吳秋靜 常 偉 潘立平 宋毅軍 趙 文

吳秋靜 常 偉 潘立平 宋毅軍 趙 文△

目的 探討癲海馬神經(jīng)元中酪氨酸激酶受體B（TrkB）不同亞型對腦源性神經(jīng)營養(yǎng)因子(BDNF)/TrkB信號通路的調(diào)控機(jī)制。方法取原代培養(yǎng)7 d后的海馬神經(jīng)元，分為鈣調(diào)蛋白抑制劑（ALLN）組和翻譯抑制劑（Anisomycin）兩大組。ALLN組又分為正常組、正常+BNDF組、癲組、癲+BDNF組、正常＋ALLN組、癲+ALLN組和癲+ALLN+BDNF組；Anisomycin組又分為正常組、正常+BDNF組、癲組、癲+BDNF組、正常＋Anisomycin組、癲+Anisomycin組和癲+Anisomycin+BDNF組。免疫熒光鑒定海馬神經(jīng)元，無鎂液處理制備癲模型，電生理鑒定細(xì)胞癇樣放電，免疫印跡技術(shù)檢測各組TrkB和磷酸化TrkB（p-TrkB）蛋白的表達(dá)變化。結(jié)果（1）ALLN組：正常+ BDNF組p-TrkB/TrkB灰度值高于正常組；癲+BDNF組高于癲組，低于正常+BDNF組；癲+ALLN+BDNF組低于癲+BDNF組，與癲ALLN組差異無統(tǒng)計學(xué)意義。（2）Anisomycin組：正常+BDNF組p-TrkB/TrkB灰度值高于正常組；癲+BDNF組高于癲組，低于正常+BDNF組；癲+Anisomycin+BDNF組高于癲+BDNF組和癲+Anisomycin組。結(jié)論通過Anisomycin降低TrkB.T表達(dá)可改善癲狀態(tài)下BDNF/TrkB受抑制的狀態(tài)，通過ALLN升高TrkB. FL表達(dá)無法改善其抑制狀態(tài)。

癲,顳葉；受體,TrkB；腦源性神經(jīng)營養(yǎng)因子；茴香霉素；海馬；神經(jīng)元；鈣調(diào)蛋白抑制劑

1 材料與方法

1.1 實驗動物 清潔級健康Sprague Dawley(SD)大鼠24只，其中雌性18只，體質(zhì)量250～280 g；雄性6只，體質(zhì)量280～320 g。實驗前于實驗室正常環(huán)境(12 h白晝、12 h夜晚，常溫26℃)按比例（雌∶雄為3∶1）合籠飼養(yǎng)，供應(yīng)足夠食水，每日注意觀察，備取24 h內(nèi)新生大鼠10只用于實驗，雌雄不限。

1.2 主要試劑 BSC系列生物安全柜(北京東聯(lián)哈爾儀器制造有限公司)；熒光倒置顯微鏡(日本OLYMPUS IX-71)；病理圖像分析軟件(美國Image-pro Plus 7.0)；ND-1000微量蛋白測定儀(美國Nanodrop)；恒溫CO2孵箱，Labofoge400R臺式低溫高速離心機(jī)，低溫高速離心機(jī)(德國Heraeus公司)；Western Blot儀(美國Bio-Rad公司)；DMEM/F12培養(yǎng)基、胎牛血清、0.25%胰蛋白酶-EDTA購自美國Gibco公司；RIPA細(xì)胞裂解液、PMSF蛋白酶抑制劑、B27購自中國索來寶公司；TrkB抗體、p-TrkB抗體購自美國SANTA CRUZ公司；山羊抗兔二抗（HRP標(biāo)記）、山羊抗小鼠二抗（HRP標(biāo)記）、β-actin抗體購自北京中杉金橋生物技術(shù)有限公司。

1.3 方法

1.3.1 海馬神經(jīng)元培養(yǎng) 取新生24 h內(nèi)SD大鼠，顯微鏡下分離雙側(cè)海馬置于D-hanks液，0.125%胰酶于37℃、5%CO2的孵箱消化21 min，以種植液(DMEM/F12+20%血清）終止消化后，調(diào)整細(xì)胞密度為4×106/mL后種植于涂有0.10 g/L多聚賴氨酸的培養(yǎng)皿里。24 h后全量換液（DMEM/F12+10%血清）繼續(xù)培養(yǎng)，至第3天時滴加終濃度為5 g/L的阿糖胞苷，第4天全量換含有B2（7DMEM/F12+2%B27）的培養(yǎng)液，根據(jù)細(xì)胞生長情況，此后3 d可不換液或半量換液。

