劉濤 綜述 竇艷玲 審校

感音神經(jīng)性聽(tīng)力損失主要源自耳蝸毛細(xì)胞和/或螺旋神經(jīng)元的損傷和退化，耳蝸螺旋神經(jīng)元(spiral ganglion neuron，SGN)是傳導(dǎo)聽(tīng)覺(jué)信息的一級(jí)神經(jīng)元，噪聲、感染、耳毒性藥物和衰老等諸多因素直接或者間接使螺旋神經(jīng)元受損，可導(dǎo)致感音神經(jīng)性聽(tīng)力損失，而哺乳動(dòng)物螺旋神經(jīng)元的再生能力非常低，因此保護(hù)螺旋神經(jīng)元或者修復(fù)受損的螺旋神經(jīng)元對(duì)恢復(fù)聽(tīng)力有關(guān)鍵性的作用。神經(jīng)營(yíng)養(yǎng)因子是神經(jīng)細(xì)胞存活的依賴因子，是成熟神經(jīng)元功能的調(diào)控因子，也是神經(jīng)元受損和病變損傷時(shí)保護(hù)和促進(jìn)其再生的必需因子。近年來(lái)，為了更好地提高人工耳蝸的效能、對(duì)神經(jīng)營(yíng)養(yǎng)因子在螺旋神經(jīng)元生長(zhǎng)和損傷修復(fù)過(guò)程中的作用研究不斷進(jìn)展，神經(jīng)營(yíng)養(yǎng)因子在螺旋神經(jīng)元發(fā)育、損傷修復(fù)、防止退化及維持其正常生理功能中的重要作用逐漸被揭示[1～3]。

與聽(tīng)覺(jué)系統(tǒng)發(fā)育密切相關(guān)的神經(jīng)營(yíng)養(yǎng)因子有神經(jīng)營(yíng)養(yǎng)因子-3(neurotrophin-3, NT-3)、腦源性神經(jīng)營(yíng)養(yǎng)因子(brain-derived neurotrophic factor, BDNF)、膠質(zhì)細(xì)胞神經(jīng)營(yíng)養(yǎng)因子(glial cell line- derived neurotrophic factor, GDNF)、白血病抑制因子(Leukaemia inhibitory factor, LIF)等[4]。目前大多學(xué)者認(rèn)為，毛細(xì)胞可以合成和分泌多種營(yíng)養(yǎng)因子支持和保護(hù)螺旋神經(jīng)元，且神經(jīng)營(yíng)養(yǎng)因子在耳蝸內(nèi)保持較高表達(dá)水平是發(fā)揮其活性作用的關(guān)鍵，毛細(xì)胞受損后，螺旋神經(jīng)元失去神經(jīng)營(yíng)養(yǎng)因子的保護(hù)作用發(fā)生退行性變。Lefebvre等(1994)體外研究表明，BDNF、NT-3、NT-4/5、GDNF、轉(zhuǎn)化生長(zhǎng)因子-β(transforming growth factor-β, TGF-β)、纖毛神經(jīng)營(yíng)養(yǎng)因子(ciliary neuroteophic factor, CNTF)等都能促進(jìn)體外培養(yǎng)的新生大鼠SGN的存活[5]。在螺旋神經(jīng)元原代培養(yǎng)過(guò)程中適時(shí)適量地加入神經(jīng)營(yíng)養(yǎng)因子已是提高螺旋神經(jīng)元的純度和活性的重要手段之一。

