GlyRS通過ELP4調(diào)控奶牛乳腺上皮細胞乳蛋白合成的機理
發(fā)布時間：2018-12-25 10:10
【摘要】：乳蛋白的合成及調(diào)控是哺乳動物乳腺上皮細胞內(nèi)非常重要的代謝過程。乳蛋白合成的機理是重要的生命科學(xué)基礎(chǔ)問題之一,近年來的研究已有一些明顯的進展。已發(fā)現(xiàn)催乳素通過JAK2/STAT5信號通路在轉(zhuǎn)錄水平控制乳蛋白生成過程,氨基酸通過m TOR/S6K1信號通路在翻譯水平調(diào)控乳蛋白合成過程。但乳腺細胞內(nèi)乳合成的詳細生化機制尚不清楚。本實驗室前期實驗發(fā)現(xiàn)甘氨酰t RNA合成酶可以在細胞核內(nèi)參與調(diào)節(jié)乳蛋白合成,然而具體調(diào)節(jié)作用方式還不知道,闡明此機制將有助于揭示乳蛋白合成的詳細分子作用機制。本實驗研究了蛋氨酸刺激下,甘氨酰t RNA合成酶通過ELP4對乳蛋白合成的影響,以闡明甘氨酰t RNA合成酶下游信號分子ELP4的作用機制。本研究為甘氨酰t RNA合成酶和ELP4對乳蛋白基因表達的調(diào)節(jié)及其作用機理提供基本理論依據(jù),并為乳蛋白合成的網(wǎng)絡(luò)信號通路提供了一個新的視野。本研究選用組織塊培養(yǎng)法,體外培養(yǎng)并得到了純化的奶牛乳腺上皮細胞,用蛋白質(zhì)免疫印記(WB)和免疫熒光(IF)檢測細胞中角蛋白18(CK18)和CSN2的表達以鑒定細胞的純度和泌乳功能。實驗通過在培養(yǎng)液中添加0.6mmol/L的蛋氨酸,建立了細胞的泌乳模型,用實時熒光定量PCR(q RT-PCR)和WB方法,檢測添加蛋氨酸泌乳模型中,ELP4的表達情況。結(jié)果表明,在蛋氨酸促進細胞泌乳時,ELP4的表達顯著上升(p0.01)。對ELP4進行過表達并利用WB檢測CSN2的表達,結(jié)果顯示,ELP4過表達后CSN2表達顯著上升(p0.01)。這說明,在蛋氨酸上調(diào)CSN2表達的過程中,ELP4可能是一個重要的調(diào)節(jié)因子。本實驗用免疫共沉淀(Co-IP)技術(shù)沉淀并鑒定了細胞中ELP4的相互作用蛋白Gly RS。構(gòu)建真核表達載體ELP4-EGFP和Gly RS-RFP,應(yīng)用激光能量共振轉(zhuǎn)移(FRET)、定量免疫共沉淀和激光共聚焦共定位等技術(shù),檢測添加蛋氨酸對Gly RS與ELP4相互作用的影響。結(jié)果顯示,添加蛋氨酸泌乳模型中,Gly RS與ELP4的結(jié)合顯著增加(p0.01)。對Gly RS進行過表達和干擾,WB檢測ELP4和CSN2的表達的變化。結(jié)果顯示,GlyRS過表達后,ELP4的表達顯著增加(p0.01),CSN2的表達顯著增加(p0.01);Gly RS干擾后,ELP4的表達顯著下降(p0.01),CSN2的表達顯著下降(p0.01)。這說明,ELP4與Gly RS結(jié)合應(yīng)答蛋氨酸直接參與CSN2合成的調(diào)節(jié)。接著研究了Gly RS過表達和干擾后對p-NFκB1與ELP4的啟動子序列結(jié)合情況的影響,結(jié)果表明,Gly RS過表達后,細胞中p-NFκB1與ELP4的啟動子序列結(jié)合顯著增加(p0.01),Gly RS干擾達后,細胞中p-NFκB1與ELP4的啟動子序列結(jié)合顯著降低(p0.01)。結(jié)果提示,在細胞中Gly RS與ELP4不僅存在相互作用,而且Gly RS還可以通過促進p-NFκB1與ELP4啟動子結(jié)合,激活ELP4的轉(zhuǎn)錄起始,從而在轉(zhuǎn)錄水平調(diào)節(jié)ELP4的表達。
[Abstract]:The synthesis and regulation of lactoprotein is a very important metabolic process in mammal mammary epithelial cells. The mechanism of milk protein synthesis is one of the important basic problems in life science. It has been found that prolactin controls the production of lactoprotein at the transcriptional level through the JAK2/STAT5 signaling pathway, while the amino acid regulates the synthesis of lactoprotein at the translation level through the m TOR/S6K1 signaling pathway. However, the detailed biochemical mechanism of breast intracellular milk synthesis is unclear. It was found that glycyl-t RNA synthase can regulate the synthesis of lactoprotein in the nucleus. However, the specific regulation mode is not known yet. The elucidation of this mechanism will help to reveal the detailed molecular mechanism of milk protein synthesis. The effects of methionine stimulated glycyl-t RNA synthase on the synthesis of lactoprotein by ELP4 were studied in order to elucidate the mechanism of the downstream signal molecule ELP4 of glycyl t RNA synthase. This study provides a theoretical basis for the regulation and mechanism of galactoprotein gene expression by glycyl-t RNA synthase and ELP4, and provides a new perspective for the network signaling pathway of milk protein synthesis. In this study, bovine mammary epithelial cells were cultured and purified by tissue mass culture in vitro. Protein imprinted (WB) and immunofluorescence (IF) were used to detect the expression of keratin 18 (CK18) and CSN2 in order to identify the cell purity and lactation function. The lactation model of cells was