醫(yī)院,黑龍江 哈爾濱 150056) 通迅作者： 王鳳軍,E-mail:fengjun19791209@163.com

1003-2754(2017)08-0718-03

R742.1

A