常 偉 潘立平 吳秋靜 宋毅軍△

實(shí)驗(yàn)研究

常 偉1潘立平2吳秋靜1宋毅軍2△

目的研究海馬神經(jīng)元癲模型中轉(zhuǎn)染miR-204后對(duì)腦源性神經(jīng)營養(yǎng)因子（BDNF）與TrkB通路調(diào)控作用的影響。方法取原代培養(yǎng)7 d后的細(xì)胞，分為正常組、正常+BDNF組、癲組、癲+BDNF組、正常轉(zhuǎn)染miR-204組、癲轉(zhuǎn)染miR-204組、癲轉(zhuǎn)染miR-204+BDNF組。癲組用無鎂液處理3 h制作癲模型。制備成功miR-204慢病毒表達(dá)載體。采用免疫熒光、膜片鉗及免疫印跡技術(shù)鑒定及觀察miR-204對(duì)BDNF/TrkB通路表達(dá)的影響。結(jié)果TrkB蛋白的磷酸化水平：正常+BDNF組高于正常組；癲+BDNF組低于正常+BDNF組，高于癲組；癲+miR-204+BDNF組高于癲+BDNF組和癲+miR-204組。結(jié)論BDNF及miR-204可以改善BDNF/TrkB受抑制的狀態(tài)，從而在癲疾病的緩解中可能發(fā)揮重要作用。

癲；海馬；神經(jīng)元；受體,trkB；微RNAs；腦源性神經(jīng)營養(yǎng)因子；miR-204

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物及分組 清潔級(jí)健康SD大鼠雌性10只，雄性2只，先取雌性6只，按比例（雌∶雄為3∶1）與雄性2只合籠飼養(yǎng)，雌性懷孕后取出，將余下4只陸續(xù)放入與雄性合籠。取24 h新生大鼠用于實(shí)驗(yàn)。分離大鼠的海馬組織，胰蛋白酶消化完全后置于DMEM/F12培養(yǎng)基終止消化，經(jīng)過吹打計(jì)數(shù)接種于培養(yǎng)皿里，孵育箱培養(yǎng)7 d。將原代培養(yǎng)7 d的神經(jīng)元細(xì)胞隨機(jī)分為7組：正常組、正常+BDNF組、癲組、癲+BDNF組、正常轉(zhuǎn)染miR-204組、癲轉(zhuǎn)染miR-204組、癲轉(zhuǎn)染miR-204+BDNF組。轉(zhuǎn)染miR-204組細(xì)胞加入慢病毒稀釋液20 μL，加BDNF組均在細(xì)胞收集前10 min加入BDNF 2 μL。

1.2 方法

1.2.1 miR-204表達(dá)載體構(gòu)建 用Trizol法及瓊脂糖凝膠電泳提取并鑒定RNA純度和完整性。將其逆轉(zhuǎn)錄合成cDNA，經(jīng)PCR擴(kuò)增并與Lentivector質(zhì)粒載體雙酶切后水浴連接，用其轉(zhuǎn)化宿主菌。將包裝質(zhì)粒導(dǎo)入293T細(xì)胞，收集上清液。

1.2.2 MAP2和DAPI雙染免疫熒光鑒定海馬神經(jīng)元 于6孔板中接種海馬神經(jīng)元細(xì)胞，經(jīng)多聚甲醛固定、Triton X-100通透后，加入山羊血清封閉液封閉（北京中杉金橋生物技術(shù)有限公司），滴加一抗MAP-2單克隆抗體（美國CST公司）及二抗FITC-山羊抗小鼠IgG（美國santacruz公司），DAPI染核，甘油封片，于倒置相差熒光顯微鏡下釆集圖像。

1.2.4 免疫印跡（Western blot）法檢測(cè)蛋白表達(dá)水平 將7組細(xì)胞經(jīng)細(xì)胞裂解液裂解，收集細(xì)胞后經(jīng)超聲離心及與蛋白上樣緩沖液煮沸制備蛋白質(zhì)樣品。取等量樣品經(jīng)十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳（SDS-PAGE）分離，偏二氟乙烯（PVDF）膜轉(zhuǎn)膜，Western blot封膜液及脫脂奶粉封閉，分別加入TrkB及磷酸化TrkB（p-TrkB）兔抗大鼠多克隆抗體及大鼠抗β-actin單克隆抗體，加入山羊抗兔及山羊抗鼠二抗，采用化學(xué)發(fā)光檢測(cè)系統(tǒng)（Pierce，USA）及SYNGENE ChemGenius系統(tǒng)成像收集數(shù)據(jù)。

