脑源性神经生长因子对mir124表达及兴奋性电流的影响

王凤军， 王洪朋， 单晶丽， 邵正凯， 姜杨薇， 陈元元， 宋婷婷， 邱丽婷

脑源性神经生长因子对mir124表达及兴奋性电流的影响

王凤军1， 王洪朋2， 单晶丽3， 邵正凯1， 姜杨薇1， 陈元元1， 宋婷婷1， 邱丽婷1

目的 探讨BDNF对海马miRNA-124表达及齿状回颗粒细胞兴奋性突触后电流(sEPSCs)的影响，以明确BDNF对颞叶内侧癫痫(MTLE)发病机制的影响。方法 选取哈尔滨医科大学附属第一临床医院神经外科2008年4月-2010年10月手术治疗的MTLE患者12例。RT-pcr技术检测BDNF孵育后MTLE患者海马组织mir-124表达、膜片钳技术检测BDNF对海马颗粒细胞sEPSCs的影响。结果 (1)BDNF降低了颞叶癫痫海马miRNA-124的表达(P<0.01)；(2)BDNF增加了颗粒细胞sEPSCs的频率和幅度(P<0.01)。结论 BDNF降低了颞叶癫痫海马mir-124的表达，提高颗粒细胞sEPSCs的频率和幅度，从而可能对人类MTLE的发病起到促进作用。

脑源性神经生长因子； 颞叶内侧癫痫； miRNA-124； 自发性兴奋性突触后电流

癫痫是中枢神经系统常见疾病，以反复痫样发作为特征，全世界大约有5000万患者，癫痫的发作类型超过40种，MTLE为最常见的一种难治性癫痫。近年来， 脑源性神经生长因子(BDNF)成为癫痫的研究热点。BDNF于1982年由德国科学家Brade从猪脑里提取的一类神经生长因子[1]，其对维持神经细胞的生理功能有重要的作用，分布广泛，海马和皮质分布最多，其它脑区也有表达[2]。BDNF能够增强大鼠海马齿状回的突触信号传导[3，4]及通过调节NMDA受体的活性来增强突触传递功能[5]和影响TrkB信号而促进了癫痫发病[6]。然而也有报道BDNF对癫痫的发生起到明显抑制作用[7]。BDNF在多种癫痫模型鼠脑内表达明显上调[8～10]。Hideo等报道癫痫动物模型惊厥发作4 h后海马区BDNF miRNA表达明显增高[11]。BDNF的多态性与不同种族之间的联系可能不同[12]及通过改变氯离子协同转运蛋白对颞叶癫痫的发生起到调节作用[13]。众多研究表明BDNF与癫痫二者关系密切，但BDNF对癫痫的发病究竟是促进还是抑制作用不得而知。miRNA对细胞基本生理过程有着重要的调控作用，miRNA的表达异常必然会导致细胞功能紊乱，引起疾病的发生。现今已发现的miRNA中，约70%在哺乳类动物的脑组织中有表达，如脑组织所特有的miRNA(miRNA-124a、miRNA-128、miRNA-101等)，脑组织富含的miRNA(如miRNA-125b)，在神经系统的不同生理过程、不同信号传导通路的基因表达调控，如神经系统发生和发育、神经干细胞分化、细胞凋亡等过程中发挥重要作用[14]。多项研究表明miRNA与癫痫发病密切相关如miRNA-124、miRNA-203能够抑制癫痫的发作[15～17]。部分miRNA在人类和癫痫的模型鼠都会不同程度表达上调[18]。BDNF与miRNA在癫痫的发病机制中可能有交互作用[19]。以上研究表明BDNF与miRNA对颞叶癫痫的发生可能存在一定的作用，探索此种机制或许会成为治疗颞叶内侧癫痫的一个新手段。

1 资料与方法

1.1 试剂与材料 脑源性神经生长因子(BDNF),甲碘荷包牡丹碱(BMI),生物胞素(Biocytin)异去甲槟榔次碱(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以上试剂均购自Sigma公司(St Louis,MO,USA)。超纯水、蒸馏水、组织胶水、PCR 引物(上海生物工程技术有限公司)。

选取哈尔滨医科大学附属第一临床医院神经外科2008年4月-2010年10月手术治疗的MTLE患者12例手术时切下的海马组织为标本，男性7例，女性5例，年龄15～35岁，平均年龄(25.91±5.51)岁。应用BDNF灌流前后对照。

