TrkB不同亚型对癫海马神经元BDNF/TrkB信号通路调控的研究*

吴秋静 常 伟 潘立平 宋毅军 赵 文

吴秋静 常 伟 潘立平 宋毅军 赵 文△

目的 探讨癫海马神经元中酪氨酸激酶受体B（TrkB）不同亚型对脑源性神经营养因子(BDNF)/TrkB信号通路的调控机制。方法取原代培养7 d后的海马神经元，分为钙调蛋白抑制剂（ALLN）组和翻译抑制剂（Anisomycin）两大组。ALLN组又分为正常组、正常+BNDF组、癫组、癫+BDNF组、正常＋ALLN组、癫+ALLN组和癫+ALLN+BDNF组；Anisomycin组又分为正常组、正常+BDNF组、癫组、癫+BDNF组、正常＋Anisomycin组、癫+Anisomycin组和癫+Anisomycin+BDNF组。免疫荧光鉴定海马神经元，无镁液处理制备癫模型，电生理鉴定细胞痫样放电，免疫印迹技术检测各组TrkB和磷酸化TrkB（p-TrkB）蛋白的表达变化。结果（1）ALLN组：正常+ BDNF组p-TrkB/TrkB灰度值高于正常组；癫+BDNF组高于癫组，低于正常+BDNF组；癫+ALLN+BDNF组低于癫+BDNF组，与癫ALLN组差异无统计学意义。（2）Anisomycin组：正常+BDNF组p-TrkB/TrkB灰度值高于正常组；癫+BDNF组高于癫组，低于正常+BDNF组；癫+Anisomycin+BDNF组高于癫+BDNF组和癫+Anisomycin组。结论通过Anisomycin降低TrkB.T表达可改善癫状态下BDNF/TrkB受抑制的状态，通过ALLN升高TrkB. FL表达无法改善其抑制状态。

癫,颞叶；受体,TrkB；脑源性神经营养因子；茴香霉素；海马；神经元；钙调蛋白抑制剂

1 材料与方法

1.1 实验动物 清洁级健康Sprague Dawley(SD)大鼠24只，其中雌性18只，体质量250～280 g；雄性6只，体质量280～320 g。实验前于实验室正常环境(12 h白昼、12 h夜晚，常温26℃)按比例（雌∶雄为3∶1）合笼饲养，供应足够食水，每日注意观察，备取24 h内新生大鼠10只用于实验，雌雄不限。

1.2 主要试剂 BSC系列生物安全柜(北京东联哈尔仪器制造有限公司)；荧光倒置显微镜(日本OLYMPUS IX-71)；病理图像分析软件(美国Image-pro Plus 7.0)；ND-1000微量蛋白测定仪(美国Nanodrop)；恒温CO2孵箱，Labofoge400R台式低温高速离心机，低温高速离心机(德国Heraeus公司)；Western Blot仪(美国Bio-Rad公司)；DMEM/F12培养基、胎牛血清、0.25%胰蛋白酶-EDTA购自美国Gibco公司；RIPA细胞裂解液、PMSF蛋白酶抑制剂、B27购自中国索来宝公司；TrkB抗体、p-TrkB抗体购自美国SANTA CRUZ公司；山羊抗兔二抗（HRP标记）、山羊抗小鼠二抗（HRP标记）、β-actin抗体购自北京中杉金桥生物技术有限公司。

1.3 方法

1.3.1 海马神经元培养 取新生24 h内SD大鼠，显微镜下分离双侧海马置于D-hanks液，0.125%胰酶于37℃、5%CO2的孵箱消化21 min，以种植液(DMEM/F12+20%血清）终止消化后，调整细胞密度为4×106/mL后种植于涂有0.10 g/L多聚赖氨酸的培养皿里。24 h后全量换液（DMEM/F12+10%血清）继续培养，至第3天时滴加终浓度为5 g/L的阿糖胞苷，第4天全量换含有B2（7DMEM/F12+2%B27）的培养液，根据细胞生长情况，此后3 d可不换液或半量换液。

1.3.3 实验分组 将海马神经元种入培养皿，培养至7 d后进行分组，7盘细胞组成ALLN组，7盘细胞组成Anisomycin组。2组中每盘细胞为1小组，每小组样本数n=5。