1 神經(jīng)營(yíng)養(yǎng)因子對(duì)螺旋神經(jīng)元的作用

在體實(shí)驗(yàn)證明了直接在耳蝸內(nèi)注入神經(jīng)營(yíng)養(yǎng)因子特別是BDNF能提高耳聾后螺旋神經(jīng)元的存活率[6]。BDNF作為神經(jīng)營(yíng)養(yǎng)因子中的一員，在神經(jīng)系統(tǒng)發(fā)育、正常生理功能維持和神經(jīng)受損修復(fù)過(guò)程中都發(fā)揮著重要作用，不僅調(diào)節(jié)著聽(tīng)覺(jué)神經(jīng)元的成熟、增殖、分化，而且對(duì)前庭神經(jīng)元和部分聽(tīng)覺(jué)神經(jīng)元的存活有促進(jìn)作用[4]。BDNF可能通過(guò)維持細(xì)胞內(nèi)鈣離子濃度、減少自由基損傷、抑制細(xì)胞凋亡和(或)促進(jìn)細(xì)胞功能恢復(fù)穩(wěn)定而起到保護(hù)作用。研究表明在螺旋神經(jīng)元上有BDNF高親和性受體，在螺旋神經(jīng)元神經(jīng)突起發(fā)育的過(guò)程中，BDNF在毛細(xì)胞內(nèi)高濃度表達(dá)，將BDNF注入耳蝸內(nèi)可阻止失去毛細(xì)胞支持后螺旋神經(jīng)元的變性退變[7]。Xie等[8，9]研究發(fā)現(xiàn)BDNF基因工程細(xì)胞具有很強(qiáng)的生物學(xué)活性，能夠促進(jìn)螺旋神經(jīng)元的生長(zhǎng)，在螺旋神經(jīng)元氧化損傷時(shí)具有明顯的保護(hù)作用。BDNF能減少聽(tīng)覺(jué)神經(jīng)元的損失，而且促進(jìn)軸突的再生，因而可能提高人工耳蝸植入的效果[10]。在體外螺旋神經(jīng)元培養(yǎng)過(guò)程中，BDNF能刺激單離培養(yǎng)的未成熟神經(jīng)元軸突的生長(zhǎng)[10]。翟所強(qiáng)等[11]對(duì)噪聲損傷模型小鼠給予外源性的BDNF后，其較對(duì)照組螺旋神經(jīng)元的存活量有顯著改變，證明BDNF能保護(hù)螺旋神經(jīng)元。Darius等[12]研究發(fā)現(xiàn)BDNF體外基因治療對(duì)螺旋神經(jīng)元的保護(hù)作用耳蝸底回較蝸?lái)敻黠@。Martijn等[13]的研究發(fā)現(xiàn)BDNF提高螺旋神經(jīng)元存活率的作用可持續(xù)到停止治療后2周，在BDNF治療后，治療組動(dòng)物的閾上電誘發(fā)聽(tīng)性腦干反應(yīng)幅值顯著大于耳聾動(dòng)物，與正常聽(tīng)覺(jué)動(dòng)物差異無(wú)統(tǒng)計(jì)學(xué)意義，在停止給予BDNF后，治療組的閾上電誘發(fā)聽(tīng)性腦干反應(yīng)幅值也不會(huì)顯著降低。應(yīng)用神經(jīng)營(yíng)養(yǎng)因子處理后的螺旋神經(jīng)元保存了電應(yīng)激性，給予神經(jīng)營(yíng)養(yǎng)因子動(dòng)物的電誘發(fā)聽(tīng)性腦干反應(yīng)潛伏期較對(duì)照組(灌入人工外淋巴液的耳聾動(dòng)物)明顯縮短[14～16]，這些均提示神經(jīng)營(yíng)養(yǎng)因子的作用可持續(xù)。在人工耳蝸的實(shí)驗(yàn)中，通過(guò)滲透泵給發(fā)育中動(dòng)物的耳蝸內(nèi)注入BDNF，可觀察到在植入電極上有包繞髓鞘的神經(jīng)軸突向鼓階萌芽[17]。

NT-3是一種多功能的神經(jīng)營(yíng)養(yǎng)因子，對(duì)神經(jīng)系統(tǒng)發(fā)育及維持其正常生理功能有重要意義，是迄今發(fā)現(xiàn)的維持螺旋神經(jīng)元存活的作用最顯著的神經(jīng)營(yíng)養(yǎng)因子之一[18]。NT-3是Ⅰ型螺旋神經(jīng)元的主要生長(zhǎng)因子，可作為小鼠螺旋神經(jīng)元離體細(xì)胞培養(yǎng)的生存因子。Zhou等[19]研究發(fā)現(xiàn)NT-3對(duì)常規(guī)神經(jīng)元有維持存活作用，對(duì)缺氧神經(jīng)元有明顯保護(hù)作用，能夠促進(jìn)神經(jīng)元軸突生長(zhǎng)，降低缺氧造成的損傷，促進(jìn)神經(jīng)元的存活，其機(jī)制可能是通過(guò)上調(diào)c-jun基因的表達(dá)而促進(jìn)神經(jīng)元的生長(zhǎng)，提高其活力。耳蝸毛細(xì)胞能分泌NT-3，當(dāng)毛細(xì)胞損傷后，NT-3分泌減少，可繼發(fā)引起神經(jīng)纖維和螺旋神經(jīng)元的變性死亡，而當(dāng)耳毒性藥物、興奮性刺激等損傷后，給予大量外源性NT-3可明顯減少螺旋神經(jīng)元的變性死亡[20]。Bibel等[21]研究發(fā)現(xiàn)在先天性缺乏BDNF和NT-3或其受體的小鼠中，感覺(jué)神經(jīng)元嚴(yán)重缺失，NT-3基因敲除小鼠的螺旋神經(jīng)元嚴(yán)重缺失，細(xì)胞數(shù)量明顯低于正常水平，由于無(wú)NT-3表達(dá)，Ⅰ型螺旋神經(jīng)元缺失。Chen等[22]證實(shí)在體外培養(yǎng)中，NT-3的濃度與螺旋神經(jīng)元的生物活性正相關(guān)。研究證明，利用單純皰疹病毒介導(dǎo)產(chǎn)生神經(jīng)營(yíng)養(yǎng)因子-3能減弱順鉑對(duì)成年老鼠外周聽(tīng)覺(jué)系統(tǒng)的毒性，證明在活體內(nèi)通過(guò)神經(jīng)營(yíng)養(yǎng)因子-3基因治療，有希望預(yù)防化學(xué)藥物導(dǎo)致的聽(tīng)力損失，并可能預(yù)防老年聽(tīng)覺(jué)退化[23]。

