GT1-7細(xì)胞及其在生殖相關(guān)研究中的應(yīng)用

馮　濤，吳曉敏，許曉玲，白佳樺，宋玉清，肖霖力，韓向敏，劉　彥*（.北京市農(nóng)林科學(xué)院畜牧獸醫(yī)研究所，北京　00097；.甘肅農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，蘭州　730070）

?

GT1-7細(xì)胞及其在生殖相關(guān)研究中的應(yīng)用

馮濤1，吳曉敏2，許曉玲1，白佳樺1，宋玉清1，肖霖力1，韓向敏2，劉彥1*
（1.北京市農(nóng)林科學(xué)院畜牧獸醫(yī)研究所，北京100097；2.甘肅農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院，蘭州730070）

摘要：GT1-7細(xì)胞是GT1細(xì)胞株的亞株，通過(guò)轉(zhuǎn)基因技術(shù)從小鼠下丘腦分離獲得的GnRH神經(jīng)元細(xì)胞系，具有高度分化的神經(jīng)內(nèi)分泌細(xì)胞典型特征。下丘腦GnRH合成和釋放對(duì)生殖功能具有重要作用，而GnRH神經(jīng)元在腦內(nèi)數(shù)量少且呈彌散分布，體內(nèi)研究較困難。目前，GT1-7細(xì)胞是研究GnRH神經(jīng)元的理想離體細(xì)胞模型，在生殖相關(guān)研究中廣泛使用。文章詳細(xì)闡述GT1-7細(xì)胞獲得過(guò)程、功能特性及其在生殖系統(tǒng)研究中應(yīng)用，以及影響GT1-7細(xì)胞活性的信號(hào)通路，以期對(duì)動(dòng)物生殖調(diào)控研究提供參考。

關(guān)鍵詞：GT1-7細(xì)胞；促性腺激素釋放激素；生殖調(diào)控；信號(hào)通路

馮濤,吳曉敏,許曉玲,等. GT1-7細(xì)胞及其在生殖相關(guān)研究中的應(yīng)用[J].東北農(nóng)業(yè)大學(xué)學(xué)報(bào), 2016, 47(1): 102-108.

Feng Tao, Wu Xiaomin, Xu Xiaoling, et al. GT1-7 cells and its application in reproduction-related researches[J]. Journal of Northeast Agricultural University, 2016, 47(1): 102-108. (in Chinese with English abstract)

雌性動(dòng)物生殖活動(dòng)受下丘腦-垂體-性腺軸（Hypothalamus-pituitary-gonad，HPG）精密調(diào)控，其中下丘腦促性腺激素釋放激素（Gonadotropin-re?leasing hormone，GnRH）神經(jīng)元是動(dòng)物發(fā)情周期啟動(dòng)關(guān)鍵部位，GnRH分泌調(diào)控是深入揭示動(dòng)物情期啟動(dòng)關(guān)鍵因素。近年來(lái)，分子遺傳學(xué)得到快速發(fā)展，特別是GnRH神經(jīng)元離體細(xì)胞模型—GT1-7細(xì)胞的發(fā)現(xiàn)，加速生殖內(nèi)分泌調(diào)控領(lǐng)域研究進(jìn)程。永生GT1-7細(xì)胞是從轉(zhuǎn)基因小鼠下丘腦視前區(qū)分離到的GnRH神經(jīng)元細(xì)胞，具有高度分化神經(jīng)內(nèi)分泌細(xì)胞特性，能模擬GnRH神經(jīng)元功能。GT1-7細(xì)胞不但具有原始GnRH神經(jīng)元多種功能，如節(jié)律性脈沖分泌GnRH，多種信號(hào)通路調(diào)節(jié)細(xì)胞活性，能表達(dá)GnRH、Kiss-1、G蛋白偶聯(lián)受體54（GPR54）、雌激素受體α（ERα）、雌激素受體β（ERβ）及卵泡抑制素樣蛋白等多種生殖相關(guān)基因、受體及其蛋白，在動(dòng)物生殖調(diào)控研究中具有重要作用。然而，GnRH神經(jīng)元作為哺乳動(dòng)物中樞生殖調(diào)控體系最終共同通路，GnRH合成和分泌受諸多因子及神經(jīng)元相互作用調(diào)控，因此利用GT1-7細(xì)胞完全模擬GnRH體內(nèi)調(diào)控存在局限性。本文從GT1-7細(xì)胞來(lái)源和特性及其在生殖相關(guān)研究中應(yīng)用等方面綜述，總結(jié)影響GT1-7細(xì)胞活性信號(hào)通路，以期對(duì)動(dòng)物生殖調(diào)控研究提供參考。

1 GT1-7細(xì)胞來(lái)源

GT1-7細(xì)胞是GT1細(xì)胞株1個(gè)亞株。GT1細(xì)胞株由美國(guó)Mellon等于1990年利用遺傳學(xué)靶向致腫瘤技術(shù)[1]，將致癌猿猴空泡病毒40（Simian vacuolat?ing virus，SV40）抗原（Tag）基因及其在GnRH細(xì)胞中表達(dá)調(diào)控基因構(gòu)建雜合基因后，整合到小鼠受精卵原核中，并將從轉(zhuǎn)基因小鼠子代下丘腦視前區(qū)分離得到腫瘤細(xì)胞經(jīng)進(jìn)一步篩選和克隆，獲得同時(shí)表達(dá)GnRH和Tag基因穩(wěn)定細(xì)胞株。GT1細(xì)胞株有3個(gè)亞株，分別是GT1-1、GT1-3和GT1-7，均具有高度分化GnRH神經(jīng)元特征。GT1-7細(xì)胞在表型形態(tài)、超微結(jié)構(gòu)、基因表達(dá)、激素儲(chǔ)存以及分泌方式上均與GnRH神經(jīng)元一致，且能在體外分裂傳代，是體外研究GnRH神經(jīng)元的理想細(xì)胞模型[2]。

