聽(tīng)覺(jué)剝奪及耳蝸內(nèi)電刺激幼鼠聽(tīng)皮層和下丘核CREB和NMDAR1蛋白表達(dá)變化*

樊碧云　盧振東　程嵐　楊軍

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·實(shí)驗(yàn)研究·

聽(tīng)覺(jué)剝奪及耳蝸內(nèi)電刺激幼鼠聽(tīng)皮層和下丘核CREB和NMDAR1蛋白表達(dá)變化*

樊碧云1盧振東1程嵐1楊軍1

【摘要】目的觀察耳聾幼鼠及其耳聾后單耳植入電極電刺激后幼鼠聽(tīng)皮層和下丘核環(huán)磷酸腺苷反應(yīng)元件結(jié)合蛋白(cyclic AMP response element- binding protein, CREB)和N-甲基-D-天冬氨酸受體-1(N-methyl-D-aspartic acid receptor one,NMDAR1)表達(dá)水平的變化。方法將66只12天齡SD幼鼠隨機(jī)分為2大組，分別為耳聾造模后4周組(33只)及耳聾造模后6周組(33只)。將耳聾造模后4周組再分為對(duì)照1組、耳聾造模后4周組(4周組)及耳聾造模后3周耳蝸內(nèi)電刺激組1(刺激時(shí)間為1周，簡(jiǎn)稱“電刺激1組”)，每組11只大鼠；將耳聾造模后6周組再分為對(duì)照2組、耳聾造模后6周組(6周組)及耳聾造模后5周耳蝸內(nèi)電刺激組2(刺激時(shí)間為1周，簡(jiǎn)稱“電刺激2組”)，每組11只大鼠；對(duì)照1、2組均正常飼養(yǎng)。除對(duì)照1、2組外，在其余4組幼鼠頸背部、兩側(cè)下腹部皮下注射慶大霉素(總量為350 mg/kg)，半小時(shí)后于相同部位注射呋塞米(總量為200 mg/kg)，兩周后行ABR檢測(cè)，于耳聾造模成功后第3、5周分別對(duì)電刺激1、2組的幼鼠植入電極，在耳蝸內(nèi)進(jìn)行電刺激，每天3小時(shí)，持續(xù)7天。于耳聾造模后4、6周分別處死4、6周組大鼠取聽(tīng)皮層和下丘組織，通過(guò)免疫組織化學(xué)方法，觀察CREB和NMDAR1表達(dá)水平的變化。結(jié)果耳聾造模成功后幼鼠ABR閾值均大于93 dB SPL，4周組聽(tīng)皮層、下丘CREB和NMDAR1的表達(dá)較對(duì)照1組增加，電刺激1組CREB和NMDAR1的表達(dá)較4周組增加。6周組聽(tīng)皮層和下丘CREB和NMDAR1的表達(dá)較對(duì)照2組下降，電刺激2組CREB和NMDAR1的表達(dá)較6周組增加。結(jié)論聽(tīng)覺(jué)剝奪可導(dǎo)致幼鼠聽(tīng)皮層和下丘CREB和NMDAR1早期表達(dá)增加而晚期表達(dá)下降。耳蝸植入電極電刺激可導(dǎo)致幼鼠聽(tīng)皮層和下丘CREB和NMDAR1的表達(dá)增加，反映這兩個(gè)部位神經(jīng)元的可塑性變化。

【關(guān)鍵詞】聽(tīng)覺(jué)可塑性；環(huán)磷酸腺苷反應(yīng)元件結(jié)合蛋白；N-甲基-D-天冬氨酸受體-1；電刺激；感音神經(jīng)性聾