1.2 方法 RT-PCR术中海马组织切下后迅速用冰冷的(4 ℃)S-ACSF保存，使用震动切片机将海马组织切成350-400 μm脑片在培养槽中孵育1 h，槽内装有A-CSF并持续的供给95%O2和5%CO2，温度控制在32 ℃。部分脑片在A-CSF含有BDNF(100 ng/ml)组孵育1 h后与A-CSF组对比检测海马组织mir-124的表达。实验方法按照试剂盒说明书，循环阈值的形式表达结果，均值加减SD，各组间的差异以△△CT=△CT(目的基因)-△CT(actin)，通过2-△△CT方法来检测各组相对miRNA的表达[20]。

Whole-cell记录 术中海马组织切下后迅速用冰冷的(4 ℃)S-ACSF保存，使用震动切片机将海马组织切成350～400 μm脑片，显微镜下齿状回颗粒细胞形成全细胞记录将膜电位钳制在-65 mV,灌流液为ACSF中加入PTX(100 μmol)或BMI(10 μmol)，电压钳下记录sEPSCs，观测应用BDNF后sEPSCs频率、幅度的变化。

2 结 果

2.1 BDNF降低了MTLE脑组织mir-124的表达(P<0.01) 术中海马组织制备350～400 μm脑片，脑片在A-CSF含有BDNF(100 ng/ml)组孵育1 h后与脑片在仅有A-CSF组比较mir-124的表达明显降低(P<0.01)(见图1)。

2.2 BDNF升高了颗粒细胞sEPSCs的频率和幅度 术中海马组织制备350-400 μm脑片，显微镜下识别齿状回颗粒细胞，膜电位钳制-65 mV， 成功记录sEPSCs，灌流液内PTX(100 μmol)或BMI(10 μmol)消除GABA影响。灌流液内加入BDNF(100 ng/ml)。记录6个颗粒细胞测量sEPSC的动力学特性。常规监测输入阻抗(10-25 MΩ)，以输入阻抗变化不超过15%为标准。统计结果表明BDNF应用前后比较sEPSC幅度明显升高，差异显著(P<0.01)；BDNF灌流前后比较sEPSC频率明显升高，差异有统计学意义(P<0.01)。洗脱组与灌流前比较，无统计学意义(P>0.05)(见图2)。

图1 BDNF应用前后mir-124的表达

与灌流前比较**P<0.01；与Washout比较**P<0.01

图2 BDNF上调了颗粒细胞sEPSCs的频率和幅度

3 讨 论

癫痫是最为常见的慢性脑疾病，病因复杂，患病率高为其特点,对癫痫的发病机制研究，在神经科学的研究中占有重要的意义。BDNF为癫痫研究的热点之一，对癫痫的发病作用结果不一甚至有些相对立。多数学者认为BDNF对癫痫的发生有促进作用，BDNF对神经递质有调节作用，可以促进神经元NPY的表达[21]和提高了兴奋性谷氨酸的浓度[22]，及胞膜NMDA受体[23]。BDNF可调节突触的形成，增加树突棘突的密度及TRPC3通道[24,25]。研究表明部分miRNA与癫痫发病密切相关[26]，在人类TLE或动物模型中mir-146a都有较高的表达[27]。有研究表明mir-134有明显的致癫痫作用[28,29]，MiR-155的拮抗剂通过激活BDNF对癫痫持续状态发作起到了保护作用。大多数研究都是以动物癫痫模型为基础，探讨其在癫痫发生发展中的作用，本实验研究以颞叶内侧癫痫患者术中切下的海马为标本。结果表明BDNF对mir-124的表达及sEPSCs的影响表明其对癫痫的发生可能有其促进作用，但二者是否具有相关性，我们将继续进行下一步研究。

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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

黑龙江省教育厅面上项目(No.12541509)

(1.哈尔滨医科大学附属第四医院神经内科，黑龙江 哈尔滨 150001；2.牡丹江市妇女儿童医院,黑龙江 牡丹江 157000;3.哈尔滨市第二医院,黑龙江 哈尔滨 150056) 通迅作者： 王凤军,E-mail:fengjun19791209@163.com

1003-2754(2017)08-0718-03

R742.1

A