2 GT1-7細(xì)胞特性

GnRH神經(jīng)元是哺乳動(dòng)物中樞生殖調(diào)控體系最終共同通路，僅分布于前腦嗅區(qū)、隔區(qū)及下丘腦等區(qū)域。下丘腦是由許多功能各異神經(jīng)核團(tuán)組成結(jié)構(gòu)復(fù)雜器官，體內(nèi)精確研究GnRH神經(jīng)元及相關(guān)功能難度較大，通過(guò)原代培養(yǎng)方法直接研究下丘腦GnRH神經(jīng)元功能也存在很大困難。因此，目前研究多利用下丘腦永生GnRH神經(jīng)元細(xì)胞系GT1-7細(xì)胞開(kāi)展相關(guān)研究。

2.1 GT1-7細(xì)胞分泌模式

GT1-7細(xì)胞表現(xiàn)GnRH神經(jīng)元形態(tài)，且保留許多原始GnRH神經(jīng)元功能[3-4]。Martinez等研究發(fā)現(xiàn)，GT1-7細(xì)胞在體外表現(xiàn)出與GnRH神經(jīng)元類(lèi)似脈沖分泌模式，下丘腦GnRH神經(jīng)元分泌GnRH平均脈沖間隔25.8 min，平均持續(xù)時(shí)間18.8 min[5]。GT1-7細(xì)胞分泌GnRH平均脈沖間隔24.8~45.4 min，平均脈沖幅度14.30~191.5 pg·min·mL-1，平均脈沖持續(xù)時(shí)間為21.30 min[4-8]?？梢?jiàn)GT1-7細(xì)胞與GnRH神經(jīng)元細(xì)胞脈沖情況基本一致，細(xì)微差異可能與培養(yǎng)液種類(lèi)、細(xì)胞系傳代次數(shù)以及培養(yǎng)時(shí)間等條件有關(guān)。

2.2時(shí)鐘基因?qū)T1-7細(xì)胞分泌調(diào)節(jié)

GnRH神經(jīng)元細(xì)胞含有分子時(shí)鐘，晝夜信號(hào)可有效分配到多個(gè)組織[9]。研究表明，GT1-7細(xì)胞能表達(dá)多種時(shí)鐘基因，包括Bmal1、Clock、mCry1、mCry2、mPer1、mPer2、mPer3和CKIε，這些基因也在下丘腦視交叉上核（SCN）和外周細(xì)胞中表達(dá)[10]。在GT1-7細(xì)胞中用顯性失活Clock-Δ19基因瞬時(shí)表達(dá)能擾亂生物鐘功能進(jìn)而破壞GnRH正常晝夜分泌模式，顯著降低平均脈沖頻率；過(guò)表達(dá)負(fù)調(diào)控Limb基因mCry1可顯著增加GnRH脈沖幅度，而對(duì)脈沖頻率無(wú)顯著變化[11]。Eileen等將GT1-7細(xì)胞暴露于0.1 U·mL-1凝血酶（誘導(dǎo)Ca2+內(nèi)流）也可顯著增加脈沖振幅，但對(duì)持續(xù)時(shí)間影響不顯著，推測(cè)可能與GT1-7細(xì)胞特定膜結(jié)合受體及細(xì)胞膜上時(shí)鐘基因有關(guān)[7]。Blackman等研究表明，GT1-7細(xì)胞內(nèi)cAMP水平通過(guò)環(huán)核苷酸門(mén)控通道（CNGs）參與內(nèi)源性生物鐘調(diào)節(jié)，影響GnRH分泌[12]?？梢?jiàn)，時(shí)鐘基因表達(dá)能夠調(diào)節(jié)GT1-7細(xì)胞分泌活性，但并不改變脈沖式釋放GnRH固有特性。

2.3離子通道對(duì)GT1-7細(xì)胞分泌調(diào)節(jié)

GT1-7細(xì)胞作為神經(jīng)內(nèi)分泌細(xì)胞，其分泌激素與細(xì)胞膜電位、胞內(nèi)鈣離子濃度和興奮性等有關(guān)[13]。GT1-7細(xì)胞能表達(dá)多種質(zhì)膜通道，有自發(fā)動(dòng)作電位能力[14]：鈉通道阻滯劑河豚毒素（TTX）敏感Na+通道[1]，三種不同類(lèi)型向外K+通道和一個(gè)K+向內(nèi)整流器[15-16]以及電壓依賴性Ca2+通道[15,17]。GT1-7細(xì)胞自發(fā)動(dòng)作電位頻率24.8 min[18]與GnRH神經(jīng)元平均脈沖頻率25.8 min[5]基本一致，因此動(dòng)作電位是GnRH釋放機(jī)制一部分。GnRH分泌和鈣振蕩是“GnRH脈沖發(fā)生器”基礎(chǔ)[19-20]，搏動(dòng)機(jī)制是Ca2+周期性交替[21]。鈣振蕩與Na+通道動(dòng)作電位和Ca2+通過(guò)電壓門(mén)控鈣通道有關(guān)[18]。K+通道阻滯劑能引起幅度或鈣振蕩頻率明顯變化[21]。GT1-7細(xì)胞能夠表達(dá)R、L、N和T型鈣通道[14]，其中R型亞基起關(guān)鍵作用，作為主要電流調(diào)節(jié)通道與L、N和T型鈣通道共同調(diào)節(jié)GnRH釋放[22]。近年來(lái)超極化激活環(huán)核苷酸調(diào)節(jié)通道（HCN）成為研究熱點(diǎn)，HCN-在超極化時(shí)被激活并產(chǎn)生內(nèi)向電流，目前發(fā)現(xiàn)有四種HCN通道亞型，即HCNl、HCN2、HCN3和HCN4，且四種亞型在GTl-7細(xì)胞上均有表達(dá)[23]。