1.3.3 實驗分組 將海馬神經(jīng)元種入培養(yǎng)皿，培養(yǎng)至7 d后進(jìn)行分組，7盤細(xì)胞組成ALLN組，7盤細(xì)胞組成Anisomycin組。2組中每盤細(xì)胞為1小組，每小組樣本數(shù)n=5。

ALLN組7個小組分別為：（1）正常組。不予處理。（2）正常+BNDF組。提取總蛋白前10 min加入2 μL 100 μg/L BDNF。（3）癲組。更換無鎂細(xì)胞外液培養(yǎng)3 h后換回正常培養(yǎng)液。（4）癲+BDNF組。無鎂細(xì)胞外液培養(yǎng)3 h，換正常培養(yǎng)液培養(yǎng)80 min后提取蛋白，于提取蛋白前10 min加入2 μL 100 μg/L BDNF。（5）正常＋ALLN組。培養(yǎng)液中加入3.83 μL 50 μmol/L ALLN孵育6.5 h。（6）癲+ALLN組。培養(yǎng)液中加入3.83 μL 50 μmol/L ALLN預(yù)孵育2 h，換無鎂細(xì)胞外液培養(yǎng)3 h，換正常培養(yǎng)液并加入3.83 μL 50 μmol/L ALLN孵育1.5 h。（7）癲+ALLN+BDNF組。培養(yǎng)液中加入3.83 μL 50 μmol/L ALLN預(yù)孵育2 h，換無鎂細(xì)胞外液培養(yǎng)3 h，換含血清培養(yǎng)液并加入3.83 μL 50 μmol/L ALLN孵育1.5 h，于提取蛋白前10 min加入2 μL 100 μg/L BDNF。

Anisomycin組7個小組分別為：（1）正常組。（2）正常+ BNDF組。（3）癲組。（4）癲+BDNF組。以上各組細(xì)胞干預(yù)方法同ALLN組。（5）正常＋Anisomycin組。培養(yǎng)液中加入2.65 μL 5 μmol/L Anisomycin孵育4.5 h。（6）癲+Anisomycin組。培養(yǎng)液中加入2.65 μL 5 μmol/L Anisomycin預(yù)孵育15 min，換無鎂細(xì)胞外液培養(yǎng)3 h，最后換正常培養(yǎng)液并加入2.65 μL 5 μmol/L Anisomycin孵育1.5 h。（7）癲+Anisomycin+BDNF組。培養(yǎng)液中加入2.65 μL 5 μmol/L Anisomycin預(yù)孵育15 min，換無鎂細(xì)胞外液培養(yǎng)3 h，換正常培養(yǎng)液并加入2.65 μL 5 μmol/L Anisomycin孵育1.5 h，于提取蛋白前10 min加入2 μL 100 μg/L BDNF。

1.3.4 Western Blot檢測TrkB.FL和p-TrkB蛋白表達(dá) 細(xì)胞培養(yǎng)7 d，ALLN及Anisomycin各實驗組分別提取海馬神經(jīng)元總蛋白，使用Nanodrop紫外分光光度計進(jìn)行蛋白濃度定量。利用Western Blot檢測海馬神經(jīng)元中TrkB.FL和p-TrkB蛋白的表達(dá)變化。將化學(xué)發(fā)光方法成像系統(tǒng)的發(fā)光圖片進(jìn)行處理，用Photoshop進(jìn)行反相，利用Quantity One進(jìn)行灰度值分析。目的蛋白灰度值與內(nèi)參β-actin進(jìn)行對比，得到相對值，進(jìn)行分析處理。

1.4 統(tǒng)計學(xué)方法 數(shù)據(jù)使用SPSS 17.0軟件進(jìn)行統(tǒng)計分析，計量資料以±s表示，多組間比較用單因素方差分析，組間多重比較用LSD-t檢驗，P＜0.05為差異有統(tǒng)計學(xué)意義。

2 結(jié)果

2.2 Western Blot結(jié)果

2.2.1 ALLN組 正常+BDNF組p-TrkB/TrkB值高于正常組；癲+BDNF組高于癲組，低于正常+ BDNF組；癲+ALLN+BDNF組低于癲+BDNF組（均P＜0.05），與癲+ALLN組差異無統(tǒng)計學(xué)意義，見表1、圖1。