1.3 統(tǒng)計(jì)學(xué)方法 采用SPSS18.0統(tǒng)計(jì)軟件包對(duì)數(shù)據(jù)進(jìn)行分析。計(jì)量資料采用±s表示，Levene方法進(jìn)行方差齊性檢驗(yàn)，成組設(shè)計(jì)2組樣本均數(shù)比較用t檢驗(yàn)，多樣本均數(shù)的比較用單因素方差分析，組間多重比較用LSD-t檢驗(yàn)，檢驗(yàn)水準(zhǔn)α=0.05。

2 結(jié)果

2.1 免疫熒光法鑒定海馬神經(jīng)元結(jié)果 鏡下觀察其神經(jīng)元純度約60%～70%。且癲狀態(tài)的神經(jīng)元細(xì)胞形態(tài)與正常組的細(xì)胞相比并無明顯改變，見圖1。

Table 1 The frequency and amplitude of cell discharge in control group and epilepsy group表1 正常組和癲組細(xì)胞放電的頻率和幅度（±s）

Table 1 The frequency and amplitude of cell discharge in control group and epilepsy group表1 正常組和癲組細(xì)胞放電的頻率和幅度（±s）

＊P＜0.05

組別正常組癲幅度（mV）67.18±1.51 75.76±1.73 10.715＊組n66 t頻率（Hz）0.28±0.11 4.41±0.37 20.422＊

Figure 2 The cell discharge in control group and epilepsy group圖2 正常組和癲組細(xì)胞放電

2.3 Western blot檢測(cè)TrkB蛋白的磷酸化水平實(shí)驗(yàn)結(jié)果 正常+BDNF組高于正常組；癲+BDNF組低于正常+BDNF組、高于癲組；癲+miR-204+BDNF組高于癲+BDNF組和癲+miR-204組，見表2、圖3。

3 討論

BDNF和TrkB是所有神經(jīng)營養(yǎng)因子中唯一一對(duì)在時(shí)間和空間上協(xié)同表達(dá)的受體配體復(fù)合物。當(dāng)BDNF與TrkB結(jié)合時(shí)，受體分子二聚化，其多個(gè)氨基酸殘基快速自動(dòng)磷酸化，磷酸化的轉(zhuǎn)錄因子移入細(xì)胞核并與特定基因的啟動(dòng)子區(qū)結(jié)合,啟動(dòng)轉(zhuǎn)錄過程[7]。酪氨酸磷酸化的TrkB作為BDNF信號(hào)轉(zhuǎn)導(dǎo)途徑的中介將信號(hào)進(jìn)一步傳遞給下游的銜接蛋白及其相關(guān)活性酶，通過水解磷脂酶C（PLCγ）、磷脂酰環(huán)己六醇激酶-3（PI3K）、細(xì)胞外信號(hào)調(diào)節(jié)激酶（ERK）、促細(xì)胞分裂活性蛋白激酶（MAPK）等信號(hào)通路激活，通過細(xì)胞內(nèi)Ca2+發(fā)揮作用，且調(diào)節(jié)BDNF、TrkB mRNA的轉(zhuǎn)化和蛋白向樹突的運(yùn)輸，從而發(fā)揮作用[8-9]。海馬神經(jīng)元在興奮性毒性損傷前與神經(jīng)營養(yǎng)蛋白共培養(yǎng)，能夠通過BDNF激活Ras/ERK和PI3-K/Akt通路的神經(jīng)保護(hù)作用[10]。miR-204位于人類9號(hào)染色體上，其可以直接與TrkB的3＇-UTR結(jié)合，通過對(duì)TrkB表達(dá)的調(diào)節(jié)，增加神經(jīng)母細(xì)胞瘤的化療藥物耐受[6]。

Table 2 Comparison of the phosphorylation levels of TrkB between seven groups表2 各組海馬神經(jīng)元TrkB蛋白的磷酸化水平比較（n=6±s）

Table 2 Comparison of the phosphorylation levels of TrkB between seven groups表2 各組海馬神經(jīng)元TrkB蛋白的磷酸化水平比較（n=6±s）

