GlyRS通过ELP4调控奶牛乳腺上皮细胞乳蛋白合成的机理
发布时间：2018-12-25 10:10
【摘要】：乳蛋白的合成及调控是哺乳动物乳腺上皮细胞内非常重要的代谢过程。乳蛋白合成的机理是重要的生命科学基础问题之一,近年来的研究已有一些明显的进展。已发现催乳素通过JAK2/STAT5信号通路在转录水平控制乳蛋白生成过程,氨基酸通过m TOR/S6K1信号通路在翻译水平调控乳蛋白合成过程。但乳腺细胞内乳合成的详细生化机制尚不清楚。本实验室前期实验发现甘氨酰t RNA合成酶可以在细胞核内参与调节乳蛋白合成,然而具体调节作用方式还不知道,阐明此机制将有助于揭示乳蛋白合成的详细分子作用机制。本实验研究了蛋氨酸刺激下,甘氨酰t RNA合成酶通过ELP4对乳蛋白合成的影响,以阐明甘氨酰t RNA合成酶下游信号分子ELP4的作用机制。本研究为甘氨酰t RNA合成酶和ELP4对乳蛋白基因表达的调节及其作用机理提供基本理论依据,并为乳蛋白合成的网络信号通路提供了一个新的视野。本研究选用组织块培养法,体外培养并得到了纯化的奶牛乳腺上皮细胞,用蛋白质免疫印记(WB)和免疫荧光(IF)检测细胞中角蛋白18(CK18)和CSN2的表达以鉴定细胞的纯度和泌乳功能。实验通过在培养液中添加0.6mmol/L的蛋氨酸,建立了细胞的泌乳模型,用实时荧光定量PCR(q RT-PCR)和WB方法,检测添加蛋氨酸泌乳模型中,ELP4的表达情况。结果表明,在蛋氨酸促进细胞泌乳时,ELP4的表达显著上升(p0.01)。对ELP4进行过表达并利用WB检测CSN2的表达,结果显示,ELP4过表达后CSN2表达显著上升(p0.01)。这说明,在蛋氨酸上调CSN2表达的过程中,ELP4可能是一个重要的调节因子。本实验用免疫共沉淀(Co-IP)技术沉淀并鉴定了细胞中ELP4的相互作用蛋白Gly RS。构建真核表达载体ELP4-EGFP和Gly RS-RFP,应用激光能量共振转移(FRET)、定量免疫共沉淀和激光共聚焦共定位等技术,检测添加蛋氨酸对Gly RS与ELP4相互作用的影响。结果显示,添加蛋氨酸泌乳模型中,Gly RS与ELP4的结合显著增加(p0.01)。对Gly RS进行过表达和干扰,WB检测ELP4和CSN2的表达的变化。结果显示,GlyRS过表达后,ELP4的表达显著增加(p0.01),CSN2的表达显著增加(p0.01);Gly RS干扰后,ELP4的表达显著下降(p0.01),CSN2的表达显著下降(p0.01)。这说明,ELP4与Gly RS结合应答蛋氨酸直接参与CSN2合成的调节。接着研究了Gly RS过表达和干扰后对p-NFκB1与ELP4的启动子序列结合情况的影响,结果表明,Gly RS过表达后,细胞中p-NFκB1与ELP4的启动子序列结合显著增加(p0.01),Gly RS干扰达后,细胞中p-NFκB1与ELP4的启动子序列结合显著降低(p0.01)。结果提示,在细胞中Gly RS与ELP4不仅存在相互作用,而且Gly RS还可以通过促进p-NFκB1与ELP4启动子结合,激活ELP4的转录起始,从而在转录水平调节ELP4的表达。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
本文编号：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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
【参考文献】
相关硕士学位论文 前1条
1 张霞;GSK3β通过mTOR/S6K1途径对奶牛乳腺上皮细胞泌乳及增殖的调控[D];东北农业大学;2014年
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