Table 1 Comparison of the gray values of p-TrkB/ TrkB in cultured hippocampal neurons between ALLN groups表1 ALLN組海馬神經(jīng)元p-TrkB/TrkB灰度值比較（n=5±s）

Table 1 Comparison of the gray values of p-TrkB/ TrkB in cultured hippocampal neurons between ALLN groups表1 ALLN組海馬神經(jīng)元p-TrkB/TrkB灰度值比較（n=5±s）

＊P＜0.05

ALLN組正常組（1）正常+BDNF組（2）癲P組（3）癲+BDNF組（4）正常+ALLN組（5）癲組比（1）∶（2）（2）∶（4）（3）∶（4）（4）∶（7）（6）∶（7）＜0.001 0.030＜0.001 0.002 0.162 +ALLN組（6）癲+ALLN+BDNF組（7）Fp-TrkB/TrkB灰度值1.00±0.00 3.23±0.16 1.15±0.11 2.61±0.19 1.01±0.08 1.33±0.19 1.71±0.31 21.100＊

Figure 1 Expressions of TrkB and p-TrkB proteins in cultured hippocampal neurons in ALLN groups圖1 ALLN組海馬神經(jīng)元中TrkB、p-TrkB蛋白的表達(dá)

2.2.2 Anisomycin組 見表2，圖2。正常+BDNF組p-TrkB/TrkB灰度值高于正常組；癲+BDNF組高于癲組，低于正常+BDNF組；癲+Anisomycin+ BDNF組高于癲+BDNF組和癲+Anisomycin組（均P＜0.05）。

Table 2 The gray values of p-TrkB/TrkB in cultured hippocampal neurons in Anisomycin groups表2 海馬神經(jīng)元Anisomycin組中p-TrkB/TrkB灰度值（n=5±s）

Table 2 The gray values of p-TrkB/TrkB in cultured hippocampal neurons in Anisomycin groups表2 海馬神經(jīng)元Anisomycin組中p-TrkB/TrkB灰度值（n=5±s）

＊P＜0.05

P Anisomycin組正常組（1）正常+BDNF組（2）癲 組（3）癲+BDNF組（4）正常+Anisomycin組（5）癲組比（1）∶（2）（2）∶（4）（3）∶（4）（4）∶（7）（6）∶（7）＜0.001 0.046 0.049＜0.001＜0.001 +Anisomycin組（6）癲+Anisomycin+BDNF組（7）Fp-TrkB/TrkB灰度值1.00±0.00 3.39±0.12 0.96±0.03 2.17±0.30 1.22±0.02 1.73±0.43 4.52±0.85 10.532＊

Figure 2 Expressions of TrkB and p-TrkB proteins in cultured hippocampal neurons in Anisomycin groups圖2 Anisomycin組海馬神經(jīng)元中TrkB、p-TrkB蛋白的表達(dá)

3 討論

Gomes等[7]通過谷氨酸鹽處理海馬神經(jīng)元造成興奮性中毒，發(fā)現(xiàn)加入Anisomycin后，TrkB.T1表達(dá)降低。本研究結(jié)果發(fā)現(xiàn)，癲+Anisomycin+BDNF組p-TrkB/TrkB灰度值是癲+BDNF組的2倍,明顯上升并恢復(fù)到正常水平。由于Anisomycin主要降低TrkB.T的蛋白表達(dá)，由此推斷通過Anisomycin降低TrkB.T表達(dá)可改善癲中BDNF/TrkB通路的抑制狀態(tài)，即癲狀態(tài)下BDNF/TrkB通路的抑制是由上調(diào)的TrkB.T引起的。

[1]Yoshii A,Constantine-Paton M.BDNF induces transport of PSD-95 to dendrites through PI3K-AKT signaling after NMDA receptor activation[J].Nat Neurosci,2007,10(6):702-711.

[2]Miyata K,Omori N,Uchino H,et al.Involvement of the brain-derived neurotrophic factor/TrkB pathway in neuroprotecive effect of cyclosporin A in forebrain ischemia[J].Neuroscience,2001,105(3): 571-578.

[3]Gill R,Chang PK,Prenosil GA,et al.Blocking brain-derived neurotrophic factor inhibits injury-induced hyperexcitability of hippocampal CA3 neurons[J].Eur J Neurosci，2013,38(11):3554-3566. doi:10.1111/ejn.12367.