以往研究觀察了耳聾幼鼠及耳聾后單耳植入電極電刺激后幼鼠聽(tīng)覺(jué)中樞的聽(tīng)皮層、下丘及耳蝸核部位的腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)和即刻早期基因之一c-fos的基因及蛋白表達(dá)水平的變化，發(fā)現(xiàn)BDNF與其受體酪氨酸蛋白激酶B(T yrosine receptor kinase-B, TrkB) 結(jié)合后, 磷酸化激活的TrkB 可進(jìn)一步激活核內(nèi)轉(zhuǎn)錄調(diào)控因子環(huán)磷酸腺苷反應(yīng)元件結(jié)合蛋白(cyclic AMP response element- binding protein, CREB)刺激BDNF的轉(zhuǎn)錄，從而促進(jìn)神經(jīng)元的生長(zhǎng)、保護(hù)受損神經(jīng)元[1]。C-fos作為即刻早期基因的一種，其表達(dá)與中樞神經(jīng)系統(tǒng)的功能可塑性相關(guān)[2]，而N-甲基-D-天冬氨酸受體(N-methyl-D-aspartic acid receptor,NMDA)通過(guò)其結(jié)構(gòu)的變化，介導(dǎo)Ca2+內(nèi)流的調(diào)控，調(diào)節(jié)神經(jīng)元內(nèi)Ca2+依賴的第二信使系統(tǒng)，作用于c-fos基因上的鈣反應(yīng)序列啟動(dòng)c-fos基因，誘導(dǎo)c-fos基因表達(dá)增高[3]；因此，在BDNF、c-fos的表達(dá)過(guò)程中其傳導(dǎo)通路中的CREB和N-甲基-D-天冬氨酸受體-1(N-methyl-D-aspartic acid receptor one,NMDAR1)發(fā)揮著重要介導(dǎo)作用；而聽(tīng)覺(jué)剝奪和耳蝸內(nèi)電刺激致幼鼠聽(tīng)皮層和下丘核CREB和NMDAR1蛋白表達(dá)的變化目前國(guó)內(nèi)外尚未見(jiàn)報(bào)道。氨基糖苷類抗生素聯(lián)合呋塞米可損傷Corti器的外毛細(xì)胞導(dǎo)致聽(tīng)力障礙[4]，SD大鼠幼鼠在出生后14天Corti器才發(fā)育成熟，才有聽(tīng)力[5]，在此期間注射耳毒性藥物可造成其類似先天性感音神經(jīng)性聾；因此，本研究擬通過(guò)皮下注射耳毒性藥物建立先天性感音神經(jīng)性聾大鼠模型，然后通過(guò)耳蝸植入電極進(jìn)行電刺激，觀察不同時(shí)間模型大鼠聽(tīng)皮層及下丘核CREB和NMDAR1的表達(dá)變化，以進(jìn)一步探討聽(tīng)覺(jué)剝奪和耳蝸內(nèi)電刺激后幼鼠聽(tīng)皮層和下丘核CREB和NMDAR1蛋白表達(dá)變化的可能機(jī)制。1材料與方法

1.1實(shí)驗(yàn)動(dòng)物及分組取出生后12天齡SD大鼠幼鼠66只(上海交通大學(xué)附屬新華醫(yī)院動(dòng)物實(shí)驗(yàn)中心提供)，雌雄不拘，耳廓反應(yīng)靈敏，體重23～28 g。隨機(jī)分為2大組，分別為耳聾造模后4周組(33只)及耳聾造模后6周組(33只)，將耳聾造模后4周組再分為對(duì)照1組、耳聾造模后4周組(4周組)及耳聾造模后3周耳蝸內(nèi)電刺激1組(刺激時(shí)間為1周，簡(jiǎn)稱“電刺激1組”)，每組11只；將耳聾造模后6周組再分為對(duì)照2組、耳聾造模后6周組(6周組)以及耳聾造模后5周接受耳蝸內(nèi)電刺激2組(刺激時(shí)間為1周，簡(jiǎn)稱“電刺激2組”)，每組11只。

1.2耳聾動(dòng)物模型的建立對(duì)照1、2組均正常飼養(yǎng)，其余4組分別在幼鼠頸背部、兩側(cè)下腹部皮下注射慶大霉素(總量350 mg/kg，上海中西制藥有限公司)，半小時(shí)后于相同部位注射呋塞米(總量200 mg/kg，上海禾豐制藥有限公司)，兩周后行ABR檢測(cè)。

1.3耳蝸內(nèi)電刺激方法于造模成功后第3、5周分別對(duì)電刺激1組、電刺激2組幼鼠行全麻后，于右側(cè)耳后切口，聽(tīng)泡骨壁鉆孔，暴露耳蝸。耳蝸底回鼓階鉆孔，電極貼近鼓階蝸軸插入大約3 mm，用肌肉封住切開(kāi)的圓窗；參考電極置于聽(tīng)泡附近的顳肌內(nèi)。用超強(qiáng)膠將刺激電極固定于動(dòng)物顳骨，頭皮縫合固定電極。刺激電極使用動(dòng)物用鉑銥合金球形電極。使用SEN-7203刺激器(日本光電公司)進(jìn)行耳蝸內(nèi)電刺激，刺激電流參數(shù)為電流強(qiáng)度0.5 mA，脈寬20～100 μs，頻率30～2 000 Hz，均為雙相脈沖[6]，每天3小時(shí)，持續(xù)7天。