Wetsel等研究發(fā)現(xiàn)，GT1-7細(xì)胞能表達(dá)縫隙連接蛋白，連接素26樣蛋白[24]，及突觸樣連接蛋白[25]。目前研究發(fā)現(xiàn)GT1-7細(xì)胞分泌GnRH主要是分子生物學(xué)和電生理機(jī)制，但精確脈沖機(jī)制尚未確定，可能還包括經(jīng)間隙連接旁分泌因子，例如一氧化氮、電生理耦合或GnRH及從GnRH前體分子加工而得到肽類(lèi)物質(zhì)，如GnRH相關(guān)肽（GAP）等。

3 GT1-7細(xì)胞在生殖相關(guān)研究中的應(yīng)用

下丘腦接受中樞神經(jīng)系統(tǒng)發(fā)布信息，通過(guò)GnRH神經(jīng)元分泌GnRH，GnRH調(diào)節(jié)垂體中促性腺激素細(xì)胞分泌促卵泡素（FSH）和黃體生成素（LH），促性腺激素（FSH，LH）作用于性腺（雄性睪丸和雌性卵巢），調(diào)節(jié)性激素分泌并影響生殖活動(dòng)。垂體分泌促性腺激素可反饋調(diào)節(jié)下丘腦GnRH分泌；性腺激素也可反饋調(diào)節(jié)下丘腦和垂體相應(yīng)激素釋放。因此，在下丘腦、垂體和性腺之間形成密切相連軸線系統(tǒng)，即HPG軸。體外GT1-7細(xì)胞不但能表現(xiàn)出與GnRH神經(jīng)元類(lèi)似脈沖分泌模式，還有GnRH、Kiss-1、GPR54、BMP受體、ERα、ERβ、抑制性Smads以及卵泡抑制素樣蛋白等表達(dá)[26-27]。目前認(rèn)為，GT1-7細(xì)胞是體外研究HPG軸調(diào)控機(jī)制的理想細(xì)胞模型。

3.1 GT1-7細(xì)胞與Kisspeptin/GPR54

Kisspeptin是Kiss-1基因編碼神經(jīng)內(nèi)分泌肽類(lèi)激素，為GPR54內(nèi)源性配體[28]，可直接作用于下丘腦GnRH神經(jīng)元，促進(jìn)GnRH分泌，激活HPG軸。Kiss-1/GPR54基因突變可導(dǎo)致特發(fā)性促性腺激素分泌不足型腺機(jī)能減退癥（Idiopathic hypogo?nadotropic hypogonadism, IHH）[29]，因此Kisspeptin/ GPR54被認(rèn)為是青春期發(fā)育啟動(dòng)“分子閥門(mén)”。Kis?speptin處理GT1-7細(xì)胞，以濃度依賴性方式增加GnRH mRNA表達(dá)及GnRH分泌[27]。Ozcan等研究表明，Kisspeptin可使GT1-7細(xì)胞過(guò)表達(dá)GPR54，激活cAMP/PKA信號(hào)通路[30]，誘導(dǎo)GnRH受體表達(dá)。同時(shí)，Kisspeptin在Kisspeptin/GPR54系統(tǒng)保護(hù)機(jī)制下升高細(xì)胞內(nèi)鈣水平，通過(guò)Ca2+/PKC信號(hào)調(diào)控GnRH分泌[30]。Terasaka等通過(guò)RT-PCR證實(shí)Kiss?peptins/GPR54系統(tǒng)在GT1-7神經(jīng)元細(xì)胞表達(dá)，但體內(nèi)GnRH神經(jīng)元并未表達(dá)，有GPR54表達(dá)[27]。因此，Kisspeptins刺激促性腺激素釋放可能通過(guò)GPR54表達(dá)實(shí)現(xiàn)。Heather等對(duì)哺乳動(dòng)物研究也證實(shí)Kisspeptins通過(guò)活化GPR54，使GnRH神經(jīng)元表達(dá)GPR54，刺激下丘腦GnRH釋放[31]。總之，Kiss?peptin/GPR54是青春期啟動(dòng)催化劑，進(jìn)一步研究可以利用GT1-7細(xì)胞為模型探究下丘腦GnRH細(xì)胞如何調(diào)控HPG軸以及青春期啟動(dòng)等現(xiàn)象。

3.2 GT1-7細(xì)胞與E2/ER

動(dòng)物體內(nèi)研究表明，溫和地持續(xù)注射雌激素（E2）（50 μg·kg-1，5 d）[32]及大劑量一次性注射雌激素（10 mg·kg-1）[33]，均能誘發(fā)大鼠真性性早熟。給卵巢摘除小鼠連續(xù)外源注射雌激素，可誘導(dǎo)GnRH和LH峰產(chǎn)生[34]。在體外用雌激素處理GT1-7細(xì)胞，pmol級(jí)濃度雌激素可對(duì)cAMP產(chǎn)生急速、持續(xù)、劑量依賴性抑制，而nmol級(jí)濃度雌激素可增加cAMP產(chǎn)生[35]。雖然GT1-7細(xì)胞表達(dá)ERα和ERβ，但小鼠GnRH神經(jīng)元表達(dá)ERβ，卻不表達(dá)ERα[36-37]。有研究報(bào)道GT1-7細(xì)胞中雌激素可能通過(guò)激活ERβ活性引起GPR54表達(dá)[38]。Tonsfeldt等研究還發(fā)現(xiàn)kiss?peptin引起GnRH分泌可能部分依賴雌激素活性，當(dāng)雌激素水平升高時(shí)，增加kisspeptin可引起ERα 和ERβ mRNA表達(dá)，雌激素也能增加GnRH神經(jīng)元對(duì)kisspeptin敏感性，加強(qiáng)GPR54表達(dá)[38]。E2能正負(fù)調(diào)控下丘腦GnRH神經(jīng)元，啟動(dòng)初情期或正常生殖周期，GT1-7細(xì)胞已廣泛用于E2生殖調(diào)控研究。