GDNF是迄今發(fā)現(xiàn)的活性最強(qiáng)的神經(jīng)元神經(jīng)營(yíng)養(yǎng)因子之一，是轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)超家族的一員，體內(nèi)很多細(xì)胞可以合成GDNF，影響神經(jīng)元的發(fā)育和存活，它可通過(guò)磷酸肌醇3激酶(PI3-K)、G蛋白、cAMP和MAP激酶等途徑介導(dǎo)GDNF信號(hào)轉(zhuǎn)導(dǎo)，維持未成熟神經(jīng)元的生存、分化和增殖，支持成熟神經(jīng)元的生存適應(yīng)，介導(dǎo)神經(jīng)元的可塑性，能抑制凋亡而延長(zhǎng)受損和退變的神經(jīng)元的生存時(shí)間，修復(fù)其功能。有研究表明，GDNF在新生大鼠內(nèi)耳的內(nèi)外毛細(xì)胞中表達(dá)，在成熟大鼠內(nèi)毛細(xì)胞中也有表達(dá)，GDNF的受體GFRα-1、酪氨酸激酶受體Ret在螺旋神經(jīng)元上也有表達(dá)[24]。Ylikoski等[25]向豚鼠鼓階內(nèi)緩慢注入GDNF后，觀察強(qiáng)噪聲下GDNF對(duì)聽(tīng)神經(jīng)的保護(hù)作用，結(jié)果發(fā)現(xiàn)GDNF可以明顯提高噪聲損傷后螺旋神經(jīng)元的存活率。噪聲暴露后，耳蝸內(nèi)GDNF的mRNA表達(dá)水平明顯加強(qiáng)[26]，GDNF對(duì)噪聲、耳毒性藥物等引起的聽(tīng)力損失具有明顯保護(hù)作用。Yagi等[27]利用腺病毒載體進(jìn)行基因治療證明應(yīng)用GDNF或其他因子進(jìn)行基因治療后，內(nèi)耳毛細(xì)胞損失后螺旋神經(jīng)元的存活率顯著提高。Stover等[28]通過(guò)RT-PCR及Western blot免疫化學(xué)的方法在螺旋神經(jīng)元內(nèi)觀察到了GDNF、GFRal、Ret三種蛋白的分布。朱雅穎等[29]研究表明耳蝸內(nèi)長(zhǎng)期灌注GDNF對(duì)耳毒性藥物損傷后殘余的螺旋神經(jīng)元有保護(hù)作用，可避免細(xì)胞進(jìn)一步丟失，對(duì)損傷的細(xì)胞可能有修復(fù)作用。

2 神經(jīng)營(yíng)養(yǎng)因子之間的相互影響

在活體外實(shí)驗(yàn)中，BNDF和CNTF協(xié)同作用較各自作用能更有效提高螺旋神經(jīng)元的存活率[30]。聯(lián)合應(yīng)用BDNF和成纖維生長(zhǎng)因子-1能更有效提高螺旋神經(jīng)元存活和促進(jìn)神經(jīng)元周圍突的生長(zhǎng)。