established by adding methionine of 0.6mmol/L to the culture medium. The expression of ELP4 in the lactation model was detected by real-time fluorescence quantitative PCR (q RT-PCR and WB. The results showed that the expression of ELP4 increased significantly when methionine promoted the lactation of cells (p 0.01). ELP4 was overexpressed and CSN2 expression was detected by WB. The results showed that the expression of CSN2 increased significantly after ELP4 overexpression (p0.01). This suggests that ELP4 may be an important regulatory factor in the up-regulation of CSN2 expression by methionine. The interaction protein Gly RS. of ELP4 in cells was precipitated and identified by immunoprecipitation (Co-IP) technique. The eukaryotic expression vectors ELP4-EGFP and Gly RS-RFP, were constructed to detect the effect of methionine on the interaction between Gly RS and ELP4 by using laser energy resonance transfer (FRET), quantitative immunoprecipitation and laser confocal co-localization. The results showed that the combination of, Gly RS and ELP4 was significantly increased in methionine lactation model (p 0.01). Gly RS was overexpressed and interfered, and ELP4 and CSN2 were detected by WB. The results showed that after overexpression of GlyRS, the expression of ELP4 increased significantly (p0.01), the expression of CSN2 increased significantly (p0.01); Gly RS interference, the expression of ELP4 decreased significantly (p0.01), the expression of CSN2 decreased significantly (p0.01). This suggests that ELP4 and Gly RS binding to methionine directly participate in the regulation of CSN2 synthesis. Then, the effects of Gly RS overexpression and interference on the binding of p-NF 魏 B1 to ELP4 promoter sequence were studied. The results showed that the binding of p-NF 魏 B1 to ELP4 promoter sequence increased significantly after, Gly RS overexpression (p0.01). After Gly RS interference, the binding of p-NF 魏 B1 to ELP4 promoter sequence was significantly decreased (p0.01). These results suggest that Gly RS and ELP4 not only interact with each other in cells, but also that Gly RS can activate the transcription initiation of ELP4 by promoting the binding of p-NF 魏 B1 to ELP4 promoter, thereby regulating the expression of ELP4 at the transcriptional level.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823
本文編號：2391037
[Abstract]:The synthesis and regulation of lactoprotein is a very important metabolic process in mammal mammary epithelial cells. The mechanism of milk protein synthesis is one of the important basic problems in life science. It has been found that prolactin controls the production of lactoprotein at the transcriptional level through the JAK2/STAT5 signaling pathway, while the amino acid regulates the synthesis of lactoprotein at the translation level through the m TOR/S6K1 signaling pathway. However, the detailed biochemical mechanism of breast intracellular milk synthesis is unclear. It was found that glycyl-t RNA synthase can regulate the synthesis of lactoprotein in the nucleus. However, the specific regulation mode is not known yet. The elucidation of this mechanism will help to reveal the detailed molecular mechanism of milk protein synthesis. The effects of methionine stimulated