3.3 GT1-7細(xì)胞與Leptin

瘦素（Leptin）作為脂肪分泌激素，作用于下丘腦，調(diào)節(jié)食物攝入和能量代謝，也是生殖系統(tǒng)重要代謝信號(hào)。只有當(dāng)體內(nèi)營(yíng)養(yǎng)狀態(tài)達(dá)到一定臨界水平，足以滿足生殖需要時(shí)，才能觸發(fā)動(dòng)物機(jī)體青春期啟動(dòng)。Leptin對(duì)生殖活動(dòng)調(diào)節(jié)主要通過(guò)調(diào)節(jié)下丘腦GnRH分泌實(shí)現(xiàn)。因肥胖基因突變而致原發(fā)性瘦素缺失ob小鼠出現(xiàn)肥胖并伴有生殖功能低下，通過(guò)單純控制飲食可使體重下降，但不能恢復(fù)生育能力，而外原性瘦素可使其恢復(fù)[39-40]。楊穎等研究表明，GT1-7細(xì)胞表達(dá)Leptin受體基因，Leptin能促使GT1-7細(xì)胞迅速釋放GnRH，呈明顯量效關(guān)系，Leptin可能通過(guò)直接作用于下丘腦GnRH神經(jīng)元，對(duì)生殖功能起調(diào)節(jié)作用[41]。雖然現(xiàn)有研究表明Leptin可直接調(diào)節(jié)GnRH分泌，但是在GnRH神經(jīng)元上并未發(fā)現(xiàn)Leptin受體表達(dá)，表明Leptin對(duì)GnRH存在中間調(diào)節(jié)機(jī)制[42]。在下丘腦視前區(qū)，nNOS神經(jīng)元上存在Leptin受體，NOS酶活性受Leptin調(diào)節(jié)。敲除Leptin基因小鼠（Lepob/ob）不育，在nNOS神經(jīng)元選擇性缺失Leptin可推遲雌鼠初情期啟動(dòng)，由于阻斷Leptin信號(hào)傳導(dǎo)經(jīng)過(guò)nNOS細(xì)胞傳至GnRH，nNOS-/-Lepob/ob小鼠在Leptin處理后缺乏LH分泌[43-44]。離體GT1-7細(xì)胞何時(shí)啟動(dòng)能量代謝調(diào)節(jié)，何時(shí)啟動(dòng)生殖機(jī)能調(diào)節(jié)，能否系統(tǒng)模擬動(dòng)物機(jī)體生長(zhǎng)發(fā)育過(guò)程尚不清楚。假設(shè)GT1-7細(xì)胞leptin受體基因缺失，通過(guò)提供外原性leptin，觀察GT1-7細(xì)胞GnRH分泌情況，可研究能量對(duì)GnRH合成分泌以及生殖機(jī)能等影響。

3.4 GT1-7細(xì)胞與褪黑激素

褪黑激素（Melatonin，MLT）是普遍存在于動(dòng)物機(jī)體的吲哚類(lèi)激素，主要由松果體（Pineal gland，PG）合成和分泌，受光照調(diào)節(jié)。MLT可降低GnRH受體數(shù)量，抑制下丘腦GnRH分泌，主要通過(guò)HPG軸調(diào)控動(dòng)物生殖活動(dòng)。研究發(fā)現(xiàn)GT1-7細(xì)胞表達(dá)G蛋白偶聯(lián)受體褪黑激素受體1（Mt1）和2（Mt2）[45]，抑制cAMP活性，激活PKC和AMPK活性而非PKA活性，抑制GT1-7細(xì)胞GnRH分泌[46]。MLT能提高立早基因c-fos和junB表達(dá)水平[46]。Kelestimur等研究結(jié)果表明MLT通過(guò)激活PKC增加細(xì)胞內(nèi)Ca2+水平，抑制GT1-7細(xì)胞GnRH釋放[47]。

在青春期與生殖功能調(diào)控研究中，胡雅婷發(fā)現(xiàn)脂聯(lián)素通過(guò)AMPK和SP1調(diào)節(jié)GT1-7細(xì)胞Kiss-1基因表達(dá)[48]。GT1-7細(xì)胞表達(dá)脂聯(lián)素受體1和2[49]、降鈣素基因相關(guān)肽（CGRP）受體[50]、垂體腺苷酸環(huán)化酶激活多肽（PACAP）Ⅰ型受體[51]、神經(jīng)激肽3受體（NKB3R）[52]等與生殖相關(guān)基因。

以上研究表明，GT1-7細(xì)胞與體內(nèi)GnRH神經(jīng)元相似，有多種生殖相關(guān)基因及其受體基因表達(dá)，可作為HPG軸調(diào)控機(jī)理理想細(xì)胞模型研究機(jī)體生殖活動(dòng)。陳名道等研究中藥補(bǔ)腎方二仙湯及其拆方對(duì)GT1-7細(xì)胞分泌GnRH影響[53]，揭示二仙湯及其拆方如何對(duì)下丘腦GnRH細(xì)胞發(fā)揮作用，調(diào)控HPG軸。

4影響GT1-7細(xì)胞活性信號(hào)通路

4.1 AMPK相關(guān)信號(hào)通路

腺苷酸活化蛋白激酶（AMPK），即AMP依賴蛋白激酶，由α、β和γ亞基組成異源三聚體復(fù)合體，是生物機(jī)體重要信號(hào)分子、細(xì)胞能量關(guān)鍵傳感器和調(diào)制器。AMPK活力主要通過(guò)調(diào)節(jié)其與催化相關(guān)結(jié)構(gòu)元件實(shí)現(xiàn)。