TrkB蛋白的磷酸化水平以各組p-TrkB/TrkB灰度值與正常組的比值表示

P組別正常組（1）正常+BDNF組（2）癲 組（3）癲+BDNF組（4）正常+miR-204組（5）癲統(tǒng)計(jì)學(xué)處理組比（1）∶（2）（2）∶（4）（3）∶（4）（4）∶（7）（6）∶（7）＜0.001＜0.001 0.034＜0.001＜0.001+miR-204組（6）癲+miR-204+BDNF組（7）F TrkB蛋白的磷酸化水平1 3.09±0.05 0.75±0.11 1.52±0.08 1.34±0.21 1.13±0.13 3.49±0.59 19.123＊＊

Figure 3 Results of Western blot for p-TrkB,TrkB and β-actin in seven groups圖3 各組海馬神經(jīng)元p-TrkB、TrkB及內(nèi)參β-actin蛋白Western blot圖

本實(shí)驗(yàn)中，TrkB蛋白的磷酸化水平在正常+BDNF組明顯高于正常組，說明正常狀態(tài)下BDNF/TrkB信號(hào)通路可以由BDNF的加入而激活。癲+BDNF組高于癲組，說明癲狀態(tài)下此信號(hào)通路也可由BDNF的加入而激活。癲+BDNF組較正常+BDNF組降低，可能是由于癲狀態(tài)下完整型的TrkB表達(dá)下降，而縮短型的升高，縮短型的TrkB對(duì)完整型的TrkB磷酸化起抑制作用，因此會(huì)出現(xiàn)BDNF/TrkB通路受到抑制。癲+miR-204+BDNF組較癲+BDNF組和癲+miR-204組升高，可見在癲狀態(tài)下高表達(dá)的縮短型TrkB表達(dá)下降，進(jìn)而解除癲中BDNF/TrkB通路的抑制狀態(tài)。以上提示癲狀態(tài)下縮短型TrkB的顯性抑制效應(yīng)可以抑制BDNF/TrkB通路活化，而miR-204可以下調(diào)縮短型TrkB表達(dá)而再次激活BDNF/TrkB通路。

Figure 1 The MAP-2 and DAPI fluorescence staining of control group and epilepsy hippocampal neuron group(×400)圖1 正常組和癲組海馬神經(jīng)元MAP-2和DAPI熒光染色（×400）

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（2013-10-10收稿 2013-11-18修回）

（本文編輯 閆娟）

The Study of miR-204 Regulates BDNF/TrkB Expression in Epileptic Neurons

CHANG Wei1,PAN Liping2,WU Qiujing1,SONG Yijun2
1 Grade 2007,7-Year Educational System,Tianjin Medical University,Tianjin 300070,China;2 Department of Neurology,General Hospital of Tianjin Medical University

ObjectiveTo study the effect of miR-204 on BDNF/TrkB signaling and pathogenesis on the neuron model of epilepsy.MethodsPrimary hippocampal neurons were cultured in vitro for 7 days,and were divided into control group,control+BDNF group,epilepsy group,epilepsy+BDNF group,control+miR204 group,epilepsy+miR204 group and epilepsy+miR204+BDNF group.The epilepsy model of hippocampal neurons was established by being exposed to Mg2+free media for 3 hours.The miR-204 lentivirus vector was constructed.The effect of miR-204 on BDNF/TrkB expression was detected by immunohistochemistry,patch clamp and Western blot technique.ResultsCompared with the control group,the TrkB phosphorylation level was higher in control+BDNF group.The TrkB phosphorylation level was lower in epilepsy+BDNF group than that of control+BDNF group,but it was higher than that of epilepsy group.The TrkB phosphorylation level was higher in epilepsy+miR204+BDNF group than that of epilepsy+BDNF group and epilepsy+miR204 group.ConclusionBDNF and miR-204 can improve the inhibitory condition of BDNF/TrkB signaling and may play an important role in alleviating epilepsy disease.

epilepsy；hippocampus；neurons；receptor,trkB；microRNAs；brain-derived neurotrophic factor；miR-204

R749.1 【

】 A 【DOI】 10.3969/j.issn.0253-9896.2014.03.007

*國家自然科學(xué)基金資助項(xiàng)目（項(xiàng)目編號(hào)：91132722、81071044）

1天津醫(yī)科大學(xué)七年制2007級(jí)（郵編300070）；2天津醫(yī)科大學(xué)總醫(yī)院神經(jīng)內(nèi)科

△通訊作者 E-mail:songyijun2000@gmail.com