TrkB不同亞型對癲海馬神經元BDNF/TrkB信號通路調控的研究*

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 探討癲海馬神經元中酪氨酸激酶受體B（TrkB）不同亞型對腦源性神經營養因子(BDNF)/TrkB信號通路的調控機制。方法取原代培養7 d后的海馬神經元，分為鈣調蛋白抑制劑（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元，無鎂液處理制備癲模型，電生理鑒定細胞癇樣放電，免疫印跡技術檢測各組TrkB和磷酸化TrkB（p-TrkB）蛋白的表達變化。結果（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組差異無統計學意義。（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。結論通過Anisomycin降低TrkB.T表達可改善癲狀態下BDNF/TrkB受抑制的狀態，通過ALLN升高TrkB. FL表達無法改善其抑制狀態。

癲,顳葉；受體,TrkB；腦源性神經營養因子；茴香霉素；海馬；神經元；鈣調蛋白抑制劑

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，體質量250～280 g；雄性6只，體質量280～320 g。實驗前于實驗室正常環境(12 h白晝、12 h夜晚，常溫26℃)按比例（雌∶雄為3∶1）合籠飼養，供應足夠食水，每日注意觀察，備取24 h內新生大鼠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，低溫高速離心機(德國Heraeus公司)；Western Blot儀(美國Bio-Rad公司)；DMEM/F12培養基、胎牛血清、0.25%胰蛋白酶-EDTA購自美國Gibco公司；RIPA細胞裂解液、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標記）、山羊抗小鼠二抗（HRP標記）、β-actin抗體購自北京中杉金橋生物技術有限公司。

1.3 方法

1.3.1 海馬神經元培養 取新生24 h內SD大鼠，顯微鏡下分離雙側海馬置于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，調整細胞密度為4×106/mL后種植于涂有0.10 g/L多聚賴氨酸的培養皿里。24 h后全量換液（DMEM/F12+10%血清）繼續培養，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）的培養液，根據細胞生長情況，此后3 d可不換液或半量換液。

1.3.3 實驗分組 將海馬神經元種入培養皿，培養至7 d后進行分組，7盤細胞組成ALLN組，7盤細胞組成Anisomycin組。2組中每盤細胞為1小組，每小組樣本數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。更換無鎂細胞外液培養3 h后換回正常培養液。（4）癲+BDNF組。無鎂細胞外液培養3 h，換正常培養液培養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。培養液中加入3.83 μL 50 μmol/L ALLN孵育6.5 h。（6）癲+ALLN組。培養液中加入3.83 μL 50 μmol/L ALLN預孵育2 h，換無鎂細胞外液培養3 h，換正常培養液并加入3.83 μL 50 μmol/L ALLN孵育1.5 h。（7）癲+ALLN+BDNF組。培養液中加入3.83 μL 50 μmol/L ALLN預孵育2 h，換無鎂細胞外液培養3 h，換含血清培養液并加入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。以上各組細胞干預方法同ALLN組。（5）正常＋Anisomycin組。培養液中加入2.65 μL 5 μmol/L Anisomycin孵育4.5 h。（6）癲+Anisomycin組。培養液中加入2.65 μL 5 μmol/L Anisomycin預孵育15 min，換無鎂細胞外液培養3 h，最后換正常培養液并加入2.65 μL 5 μmol/L Anisomycin孵育1.5 h。（7）癲+Anisomycin+BDNF組。培養液中加入2.65 μL 5 μmol/L Anisomycin預孵育15 min，換無鎂細胞外液培養3 h，換正常培養液并加入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 細胞培養7 d，ALLN及Anisomycin各實驗組分別提取海馬神經元總蛋白，使用Nanodrop紫外分光光度計進行蛋白濃度定量。利用Western Blot檢測海馬神經元中TrkB.FL和p-TrkB蛋白的表達變化。將化學發光方法成像系統的發光圖片進行處理，用Photoshop進行反相，利用Quantity One進行灰度值分析。目的蛋白灰度值與內參β-actin進行對比，得到相對值，進行分析處理。

1.4 統計學方法 數據使用SPSS 17.0軟件進行統計分析，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為差異有統計學意義。

2 結果

2.2 Western Blot結果

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組差異無統計學意義，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組海馬神經元中TrkB、p-TrkB蛋白的表達

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組海馬神經元中TrkB、p-TrkB蛋白的表達

3 討論

Gomes等[7]通過谷氨酸鹽處理海馬神經元造成興奮性中毒，發現加入Anisomycin后，TrkB.T1表達降低。本研究結果發現，癲+Anisomycin+BDNF組p-TrkB/TrkB灰度值是癲+BDNF組的2倍,明顯上升并恢復到正常水平。由于Anisomycin主要降低TrkB.T的蛋白表達，由此推斷通過Anisomycin降低TrkB.T表達可改善癲中BDNF/TrkB通路的抑制狀態，即癲狀態下BDNF/TrkB通路的抑制是由上調的TrkB.T引起的。

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（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

*國家自然科學基金資助項目（項目編號：81071044,91132722）

天津醫科大學總醫院神經內科（郵編300052）

△通訊作者 E-mail：1654167477@qq.com