ALLN组7个小组分别为：（1）正常组。不予处理。（2）正常+BNDF组。提取总蛋白前10 min加入2 μL 100 μg/L BDNF。（3）癫组。更换无镁细胞外液培养3 h后换回正常培养液。（4）癫+BDNF组。无镁细胞外液培养3 h，换正常培养液培养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组。培养液中加入3.83 μL 50 μmol/L ALLN孵育6.5 h。（6）癫+ALLN组。培养液中加入3.83 μL 50 μmol/L ALLN预孵育2 h，换无镁细胞外液培养3 h，换正常培养液并加入3.83 μL 50 μmol/L ALLN孵育1.5 h。（7）癫+ALLN+BDNF组。培养液中加入3.83 μL 50 μmol/L ALLN预孵育2 h，换无镁细胞外液培养3 h，换含血清培养液并加入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组7个小组分别为：（1）正常组。（2）正常+ BNDF组。（3）癫组。（4）癫+BDNF组。以上各组细胞干预方法同ALLN组。（5）正常＋Anisomycin组。培养液中加入2.65 μL 5 μmol/L Anisomycin孵育4.5 h。（6）癫+Anisomycin组。培养液中加入2.65 μL 5 μmol/L Anisomycin预孵育15 min，换无镁细胞外液培养3 h，最后换正常培养液并加入2.65 μL 5 μmol/L Anisomycin孵育1.5 h。（7）癫+Anisomycin+BDNF组。培养液中加入2.65 μL 5 μmol/L Anisomycin预孵育15 min，换无镁细胞外液培养3 h，换正常培养液并加入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检测TrkB.FL和p-TrkB蛋白表达 细胞培养7 d，ALLN及Anisomycin各实验组分别提取海马神经元总蛋白，使用Nanodrop紫外分光光度计进行蛋白浓度定量。利用Western Blot检测海马神经元中TrkB.FL和p-TrkB蛋白的表达变化。将化学发光方法成像系统的发光图片进行处理，用Photoshop进行反相，利用Quantity One进行灰度值分析。目的蛋白灰度值与内参β-actin进行对比，得到相对值，进行分析处理。

1.4 统计学方法 数据使用SPSS 17.0软件进行统计分析，计量资料以±s表示，多组间比较用单因素方差分析，组间多重比较用LSD-t检验，P＜0.05为差异有统计学意义。

2 结果

2.2 Western Blot结果

2.2.1 ALLN组 正常+BDNF组p-TrkB/TrkB值高于正常组；癫+BDNF组高于癫组，低于正常+ BDNF组；癫+ALLN+BDNF组低于癫+BDNF组（均P＜0.05），与癫+ALLN组差异无统计学意义，见表1、图1。

Table 1 Comparison of the gray values of p-TrkB/ TrkB in cultured hippocampal neurons between ALLN groups表1 ALLN组海马神经元p-TrkB/TrkB灰度值比较（n=5±s）

Table 1 Comparison of the gray values of p-TrkB/ TrkB in cultured hippocampal neurons between ALLN groups表1 ALLN组海马神经元p-TrkB/TrkB灰度值比较（n=5±s）

＊P＜0.05

ALLN组正常组（1）正常+BDNF组（2）癫P组（3）癫+BDNF组（4）正常+ALLN组（5）癫组比（1）∶（2）（2）∶（4）（3）∶（4）（4）∶（7）（6）∶（7）＜0.001 0.030＜0.001 0.002 0.162 +ALLN组（6）癫+ALLN+BDNF组（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图1 ALLN组海马神经元中TrkB、p-TrkB蛋白的表达

2.2.2 Anisomycin组 见表2，图2。正常+BDNF组p-TrkB/TrkB灰度值高于正常组；癫+BDNF组高于癫组，低于正常+BDNF组；癫+Anisomycin+ BDNF组高于癫+BDNF组和癫+Anisomycin组（均P＜0.05）。

Table 2 The gray values of p-TrkB/TrkB in cultured hippocampal neurons in Anisomycin groups表2 海马神经元Anisomycin组中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 海马神经元Anisomycin组中p-TrkB/TrkB灰度值（n=5±s）

＊P＜0.05

P Anisomycin组正常组（1）正常+BDNF组（2）癫 组（3）癫+BDNF组（4）正常+Anisomycin组（5）癫组比（1）∶（2）（2）∶（4）（3）∶（4）（4）∶（7）（6）∶（7）＜0.001 0.046 0.049＜0.001＜0.001 +Anisomycin组（6）癫+Anisomycin+BDNF组（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图2 Anisomycin组海马神经元中TrkB、p-TrkB蛋白的表达

3 讨论

Gomes等[7]通过谷氨酸盐处理海马神经元造成兴奋性中毒，发现加入Anisomycin后，TrkB.T1表达降低。本研究结果发现，癫+Anisomycin+BDNF组p-TrkB/TrkB灰度值是癫+BDNF组的2倍,明显上升并恢复到正常水平。由于Anisomycin主要降低TrkB.T的蛋白表达，由此推断通过Anisomycin降低TrkB.T表达可改善癫中BDNF/TrkB通路的抑制状态，即癫状态下BDNF/TrkB通路的抑制是由上调的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]常伟,潘立平,吴秋静,等.miR-204对癫痫神经元中BDNF-TrkB信号通路表达的研究[J].天津医药,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）

（本文编辑 陈丽洁）

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

*国家自然科学基金资助项目（项目编号：81071044,91132722）

天津医科大学总医院神经内科（邮编300052）

△通讯作者 E-mail：1654167477@qq.com