AMPK通過(guò)接受上游信號(hào)調(diào)控活力，影響GT1-7細(xì)胞GnRH分泌。研究發(fā)現(xiàn)，GT1-7細(xì)胞中，胰高血糖素樣肽-1受體（GLP-1R）活化后以時(shí)間依賴方式減少Akt磷酸化，可能通過(guò)PI3K-Akt信號(hào)通路調(diào)控AMPK活性，影響食物攝取[54]。在GT1-7細(xì)胞中，α-黑色素細(xì)胞刺激素（α-MSH）通過(guò)PKA-AMPK途徑以調(diào)節(jié)能量消耗和食物攝取[55]；葡萄糖轉(zhuǎn)運(yùn)蛋白2（GLUT2）[56]通過(guò)改變細(xì)胞能量狀態(tài)或AMP/ATP比率，調(diào)控食欲相關(guān)肽（AgRP）mRNA表達(dá)并使AMPK磷酸化葡萄糖；CGRP通過(guò)其受體下調(diào)GnRH mRNA表達(dá)[50]，激活cAMP-PKA通路而非cAMP-PKC通路抑制AMPK活性[12]，降低GnRH mRNA表達(dá)。

通過(guò)激活A(yù)MPK激動(dòng)其下游信號(hào)調(diào)控GT1-7細(xì)胞GnRH分泌。球狀域脂聯(lián)素[57]通過(guò)AMPK-ERK，增加轉(zhuǎn)錄因子SPl表達(dá)或磷酸化，抑制GTl-7細(xì)胞Kiss-1基因啟動(dòng)子活性及其表達(dá)，影響GnRH分泌。GT1-7細(xì)胞中，球狀脂連蛋白能激活A(yù)MPK磷酸化使乙酰輔酶A羧化酶（ACC）失活，使丙二酰CoA降低，增加AgRP mRNA表達(dá)，證明在下丘腦球狀脂連蛋白調(diào)節(jié)能量平衡通過(guò)AMPK-ACC信號(hào)通路而非JAK-STAT3通路[58]，在泌乳奶牛飼喂苜蓿青貯粗飼料研究中發(fā)現(xiàn)，血清中胰島素和脂聯(lián)素濃度提高，激活奶牛乳腺胰島素信號(hào)通路及JAK2-STAT信號(hào)通路[59]。Beall等研究發(fā)現(xiàn)，GT1-7神經(jīng)元能表現(xiàn)出GE型葡萄糖傳感行為并通過(guò)AMPKα2-UCP2通路實(shí)現(xiàn)[60]。

4.2 MAPK相關(guān)信號(hào)通路

絲裂原活化蛋白激酶（MAPKs）是一組可被不同細(xì)胞外刺激，如細(xì)胞因子、神經(jīng)遞質(zhì)、激素、細(xì)胞應(yīng)激及細(xì)胞黏附等激活絲氨酸-蘇氨酸蛋白激酶。MAPK信號(hào)轉(zhuǎn)導(dǎo)通路存在于大多數(shù)細(xì)胞內(nèi)，能將細(xì)胞外刺激信號(hào)轉(zhuǎn)導(dǎo)至細(xì)胞及其核內(nèi)，并引起細(xì)胞生物學(xué)反應(yīng)。研究發(fā)現(xiàn)，在GT1-7細(xì)胞中，E2能活化ERK1/2和SAPK/JNK信號(hào)通路，但不激活p38MAPK信號(hào)通路[26]。Hayes等研究發(fā)現(xiàn)[61]，利用胰高血糖素樣肽1（GLP-1）類(lèi)似物Exendin-4，能通過(guò)激活PKA抑制AMPK并激活MAPK信號(hào)通路誘導(dǎo)胰高血糖素樣肽1受體（GLP-1R）表達(dá)，抑制食物攝入量。研制藥物抑制GLP-1R表達(dá)，可有效治療肥胖癥。Terasaka等研究表明，在GT1-7神經(jīng)元細(xì)胞中，Kisspeptin能夠刺激MAPK-AKT信號(hào)，與ERK信號(hào)在功能上參與GnRH mRNA表達(dá)[27]。

在GT1-7細(xì)胞中，是否存在除AMPK及MAPK相關(guān)信號(hào)通路以外信號(hào)轉(zhuǎn)導(dǎo)，使一些神經(jīng)遞質(zhì)或激素能通過(guò)多條信號(hào)通路傳導(dǎo)共同作用影響GT1-7細(xì)胞分泌活性，尚需要深入研究。

5展　望

在脊椎動(dòng)物腦組織中，一些激素和神經(jīng)遞質(zhì)通過(guò)作用于GnRH神經(jīng)元調(diào)控性腺軸。因此，GnRH神經(jīng)元是這些物質(zhì)調(diào)控性腺軸共同通路。劉金玲等研究發(fā)現(xiàn)，蛋氨酸腦啡肽（MENK）能促進(jìn)小鼠骨髓來(lái)源樹(shù)突狀細(xì)胞TLR-4和TNF-α表達(dá)[62]，GT1-7細(xì)胞能否表達(dá)MENK受體，如何調(diào)控免疫系統(tǒng)有待進(jìn)一步探討。在體內(nèi)研究中，神經(jīng)元之間聯(lián)系復(fù)雜，難以判斷其直接作用抑或通過(guò)中間神經(jīng)元間接作用于該GnRH神經(jīng)元，利用下丘腦永生GnRH神經(jīng)元細(xì)胞系GT1-7細(xì)胞可解決該問(wèn)題。GT1-7細(xì)胞作為GnRH神經(jīng)元理想細(xì)胞模型，在研究過(guò)程中取得突破，體外培養(yǎng)GT1-7細(xì)胞也更易于試驗(yàn)。但隨著研究深入，GT1-7細(xì)胞模型局限性逐漸凸顯。該細(xì)胞保持單一細(xì)胞克隆特性，只能代表一種GnRH神經(jīng)元行為特點(diǎn)，激酶或細(xì)胞因子參與細(xì)胞活性與細(xì)胞類(lèi)型、細(xì)胞成熟程度有關(guān)，某些基因或受體表達(dá)與體內(nèi)GnRH神經(jīng)元不一致，能否系統(tǒng)而精確模擬動(dòng)物機(jī)體生長(zhǎng)發(fā)育過(guò)程中各種神經(jīng)遞質(zhì)及調(diào)控因子相互作用有待探索。

[參考文獻(xiàn)]

[ 1 ] Mellon P L, Windle J J, Goldsmith P C, et al. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigene?sis[J]. Neuron, 1990(5): 1-10.

