駱超超　王春梅　李慶章　高學(xué)軍

摘 要：乳蛋白合成機(jī)理是重要的生命科學(xué)基礎(chǔ)問(wèn)題之一，目前已發(fā)現(xiàn)催乳素通過(guò)JAK/STAT5信號(hào)通路在轉(zhuǎn)錄水平調(diào)控乳蛋白合成，氨基酸通過(guò)mTOR/S6K1信號(hào)通路在翻譯水平調(diào)控乳蛋白合成。但乳腺細(xì)胞內(nèi)乳蛋白合成還有哪些重要的信號(hào)分子參與轉(zhuǎn)錄和翻譯調(diào)控，什么分子是氨基酸等營(yíng)養(yǎng)素的“分子傳感器”，細(xì)胞核和細(xì)胞液之間的串話如何應(yīng)答氨基酸信號(hào)從而調(diào)節(jié)乳蛋白的合成等，仍是尚待解決的重要科學(xué)問(wèn)題。該實(shí)驗(yàn)較系統(tǒng)的研究了甘氨酰胺合成酶作為一個(gè)新的信號(hào)分子，接受蛋氨酸信號(hào)，從轉(zhuǎn)錄水平和翻譯水平對(duì)奶牛乳腺上皮細(xì)胞中β-酪蛋白（CSN2）合成的調(diào)節(jié)作用及機(jī)理。實(shí)驗(yàn)既為人們弄清楚氨基酸對(duì)乳蛋白的調(diào)節(jié)及作用機(jī)理提供基本理論依據(jù)，也為人們?nèi)媪私馊榈鞍缀铣傻木W(wǎng)絡(luò)信號(hào)通路提供新的視野。該研究采用組織塊培養(yǎng)法培養(yǎng)并純化體外培養(yǎng)的奶牛乳腺上皮細(xì)胞，用蛋白質(zhì)免疫印記和免疫熒光檢測(cè)細(xì)胞中角蛋白18（CK18）和CSN2的表達(dá)以鑒定細(xì)胞純度和泌乳功能。實(shí)驗(yàn)通過(guò)在培養(yǎng)液中添加0.6 mmol/L的蛋氨酸，建立細(xì)胞泌乳模型用實(shí)時(shí)熒光定量PCR、WB和IF等方法，檢測(cè)添加蛋氨酸泌乳模型中，GlyRS的表達(dá)與定位情況。結(jié)果表明蛋氨酸在促進(jìn)細(xì)胞泌乳時(shí)，GlyRS表達(dá)顯著上升（P<0.01），并且GlyRS入核也顯著增加（P<0.01）。這說(shuō)明，在蛋氨酸上調(diào)CSN2表達(dá)過(guò)程中，GlyRS可能是一個(gè)重要的調(diào)節(jié)因子。對(duì)GlyRS進(jìn)行過(guò)表達(dá)與干擾，利用qRT-PCR、WB和IF等方法檢測(cè)mTOR、p-mTOR、Stat5a、p-Stat5a、S6K1、p-S6K1、4EBP1、p-4EBP1和CSN2的表達(dá)，用CASY細(xì)胞活力分析儀和流式細(xì)胞儀分析測(cè)定細(xì)胞活力、細(xì)胞數(shù)和細(xì)胞的增殖率。結(jié)果顯示，GlyRS過(guò)表達(dá)和干擾后，檢測(cè)的磷酸化蛋白和CSN2均分別顯著上升和下降（P<0.01），細(xì)胞活力、細(xì)胞數(shù)和細(xì)胞的增殖率也分別顯著增加和減少（P<0.01），而非磷酸化蛋白無(wú)顯著變化（P>0.05）；添加蛋氨酸后，檢測(cè)各蛋白的表達(dá)均顯著上升（P<0.01），但GlyRS干擾組，添加蛋氨酸不能使磷酸化蛋白和CSN2的表達(dá)恢復(fù)。這說(shuō)明，GlyRS能接受蛋氨酸的信號(hào)，上調(diào)CSN2合成相關(guān)信號(hào)分子的磷酸化、CSN2的合成及促進(jìn)細(xì)胞增殖，且蛋氨酸上調(diào)CSN2合成和促進(jìn)細(xì)胞增殖的信號(hào)很大一部分是由GlyRS接受并傳遞的。綜上所述，GlyRS是蛋氨酸調(diào)節(jié)奶牛乳腺上皮細(xì)胞泌乳過(guò)程的重要信號(hào)分子，其可能調(diào)控機(jī)制如下：GlyRS在胞漿中接受蛋氨酸信號(hào)后，544位蘇氨酸和704位絲氨酸發(fā)生磷酸化，磷酸化后一部分在胞漿中通過(guò)與eIF2D結(jié)合，在翻譯水平上上調(diào)CSN2的表達(dá)；一部分由NLS引導(dǎo)入核，同時(shí)發(fā)生分子剪切，剪切后的C端與NFκB1結(jié)合，促進(jìn)NFκB1與CSN2啟動(dòng)子結(jié)合，從轉(zhuǎn)錄水平上上調(diào)CSN2的表達(dá)。

關(guān)鍵詞：甘氨酰胺合成酶 泌乳信號(hào)通路 β-酪蛋白 奶牛乳腺上皮細(xì)胞 蛋氨酸

Abstract：The mechanism of milk protein synthesis is one of the important foundation problems of life science. In recent years， obvious progress of this problem has been made by some research. At present， we found that the transcription process of milk protein synthesis was regulated by prolactin through the JAK/STAT5 signaling pathways and the translation process of it was regulated by amino acids through the mTOR/S6K1 signaling pathways. But， which other important signaling molecules involved in transcription and translation of milk protein synthesis in mammary gland cells？ What molecule are the "molecular sensors" of amino acids and other nutrients and how the nucleus and cytoplasm reply the amino acid signal are still the important scientific problems that needed to be solved in lactation biology nowadays. In this study， a comprehensive study of glycyl-tRNA synthetase （GlyRS） regulation the synthesis of β-casein （CSN2） as a new signal molecule reply the methionine signal in dairy cow mammary epithelial cells and its mechanism were completed. Both the basic theory of milk protein synthesis regulated by amino acids and the new horizon of understanding the signaling pathway network of milk protein synthesis were provided by this study. Comprehensive these experimental results， GlyRS was an important signal molecule in the process of methionine regulated the lactation in dairy cow mammary gland epithelial cells. The mechanism of regulation by GlyRS was as follows：The T544 and S704 of GlyRS were phosphorylated after GlyRS accept the methionine signal in the cytoplasm，then a part of GlyRS combined with eIF2D in the cytoplasm and raised the expression of CSN2 at translation level， the other part of GlyRS were guided into the nucleus by NLS， and these GlyRS were spliced at the same time. Then， the C-terminal parts of the spliced GlyRS combined with NFκB1 in the nucleus and promote the combination of NFκB1 with CSN2 promoter， and finally raised the expression of CSN2 at transcription level.

Key Words：Glycyl-tRNA synthetase；Lactation signaling pathway；β-casein；Bovine mammary epithelial cells；Methionine

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