研究發(fā)現(xiàn)神經(jīng)營(yíng)養(yǎng)因子BDNF和NT-3在活體和離體培養(yǎng)中都是維持聽(tīng)覺(jué)上皮和聽(tīng)覺(jué)神經(jīng)元生存的重要物質(zhì)。內(nèi)耳毛細(xì)胞釋放神經(jīng)營(yíng)養(yǎng)因子-3和腦源性神經(jīng)生長(zhǎng)因子對(duì)螺旋神經(jīng)元存活是基本必須的[31]，并且BDNF和NT-3對(duì)螺旋神經(jīng)元的存活、生長(zhǎng)和功能有調(diào)節(jié)作用，并相互協(xié)調(diào)相互影響[32，33]。BDNF和NT-3可通過(guò)結(jié)合其高親和力受體影響螺旋神經(jīng)元生長(zhǎng)和發(fā)育過(guò)程，均能促進(jìn)神經(jīng)元的存活，其中BDNF效果更明顯。近年來(lái)發(fā)現(xiàn)BDNF在螺旋神經(jīng)元生長(zhǎng)和發(fā)育過(guò)程中主要促進(jìn)胞體發(fā)育，而NT-3主要促進(jìn)軸突分化和生長(zhǎng)，并且發(fā)現(xiàn)BDNF、NT-3均能刺激分離培養(yǎng)的未成熟螺旋神經(jīng)元的軸突生長(zhǎng)，但對(duì)成熟的螺旋神經(jīng)元，無(wú)論是BDNF還是NT-3均不能促進(jìn)軸突生長(zhǎng)，可能在發(fā)育成熟的神經(jīng)系統(tǒng)中，神經(jīng)營(yíng)養(yǎng)因子的作用發(fā)生了改變。

在GDNF對(duì)內(nèi)耳保護(hù)作用的研究中發(fā)現(xiàn)NT3對(duì)GDNF的增強(qiáng)作用具有特異性[36]，聯(lián)合應(yīng)用GDNF和NT-3能夠顯著提高SGN體外培養(yǎng)的存活率，NT-3能增強(qiáng)GDNF誘導(dǎo)的Ret酪氨酸磷酸化，因而使螺旋神經(jīng)元發(fā)育存活所需的Akt磷酸化反應(yīng)大大增強(qiáng)。有研究表明在耳毒性藥物制造的耳聾動(dòng)物模型中，應(yīng)用NT-3和GDNF在耳蝸共同轉(zhuǎn)導(dǎo)，不僅能夠增強(qiáng)神經(jīng)營(yíng)養(yǎng)因子對(duì)耳蝸的保護(hù)，而且能降低耳毒性藥物對(duì)聽(tīng)力的損傷[34]。GDNF和NT-3的聯(lián)合作用是協(xié)同效應(yīng)，可顯著提高螺旋神經(jīng)元的存活率[35]。

CNTF和LIF也具有促進(jìn)螺旋神經(jīng)元的軸突生長(zhǎng)的作用，而同時(shí)應(yīng)用LIF與BDNF時(shí)，兩者具有協(xié)同作用，可促進(jìn)神經(jīng)元軸突的生長(zhǎng)[37]。在螺旋神經(jīng)元培養(yǎng)過(guò)程中，LIF與NT-3因子也相互影響其作用的發(fā)揮，LIF能間接增強(qiáng)NT-3的誘導(dǎo)效應(yīng)，促進(jìn)神經(jīng)元的存活[11]。

3 展望

進(jìn)行性的SGN變性部分原因是因?yàn)槭ッ?xì)胞支持后缺少神經(jīng)營(yíng)養(yǎng)因子，因此，神經(jīng)營(yíng)養(yǎng)因子被認(rèn)為可以阻止和延緩SGN的變性。噪聲損傷后，內(nèi)耳內(nèi)BDNF、GDNF、NT-3等神經(jīng)因子mRNA表達(dá)均有增強(qiáng)，內(nèi)耳毛細(xì)胞及螺旋神經(jīng)元損傷時(shí)，各種神經(jīng)營(yíng)養(yǎng)因子可能協(xié)同作用，而非一種因子的作用。近年來(lái)，對(duì)神經(jīng)營(yíng)養(yǎng)因子在內(nèi)耳的研究已廣泛開(kāi)展，但仍然處于初步階段，對(duì)內(nèi)耳興奮性刺激、耳毒性藥物、缺血性損傷后螺旋神經(jīng)元的保護(hù)和損傷的研究尚有待進(jìn)一步發(fā)展。利用耳蝸內(nèi)持續(xù)灌注、通過(guò)病毒載體的基因治療等方法，均在動(dòng)物實(shí)驗(yàn)中證明了神經(jīng)營(yíng)養(yǎng)因子能保護(hù)感音神經(jīng)性聾殘余神經(jīng)細(xì)胞，可避免更多的細(xì)胞丟失，也能提高靶神經(jīng)元的存活。螺旋神經(jīng)元的存活數(shù)量和生物活性尤為重要，利用神經(jīng)營(yíng)養(yǎng)因子的治療，很可能使人工耳蝸植入更有效發(fā)揮作用[9]。研究神經(jīng)營(yíng)養(yǎng)因子對(duì)螺旋神經(jīng)元的保護(hù)與細(xì)胞修復(fù)、分子機(jī)制以及將來(lái)可行的治療方法將為治療感音神經(jīng)性聾開(kāi)辟新途徑。

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