1.4免疫組織化學(xué)染色法 分別于耳聾造模后4周、6周將4周組、6周組大鼠行心臟灌注，處死動(dòng)物，斷頭，根據(jù)大鼠腦立體定位圖[7]，取左側(cè)聽(tīng)皮層和下丘，以4%多聚甲醛固定，經(jīng)脫水、透明、浸蠟、石蠟包埋，連續(xù)冠狀位切片，片厚5 μm。分別進(jìn)行免疫組織化學(xué)染色；按SABC 免疫組化檢測(cè)聽(tīng)皮層和下丘的CREB和NMDAR1的表達(dá)，嚴(yán)格按試劑盒說(shuō)明書操作，兔抗鼠NMDAR1抗體和兔抗鼠CREB抗體均取自Abcam公司(英國(guó))。采集圖像時(shí)均在同一光強(qiáng)度、同一放大倍數(shù)下進(jìn)行，每個(gè)標(biāo)本取5張切片，每張切片取5個(gè)不同的視野，進(jìn)行積分光密度值(IOD)分析，取平均值為每張切片的平均IOD值，計(jì)算每組動(dòng)物的平均IOD值。

圖1　耳聾造模4周后對(duì)照1組、4周組、電刺激1組下丘內(nèi)NMDAR1蛋白(a～c)和CREB蛋白(d～f)的表達(dá) (DAB×400)

圖2　耳聾造模6周后對(duì)照2組、6周組、電刺激2組下丘內(nèi)NMDAR1蛋白(a～c)和CREB蛋白(d～f)的表達(dá)(DAB×400)

1.5統(tǒng)計(jì)學(xué)方法所有數(shù)據(jù)采用SPSS17.0 軟件包進(jìn)行統(tǒng)計(jì)學(xué)分析，組間比較采用單因素方差分析，P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1ABR檢測(cè)結(jié)果耳聾造模完成后幼鼠ABR閾值均大于93 dB SPL，根據(jù)Tan等[8]建立的耳聾模型標(biāo)準(zhǔn)，本實(shí)驗(yàn)耳聾動(dòng)物模型建立成功。

2.2各組聽(tīng)皮層及下丘CREB、NMDAR1蛋白表達(dá)耳聾造模后4周，在聽(tīng)皮層和下丘中，4周組與對(duì)照1組相比、電刺激1組與4周組相比，CREB、NMDAR1蛋白的表達(dá)量均增加(P<0.05)；耳聾造模后6周，在聽(tīng)皮層和下丘中，6周組與對(duì)照2組相比，CREB、NMDAR1蛋白的表達(dá)量均降低(P<0.05)，電刺激2組與6周組相比CREB、NMDAR1蛋白的表達(dá)量均增加(P<0.05)(表1、2，圖1～4)。

圖3　耳聾造模4周后對(duì)照1組、4周組、電刺激1組聽(tīng)皮層內(nèi)NMDAR1蛋白(a～c)和CREB蛋白(d～f)的表達(dá) (DAB×400)

圖4　耳聾造模6周后對(duì)照2組、6周組、電刺激2組聽(tīng)皮層內(nèi)NMDAR1蛋白(a～c)和CREB蛋白(d～f)的表達(dá) (DAB×400)

組別NMDAR1 聽(tīng)皮層 下丘 CREB 聽(tīng)皮層 下丘 對(duì)照1組53.20±1.08*36.23±0.89*95.88±1.02*71.56±2.31*4周組68.44±1.7844.76±0.46111.96±0.52110.42±1.65電刺激1組87.80±1.36*80.50±1.06*144.72±1.30*123.90±1.21*