[ 2 ] Weiner R I, Wetsel W, Goldsmith P, et al. Gonadotropin-releas?ing hormone neuronal cell lines[J]. Front Neuroendocrin, 1992, (13): 95-119.

[ 3 ] Kersmanovic L Z, Stojilkovic S S, Merelli F, et al. Calcium signal?ing and episodic secretion of gonadotropin releasing hormone in hypothalamic neurons[J]. Proc Natl Acad Sci USA, 1992, 89: 8462-8466.

[ 4 ] Wetsel W C, Valenca M M, Merchenthaler I, et al. Intrinsic pulsa?tile secretory activity of immortalized luteinizing hormone-releas?ing hormone-secreting neurons[J]. Proc Natl Acad Sci USA, 1992, 89: 4149-4153.

[ 5 ] Martinez de la, Escalera G, Choi L H, et al. Generation and syn?chronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line[J]. Proc Natl Acad Sci USA 1992, 89: 1852-1855.

[ 6 ] King T S, Potter D, Kang I S, et al. Concentration-dependent ef?fects of muscimol to enhance pulsatile GnRH release from GT1-7 neurons in vitro[J]. Brain research, 1999, 824(1): 56-62.

[ 7 ] Chen E C, King T S, Chang X, et al. Thrombin-stimulated in?creases in cytosolic Ca2 +level and gonadotropin-releasing hor?mone release in GT1-7 neurons[J]. Peptides, 1999, 20(7): 859-864.

[ 8 ] Vazquez-Martinez R, Shorte S L, Boockfor F R, et al. Synchro?nized exocytotic bursts from gonadotropin-releasing hormone-ex?pressing cells: dual control by intrinsic cellular pulsatility and gap junctional communication[J]. Endocrinology, 2001, 142(5): 2095-2101.

[ 9 ] Balsalobre A. Clock genes in mammalian peripheral tissues[J]. Cell Tissue Res, 2002, 309(1): 193-199.

[10] Balsalobre A, Damiola F, Schibler U. A serum shock induces cir?cadian gene expression in mammalian tissue culture cells[J]. Cell, 1998, 93(6): 929-937.

[11] Chappell P E, White R S, Mellon P L. Circadian gene expression regulates pulsatile gonadotropin-releasing hormone(GnRH) secre?tory patterns in the hypothalamic GnRH-secreting GT1-7 cell line[J]. J Neurosci, 2003, 23(35): 11202-11213.

[12] Blackman B E, Yoshida H, Paruthiyil S, et al. Frequency of intrin?sic pulsatile gonadotropin-releasing hormone secretion is regulat?ed by the expression of cyclic nucleotide-gated channels in GT1 cells[J]. Endocrinology, 2007, 148(7): 3299-3306.

[13] Charles A C, Hales T G. Mechanisms of spontaneous calcium os?cillations and action potentials in immortalized hypothalamic(GTl-7) neurons[J]. Neurophysiol, 1995, 73(1): 56-64.

[14] Nunemaker C S, Defazio R A, Geusz M E, et al. Long-term record?ings of networks of immortalized GnRH neurons reveal episodic patterns of electrical activity[J]. J Neurophysiol, 2001, 86(1): 86-93.

[15] Bosma M M. Ion channel properties and episodic activity in isolat?ed immortalized gonadotropin-releasing hormone (GnRH) neurons [J]. J Membrane Biol, 1993, 136(1): 85-96.

[16] Spergel D J, Catt K J, Rojas E. Immortalized GnRH neurons ex?press large-conductance calcium-activated potassium channels [J]. Neuroendocrinology, 1996, 63(2): 101-111.

[17] Javors M A, King T S, Chang X, et al. Partial characterization of K+-induced increase in [Ca2+] cyt and GnRH release in GT1-7 neurons[J]. Brain Res, 1995, 694(1-2): 49-54.

[18] Schmitt K E. Optical neuronal guiding on the hypothalamic GnRH cell line GT1[D]. Texas: The University of Texas at Austin, 2003.

[19] Costantin J L, Charles A C. Modulation of Ca2 +signaling by K+channels in a hypotha lamic neuronal cell line (GT1-1) [J]. Jour?nal of Neurophysiology, 2001, 85(1): 295-304.

[20] Van G F, Krsmanovic L Z, Catt K J, et al. Control of action poten?tial-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels[J]. Molecular Endocrinology, 1999, 13(4): 587-603.

[21] Nunez L, Villalobos C, Boockfor F R, et al. The relationship be?tween pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons[J]. Endocrinology, 2000, 141(6): 2012-2017.

[22] Watanabe M, Sakuma Y, Kato M. High expression of the R-type voltage-gated Ca2 +channel and its involvement in Ca2 +-depen?dent gonadotropin-releasing hormone release in GT1-7 cells[J]. Endocrinology, 2004, 145(5): 2375-2383.

[23] Arroyo A, Kim B, Rasmusson R L, et al. Hyperpolarization-acti?vated cation channels are expressed in rat hypothalamic gonado?tropin-releasing hormone(GnRH) neurons and immortalized GnRH neurons[J]. J Soc Gynecol Investig, 2006, 13: 442-450.

[24]楊穎,陳名道,陳家倫. GT1細(xì)胞株在研究GnRH釋放機(jī)理中的應(yīng)用[J].國(guó)外醫(yī)學(xué)內(nèi)分泌學(xué)分冊(cè), 1999, 19(3): 110-113.