glycyl-t RNA synthase on the synthesis of lactoprotein by ELP4 were studied in order to elucidate the mechanism of the downstream signal molecule ELP4 of glycyl t RNA synthase. This study provides a theoretical basis for the regulation and mechanism of galactoprotein gene expression by glycyl-t RNA synthase and ELP4, and provides a new perspective for the network signaling pathway of milk protein synthesis. In this study, bovine mammary epithelial cells were cultured and purified by tissue mass culture in vitro. Protein imprinted (WB) and immunofluorescence (IF) were used to detect the expression of keratin 18 (CK18) and CSN2 in order to identify the cell purity and lactation function. The lactation model of cells was established by adding methionine of 0.6mmol/L to the culture medium. The expression of ELP4 in the lactation model was detected by real-time fluorescence quantitative PCR (q RT-PCR and WB. The results showed that the expression of ELP4 increased significantly when methionine promoted the lactation of cells (p 0.01). ELP4 was overexpressed and CSN2 expression was detected by WB. The results showed that the expression of CSN2 increased significantly after ELP4 overexpression (p0.01). This suggests that ELP4 may be an important regulatory factor in the up-regulation of CSN2 expression by methionine. The interaction protein Gly RS. of ELP4 in cells was precipitated and identified by immunoprecipitation (Co-IP) technique. The eukaryotic expression vectors ELP4-EGFP and Gly RS-RFP, were constructed to detect the effect of methionine on the interaction between Gly RS and ELP4 by using laser energy resonance transfer (FRET), quantitative immunoprecipitation and laser confocal co-localization. The results showed that the combination of, Gly RS and ELP4 was significantly increased in methionine lactation model (p 0.01). Gly RS was overexpressed and interfered, and ELP4 and CSN2 were detected by WB. The results showed that after overexpression of GlyRS, the expression of ELP4 increased significantly (p0.01), the expression of CSN2 increased significantly (p0.01); Gly RS interference, the expression of ELP4 decreased significantly (p0.01), the expression of CSN2 decreased significantly (p0.01). This suggests that ELP4 and Gly RS binding to methionine directly participate in the regulation of CSN2 synthesis. Then, the effects of Gly RS overexpression and interference on the binding of p-NF 魏 B1 to ELP4 promoter sequence were studied. The results showed that the binding of p-NF 魏 B1 to ELP4 promoter sequence increased significantly after, Gly RS overexpression (p0.01). After Gly RS interference, the binding of p-NF 魏 B1 to ELP4 promoter sequence was significantly decreased (p0.01). These results suggest that Gly RS and ELP4 not only interact with each other in cells, but also that Gly RS can activate the transcription initiation of ELP4 by promoting the binding of p-NF 魏 B1 to ELP4 promoter, thereby regulating the expression of ELP4 at the transcriptional level.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823
【參考文獻】
相關(guān)碩士學(xué)位論文 前1條
1 張霞;GSK3β通過mTOR/S6K1途徑對奶牛乳腺上皮細胞泌乳及增殖的調(diào)控[D];東北農(nóng)業(yè)大學(xué);2014年
，本文編號：2391037
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