[4]Sharma AV,Nargang FE,Dickson CT.Neurosilence:profound suppression of neural activity following intracerebral administration of the protein synthesis inhibitor anisomycin[J].J Neurosi,2012,32(7): 2377-2387.doi:10.1523/JNEUROSCI.3543-11.2012.

[5]常偉,潘立平,吳秋靜,等.miR-204對癲癇神經(jīng)元中BDNF-TrkB信號通路表達(dá)的研究[J].天津醫(yī)藥,2014,42(3):214-216.doi: 10.3969/j.issn.0253-9896.2014.03.007.

[6]Vidaurre OG,Gascón S,Deogracias R,et al.Imbalance of neurotrophin receptor isoforms TrkB-FL/TrkB-T1 induces neuronal death in excitotoxicity[J].Cell Death Dis,2012,3(1):e256.doi: 10.1038/cddis.2011.143.

[7]Gomes JR,Costa JT,Melo CV,et al.Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons[J].J Neurosci,2012,32(13):4610-4622.doi:10.1523/JNEUROSCI.0374-12.2012.

[8]Gao H,Geng Z.Calpain I activity and its relationship with hippocampal neuronal death in pilocarpine-induced status epilepticus rat model[J].Cell Biochem Biophys,2013,66(2):371-377.doi: 10.1007/s12013-012-9476-5.

（2013-10-01收稿 2014-02-24修回）

（本文編輯 陳麗潔）

An Experimental Study of the Regulation of BDNF/TrkB Signal Pathway by Different Isoforms of TrkB in Epileptic Hippocampal Neurons

WU Qiujing,CHANG Wei,PAN Liping,SONG Yijun，ZHAO Wen
Department of Neurology，the General Hospital of Tianjin Medical University,Tianjin 300052,China

ObjectiveTo investigate the mechanism of brain derived neurotrophic factor(BDNF)regulated by different isoforms of tyrosine kinase receptor B(TrkB)in epileptic hippocampal neurons.MethodsPrimary hippocampal neurons were cultured in vitro for 7 days,and divided into two groups,ALLN(calcineurin inhibitor)group and Anisomycin(translation inhibitor)group.ALLN group included control group,control+BDNF group,epilepsy group,epilepsy+BDNF group, control+ALLN group,epilepsy+ALLN group and epilepsy+ALLN+BDNF group.Anisomycin group was sub-divided into control group,control+BDNF group,epilepsy group,epilepsy+BDNF group,control+Anisomycin group,epilepsy+Anisomycin group and epilepsy+Anisomycin+BDNF group.The immunofluorescent technique was used to identificate the hippocampal neurons.Epileptiform discharges were detected by electrophysiological techniques.Western blot assay was used to determine the protein expression of TrkB and phosphorylated TrkB(p-TrkB)in all cell groups.Results(1)In ALLN group,the gray value of p-TrkB/TrkB was higher in control+BDNF group compared with that of control group,the value was higher in epilepsy+BDNF group than that of epilepsy group but was lower than that of control+BDNF group.The gray value of p-TrkB/ TrkB was lower in epilepsy+ALLN+BDNF group than that of epilepsy+BDNF group,but no significant difference compared with that of epilepsy+ALLN group.(2)In Anisomycin group:the gray value of p-TrkB/TrkB was higher in control+BDNF group than that of control group.The gray value of p-TrkB/TrkB was higher in epilepsy+BDNF group than that of epilepsy group,but which was lower than that of control+BDNF group.The gray value of p-TrkB/TrkB was higher in epilepsy+Anisomycin+BDNF group than that of epilepsy+BDNF group and epilepsy+Anisomycin group.ConclusionThe decreased expression of TrkB.T can improve the inhibition of BDNF/TrkB signaling,and BDNF can activate BDNF/TrkB signal pathway in epileptic hippocampal neurons.The increased TrkB.FL protein level by ALLN can’t improve the inhibition of BDNF/ TrkB signal pathway.

epilepsy,temporal lobe;receptor,TrkB;brain-derived neurotrophic factor;ANISOMYCIN;hippocampus;neurons;N-Acetyl-L-leucyl-L-leucyl-L-norleucinal

R742.1

A

10.3969/j.issn.0253-9896.2014.05.002

*國家自然科學(xué)基金資助項目（項目編號：81071044,91132722）

天津醫(yī)科大學(xué)總醫(yī)院神經(jīng)內(nèi)科（郵編300052）

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