[25] Wetsel W C, Valenca M M, Merchenthaler I. Intrinsic pulsatile se?cretory activity of immortalized luteinizing hormone-releasing hor?mone-secreting neurons[J]. Proc Natl Acad Sci USA, 1992, 89(9): 4149-4153.

[26] Otani H, Otsuka F, Takeda M, et al. Regulation of GnRH produc? tion by estrogen and bone morphogenetic proteins in GT1-7 hypo?thalamic cells[J]. The Journal of Endocrinology, 2009, 203(1): 87-97.

[27] Terasaka T, Otsuka F, Tsukamoto N, et al. Mutual interaction of kisspeptin, estrogen and bone morphogenetic protein-4 activity in GnRH regulation by GT1-7 cells[J]. Mol Cell Endocrinol, 2013, 381(1-2): 8-15.

[28] Ohaki T, Shintani Y, Honda S, et al. Metastasis suppressor gene Kiss-1 encodes peptide ligand of a G-protein-coupled receptor [J]. Nature, 2001, 411(6837): 613-617.

[29] Funes S, Hedrick J A, Vassileva G, et al. The Kiss-1 receptor GPR54 is essential for the development of the murine reproduc?tive system[J]. Biochem Biophys Res Commun, 2003, 312(4): 1357-1363.

[30] Ozcan M, Alcin E, Ayar A, et al. Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neu?rones[J]. Neurosci Lett, 2011, 492(1): 55-58.

[31] Heather M D, Donald K C, Robert A S. Kisspeptin neurons as cen?tral processors in the regulation of gonadotropin-releasing hor?mone secretion[J]. Endocrinology, 2006, 147(3): 1154-1158.

[32]楊嶸,張立實(shí),王以美,等.雌二醇誘導(dǎo)雌性大鼠真性性早熟模型的建立[J].中國(guó)藥理學(xué)與毒理學(xué)雜志, 2013, 27(2): 279-283.

[33] Matagne V, Rasier G, Lebrethon M C, et al. Estradiol stimulation of pulsatile gonadotropin-releasing hormone secretion in vitro: correlation with perinatal exposure to sex steroids and induction of sexual precocity in vivo[J]. Endocrinology, 2004, 145(6): 2775-2783.

[34] Christlan C A, Mobley J L, Moenter S M. Diurnal and estradi?ol-dependent changes in gonadotropin-releasing hormone neuron firing activity[J]. Proceedings of the National Academy of Scienc?es of the United States of America, 2005, 102(43): 15682-15687.

[35] Navarro C E, Saeed S A, Murdock C, et al. Regulation of cyclic ad?enosine 3', 5'-monophosphate signaling and pulsatile neurosecre?tion by Gi-coupled plasma membrane estrogen receptors in im?mortalized gonadotrophin-releasing hormone neurons[J]. Mol En?docrinol, 2003, 17: 1792-1804.

[36] Sharifi N, Reuss A E, Wray S. Prenatal LHRH neurons in nasal explant cultures express estrogen receptor beta transcript[J]. En?docrinology, 2002, 143(7): 2503-2507.

[37] Herbison A E, Pape J R. New evidence for estrogen receptors in gonadotropin-releasing hormone neurons[J]. Front Neuroendocri?nol, 2001, 22(4): 292-308.

[38] Tonsfeldt K J, Goodall C P, Latham K L, et al. Oestrogen induces rhythmic expression of the Kisspeptin-1 receptor GPR54 in hypo?thalamic gonadotrophin-releasing hormone-secreting GT1-7 cells [J]. J Neuroendocrinol, 2011, 23(9): 823-830.

[39] Chehab F F, Mounzih K, Lu R, et al. Early onset of reproduct ive function in normal female mice treated with leptin[J]. Science, 1997, 275(5296): 88-90.

[40] Hileman S M, Pierroz D D, Flier J S. Leptin, nutrition, and repro?duction: timing is everything[J]. J Clin Endocrinol Metab, 2000, 85 (2): 804-807.

[41]楊穎,陳名道,李鳳英,等.瘦素對(duì)GT1-7細(xì)胞GnRH釋放的影響[J].放射免疫學(xué)雜志, 2002, 15: 65-67.

[42]王軍,婁玉杰.能量平衡與哺乳動(dòng)物生殖:瘦素和KiSS-1/ GPR54系統(tǒng)的最新研究進(jìn)展[J].動(dòng)物營(yíng)養(yǎng)學(xué)報(bào), 2011, 23: 1-4.

[43] Bellefontaine N, Chachlaki K, Parkash J, et al. Leptin-dependent neuronal NO signaling in the preoptic hypothalamus facilitates re?production[J]. The Journal of Clinic Investigation, 2014, 124(6): 2550-2559.

[44] Leshan R L, Greenwald-Yarnell M, Patterson C M, et al. Leptin action through hypothalamic nitric oxide synthase-1-expressing neurons controls energy balance[J]. Nature Medicine, 2012, 18(5): 820-823.

[45] Roy D, Angelini N L, Frieda H, Brown G M, et a1. Cyclical regu?lation of GnRH gene expression in GT1-7 GnRH-secreting neu?rons by melatonin[J]. Endocrinology 2001, 142(11): 4711-4720.

[46] Roy D, Belshan D D. Melatonin receptor activation regulates GnRH gene expression and secretion in GT1-7 GnRH neurons signal transduction mechanisms[J]. J Biol Chem, 2002, 277(1): 251-258.

[47] Kelestimur H, Ozcan M, Kacar E, et a1. Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1-7 GnRH neurons[J]. Brain Res, 2012, 1435: 24-28.

[48]胡雅婷.脂聯(lián)素通過(guò)AMPK和SP1抑制下丘腦GT1-7神經(jīng)細(xì)KISS-1基因轉(zhuǎn)錄和表達(dá)[D].福州:福建醫(yī)科大學(xué), 2010.