注：*與4周組比較，P<0.05

表2　耳聾造模后6周各組聽(tīng)皮層、下丘NMDAR1和CREB

注：*與6周組比較，P<0.05

3討論

CREB是真核生物細(xì)胞核內(nèi)的一種蛋白質(zhì)，它的功能是調(diào)節(jié)基因轉(zhuǎn)錄，被稱為調(diào)節(jié)轉(zhuǎn)錄的核因子，它的存在能刺激BDNF等基因的轉(zhuǎn)錄，所以又稱之為轉(zhuǎn)錄增強(qiáng)因子[9]。此外，Alvarado研究發(fā)現(xiàn)BDNF為CREB的靶基因，被激活的CREB能夠結(jié)合CRE并增強(qiáng)BDNF的轉(zhuǎn)錄，使其mRNA表達(dá)量增高，并且加強(qiáng)BDNF翻譯，使BDNF蛋白表達(dá)上調(diào)[10]。BDNF作為神經(jīng)營(yíng)養(yǎng)因子家族中重要成員，可以調(diào)節(jié)神經(jīng)細(xì)胞間突觸的傳遞，修復(fù)受損神經(jīng)元，維持神經(jīng)元存活，促進(jìn)其生長(zhǎng)[11]。尤其在內(nèi)耳神經(jīng)細(xì)胞和感覺(jué)上皮細(xì)胞的發(fā)生發(fā)育過(guò)程中起重要作用，能夠減少耳毒性藥物、噪聲等因素對(duì)毛細(xì)胞的損害，維持SGNs的存活[12～14]。前文也提到NMDAR1蛋白可以誘導(dǎo)c-fos基因表達(dá)增高，促進(jìn)神經(jīng)元修復(fù)。文中結(jié)果顯示，SD幼鼠耳聾造模后4周，在聽(tīng)皮層和下丘中，與對(duì)照1組相比耳聾4周組CREB、NMDAR1蛋白的表達(dá)量均增加；而耳聾造模后6周，與對(duì)照2組相比耳聾6周組聽(tīng)皮層和下丘中，CREB、NMDAR1蛋白的表達(dá)量均降低。因此推測(cè)耳聾造模后4周CREB、NMDAR1表達(dá)增多可能是由于耳毒性藥物損傷外毛細(xì)胞，導(dǎo)致聽(tīng)神經(jīng)傳入減少，具有活性的神經(jīng)元代償性表達(dá)CREB和NMDAR1，使其表達(dá)上升，導(dǎo)致BDNF和c-fos的表達(dá)上升，來(lái)維持正常的突觸傳遞、發(fā)育；而耳聾造模后6周CREB、NMDAR1表達(dá)降低可能是由于長(zhǎng)時(shí)間的代償作用加劇神經(jīng)元的死亡，失去神經(jīng)元活動(dòng)的控制后，兩種蛋白的表達(dá)量下降，導(dǎo)致BDNF和c-fos的表達(dá)下降，神經(jīng)元不能獲得足夠的營(yíng)養(yǎng)支持，進(jìn)一步加劇神經(jīng)元的死亡。

有學(xué)者研究發(fā)現(xiàn)電針刺激可使慢性應(yīng)激大鼠海馬CREB的表達(dá)升高[15]，本研究發(fā)現(xiàn)耳蝸內(nèi)植入電極電刺激亦可使CREB在聽(tīng)皮層及下丘核表達(dá)增多。CREB表達(dá)的上調(diào)可作用于其靶基因BDNF，導(dǎo)致BDNF表達(dá)增多。BDNF對(duì)突觸前興奮性谷氨酸遞質(zhì)的釋放及突觸后膜NMDA受體均有調(diào)控作用，可上調(diào)NMDA受體的功能[16]。NMDA 受體有NMDAR1和NMDAR2兩個(gè)亞單位，NMDAR1是主要的功能單位。激活后的NMDA受體可引起神經(jīng)元細(xì)胞膜對(duì)Ca2+等離子的通透性增強(qiáng)，產(chǎn)生興奮性突觸后電位[17]。

耳蝸電刺激可引起NMDA的受體NMDAR1在耳蝸神經(jīng)后核、腹側(cè)核及中央核等處表達(dá)增多[18]。NMDAR1受體介導(dǎo)的Ca2+內(nèi)流的調(diào)控，調(diào)節(jié)神經(jīng)元內(nèi)Ca2+依賴的第二信使系統(tǒng)，最終實(shí)現(xiàn)對(duì)中樞神經(jīng)系統(tǒng)發(fā)育過(guò)程的復(fù)雜調(diào)控[19]。從本研究結(jié)果看，電刺激亦可引起聽(tīng)皮層和下丘核NMDAR1表達(dá)增多。推測(cè)是因?yàn)槎亙?nèi)的電刺激使CREB的表達(dá)上調(diào)促使BDNF表達(dá)增多，同時(shí)，BDNF促進(jìn)NMDAR1表達(dá)上調(diào)恢復(fù)神經(jīng)元的興奮性，使得電刺激在突觸傳遞、突觸發(fā)育及可塑性方面發(fā)揮重要作用[20，21]。