[49] Rodriguez-Pacheco F, Martinez-Fuentes A J, Toval S, et a1. Reg?ulation of pituitary cell function by adiponectin[J]. Endocrinology, 2007, 148(1): 401-410.

[50] Kinsey-Jones J S, Ll X F, Bowe J E, et al. Effect of calcitonin gene-related peptide on gonadotrophin-releasing hormone mRNA expression in GT1-7 cells[J]. J Neuroendocrinol, 2005, 17(9): 541-544.

[51] Kanasaki H, Mijiddorj T, Sukhbaatar U, et al. Pituitary adenylate cyclaseactivating polypeptide(PACAP) increases expression of the gonadotropin-releasing hormone(GnRH) receptor in GnRH-pro?ducing GT1-7 cells overexpressing PACAP type I receptor[J]. Gen Comp Endocrinol, 2013, 193: 95-102.

[52] Glidewell-Kenney C A, Shao P P, Iyer A K, et al. Neurokinin B causes acute GnRH secretion and repression of GnRH transcrip?tion in GT1-7 GnRH neurons[J]. Mol Endocrinol, 2013, 27(3): 437-454.

[53]陳名道,楊穎,李鳳英,等.二仙湯及其拆方對(duì)GT1-7細(xì)胞株GnRH基因轉(zhuǎn)錄和表達(dá)的影響[J].中國(guó)中西醫(yī)結(jié)合雜志, 2002, 22: 220-222.

[54] Rupprecht L E, Mietlicki-Baase E G, Zimmer D J, et al. Hind?brain GLP-1 receptor-mediated suppression of food intake re?quires a PI3K-dependent decrease in phosphorylation of mem?brane-bound Akt[J]. Am J Physiol Endocrinol Metab, 2013, 305 (6): 751-759.

[55] Damm E, Buech T R, Gudermann T, et al. Melanocortin-induced PKA Activation Inhibits AMPK activity via ERK-1/2 and LKB-1 in hypothalamic GT1-7 cells[J]. Mol Endocrinol, 2012, 26(4): 643-654.

[56] Li B, Lee K, Martin R J. Overexpression of glucose transporter 2 in GT1-7 cells inhibits AMP-activated protein kinase and agou?ti-related peptide expression[J]. Brain Res, 2006 , 1118(1): 1-5.

[57]溫俊平.脂聯(lián)素對(duì)下丘腦生殖和能量代謝的調(diào)控作用[D].上海:上海交通大學(xué), 2000.

[58] Wen J P, Liu C E, Hu Y T, et al. Globular adiponectin regulates energy homeostasis through AMP-activated protein kinase-ace?tyl-CoA carboxylase（AMPK/ACC）pathway in the hypothala?mus[J]. Mol Cell Biochem, 2010, 344(1-2): 109-115.

[59]袁國(guó)鋮,卜登攀,南雪梅,等.不同類(lèi)型日糧對(duì)泌乳奶牛乳腺組織胰島素通路及JAK2-STAT信號(hào)通路[J].東北農(nóng)業(yè)大學(xué)學(xué)報(bào), 2013, 44(6): 44-53.

[60] Beall C, Hamilton D L, Gallagher J, et al. Mouse hypothalamic GT1-7 cells demonstrate AMPK-dependent intrinsic glucosesensing behavior[J]. Diabetologia, 2012, 55(9): 2432-2444.

[61] Hayes M R, Leichner T M, Zhao S, et al. Intracellular signals me?diating the food intake-suppressive effects of hindbrain gluca?gon-like peptide-1 receptor activation[J]. Cell Metab, 2011, 13 (3): 320-330.

[62]劉金玲,單風(fēng)平.蛋氨酸腦啡肽對(duì)GM-CSF誘導(dǎo)的小鼠骨髓來(lái)源樹(shù)突狀細(xì)胞TLR-4和TNF-α表達(dá)影響[J].東北農(nóng)業(yè)大學(xué)學(xué)報(bào), 2013, 44(3): 5-10.

GT1-7 cells and its application in reproduction-related researches

FENG Tao1, WU Xiaomin1,2, XU Xiaoling1, BAI Jiahua1, SONG Yuqing1, XIAO Linli1, HAN Xiangmin2, LIU Yan1(1. Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China; 2. School of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China)

Abstract:GT1-7 cells were subset strains of GT1 cell lines, which were GnRH neuron cell line isloated from the hypothalamus of transgenic mice and had the typical characteristics of highly differentiated neuroendocrine cells. GnRH synthesis and release in hypothalamus played an important role in reproductive function. However, GnRH neurons were only thousands and discrete distribution in the brain, which leads more difficulties in studying in vivo. Currently, GT1-7 cell was widely using in reproduction-related research works as an ideal GnRH cell model in vitro. In this review, the source, characteristics, applications as well as signaling pathways influencing ability of GT1-7 cells were summarized to provide references for GnRH related researches.

Key words:GT1-7 cells; gonadotropin-releasing hormone; reproductive regulation；signaling pathway

*通訊作者:劉彥，研究員，研究方向?yàn)閯?dòng)物繁殖學(xué)。E-mail: liuyanxms@163.com

作者簡(jiǎn)介：馮濤（1980-），男，副研究員，博士，研究方向?yàn)閯?dòng)物生殖營(yíng)養(yǎng)調(diào)控。E-mail: fengtao_gs@163.com

基金項(xiàng)目：國(guó)家自然科學(xué)基金項(xiàng)目（31501950）；北京市科技新星計(jì)劃項(xiàng)目（Z141105001814046）；奶牛產(chǎn)業(yè)技術(shù)體系北京市創(chuàng)新團(tuán)隊(duì)

收稿日期：2015-06-29

中圖分類(lèi)號(hào)：R58

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1005-9369（2016）01-0102-07