CREB和NMDAR1作為分子工具被用于監(jiān)測(cè)聽(tīng)覺(jué)剝奪后聽(tīng)覺(jué)神經(jīng)系統(tǒng)的變化，其表達(dá)水平反映了神經(jīng)元及突觸的生長(zhǎng)發(fā)育可塑性變化，可用于監(jiān)測(cè)損傷誘導(dǎo)的神經(jīng)活動(dòng)變化。本研究著重于觀察幼年大鼠聽(tīng)覺(jué)剝奪后及電刺激后聽(tīng)皮層、下丘核CREB和NMDAR1的表達(dá)變化，至于CREB和NMDAR1在聽(tīng)覺(jué)中樞其它核團(tuán)中如何變化及其變化具體機(jī)制尚待進(jìn)一步研究。

4參考文獻(xiàn)

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(2015-12-09收稿)

(本文編輯周濤)

網(wǎng)絡(luò)出版時(shí)間：2016-4-2615：53

網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/42.1391.R.20160426.1553.034.html

Variation of CREB and NMDAR1 Proteins Induced by Auditory Deprivation and Intracochlear Electrical Stimulation in Infant Rats

Fan Biyun， Lu Zhendong， Cheng Lan， Yang Jun

(Department of Otolaryngology-Head and Neck Surgery，Shanghai Xinhua Hospital，Affiliated to Shanghai Jiaotong University of Medicine，Ear Institute，Shanghai，200092，China)

【Abstract】ObjectiveTo observe the expression of cAMP-response element binding protein (CREB) and N-methyl-D-aspartic acid receptor (NMDA) after intracochlear electrical stimulation in the auditory cortex and inferior colliculus in infant rats with auditory deprivation. MethodsSixty six SD infant rats were randomly divided into 6 groups (11 rats each group): 4 weeks, and 6 weeks after injection of ototoxic drug, the control group, and 3 weeks and 5 weeks after injection of ototoxic drug with intra-cochlear electrical stimulation for one week. Gentamicin sulphate (350 mg/kg body weight) and frusemide (200 mg/kg body weight) were injected subcutaneously in the skin folds on the lateral abdominal side and the dorsal neck area, respectively. The expression of CREB and NMDAR1 protein were detected by immunohistological staining. ResultsThe results of immunohisto-chemistry revealed that protein expression of CREB and NMDAR1 in 4 week group of injection increased as compared to the control group, while decreasing as compared to intracochlear electrical stimulation group, significantly. However, protein expression of CREB and NMDAR1 in 6 week group of injection decreased as compared to the control group and intracochlear electrical stimulation group, significantly. ConclusionAuditory deprivation could result in the expression of protein of CREB and NMDAR1 in auditory cortex and inferior colliculus increasing in an early stage and then decreasing in infant rats. Intracochlear electrical stimulation could result in the expression of proteins of CREB and NMDAR1 in auditory cortex and inferior colliculus increasing in infant rats. The dynamic variation of CREB and NMDAR1 expression in rat auditory cortex and inferior colliculus reflects synaptic plasticity in neurons of auditory pathway．

【Key words】Auditory plasticity;Cyclic AMP response element- binding protein(CREB);N-methyl-D-aspartic acid receptor one(NMDAR1);Electrical stimulation;Sensorineural hearing loss

【中圖分類號(hào)】R764.4

【文獻(xiàn)標(biāo)識(shí)碼】A

【文章編號(hào)】1006-7299(2016)03-0245-06

DOI：10.3969/j.issn.1006-7299.2016.03.007

作者簡(jiǎn)介：樊碧云，女，上海人，博士研究生，主要研究方向?yàn)槎伮?tīng)覺(jué)植入。通訊作者：楊軍(Email：13764981808@126.com)

*上海市衛(wèi)生局項(xiàng)目(2010240)、上海市科委醫(yī)學(xué)引導(dǎo)項(xiàng)目(114119a6300)聯(lián)合資助

1上海交通大學(xué)醫(yī)學(xué)院附屬新華醫(yī)院耳鼻咽喉-頭頸外科上海交通大學(xué)醫(yī)學(xué)院耳科學(xué)研究所(上海200092)