小G蛋白Ran与GlyRS相互作用调节乳蛋白合成的初步研究
发布时间：2018-08-22 19:53
【摘要】：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。细胞核和细胞液之间如何应答氨基酸信号从而调节乳蛋白的合成,仍是现今泌乳生物学领域尚待解决的重要科学问题。Ran作为一个重要的细胞增殖因子,在多种细胞活动中发挥作用,包括DNA复制,细胞核膜重建,纺锤体的形成,物质的核浆转运等。国内外关于Ran基因与甘氨酰tRNA合成酶在奶牛乳腺发育过程中表达及相互作用的研究鲜有报道。本研究以泌乳期奶牛乳腺上皮细胞为模型。探索Ran基因与甘氨酰tRNA合成酶的相互作用,在奶牛乳腺中的调节作用,为动物乳腺发育和泌乳调节机制的研究提供基础资料,为人工调节乳品质和乳产量提供理论依据。本研究以中国荷斯坦奶牛作为实验动物,采用组织块培养法,培养并纯化体外培养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)和酪蛋白(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0.6 mmol/L的蛋氨酸,建立了细胞的泌乳模型。检测添加蛋氨酸泌乳模型中,Ran过表达后GlyRS的表达显著上升(p0.01)。以体外培养的泌乳期奶牛乳腺上皮细胞为研究对象,采用共定位技术初步检测Ran与GlyRS是否相互作用,采用Western blotting技术探索在泌乳过程中Ran的与GlyRS成正相关。构建重组质粒PEX-7-Ran(Ran-RFP)和实验室构建成功的GlyRS-GFP,对细胞进行瞬时转染,进行荧光能量共振转移实验,用相关软件进行结果分析,证实Ran与GlyRS之间的相互作用。综上所述,Ran与GlyRS在乳腺上皮细胞泌乳过程中相互作用,并且在泌乳过程中,呈正相关。
[Abstract]:The mechanism of milk protein synthesis is one of the important basic problems in life science. How the nucleus and cell fluid respond to amino acid signals and regulate the synthesis of lactoprotein is still an important scientific problem in the field of lactation biology. Ran is an important cell proliferation factor. It plays a role in many cell activities, including DNA replication, nuclear membrane reconstruction, spindle formation, nuclear and cytoplasmic transport. There are few reports on the expression and interaction of Ran gene and glycosyl tRNA synthase during the development of mammary gland in dairy cattle. In this study, milk cow mammary epithelial cells were used as model. To explore the interaction of Ran gene with glycosyl tRNA synthase and its regulatory role in dairy cow mammary gland, to provide basic data for the study of mammary gland development and lactation regulation mechanism, and to provide theoretical basis for artificial regulation of milk quality and milk yield. In this study, Chinese Holstein cows were used as experimental animals to culture and purify bovine mammary epithelial cells in vitro by tissue mass culture. Protein imprinted (WB) and immunofluorescence (IF) were used to detect the expression of keratin 18 (CK18) and casein (CSN2) in order to identify the cell purity and lactation function. The lactation model of cells was established by adding 0. 6 mmol/L methionine to the culture medium. The overexpression of ran in methionine lactation model was detected and the expression of GlyRS was significantly increased (p0.01). The colocalization technique was used to detect the interaction between Ran and GlyRS, and Western blotting technique was used to explore the positive correlation between Ran and GlyRS during lactation. The recombinant plasmid PEX-7-Ran (Ran-RFP) and the successfully constructed GlyRS-GFPs were constructed. The transient transfection of the cells was carried out, and the fluorescence energy resonance transfer experiment was carried out. The results were analyzed with the relevant software to confirm the interaction between Ran and GlyRS. In conclusion, ran and GlyRS interact with each other during lactation of mammary epithelial cells, and there is a positive correlation during lactation.
【学位授予单位】：哈尔滨师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
[Abstract]:The mechanism of milk protein synthesis is one of the important basic problems in life science. How the nucleus and cell fluid respond to amino acid signals and regulate the synthesis of lactoprotein is still an important scientific problem in the field of lactation biology. Ran is an important cell proliferation factor. It plays a role in many cell activities, including DNA replication, nuclear membrane reconstruction, spindle formation, nuclear and cytoplasmic transport. There are few reports on the expression and interaction of Ran gene and glycosyl tRNA synthase during the development of mammary gland in dairy cattle. In this study, milk cow mammary epithelial cells were used as model. To explore the interaction of Ran gene with glycosyl tRNA synthase and its regulatory role in dairy cow mammary gland, to provide basic data for the study of mammary gland development and lactation regulation mechanism, and to provide theoretical basis for artificial regulation of milk quality and milk yield. In this study, Chinese Holstein cows were used as experimental animals to culture and purify bovine mammary epithelial cells in vitro by tissue mass culture. Protein imprinted (WB) and immunofluorescence (IF) were used to detect the expression of keratin 18 (CK18) and casein (CSN2) in order to identify the cell purity and lactation function. The lactation model of cells was established by adding 0. 6 mmol/L methionine to the culture medium. The overexpression of ran in methionine lactation model was detected and the expression of GlyRS was significantly increased (p0.01). The colocalization technique was used to detect the interaction between Ran and GlyRS, and Western blotting technique was used to explore the positive correlation between Ran and GlyRS during lactation. The recombinant plasmid PEX-7-Ran (Ran-RFP) and the successfully constructed GlyRS-GFPs were constructed. The transient transfection of the cells was carried out, and the fluorescence energy resonance transfer experiment was carried out. The results were analyzed with the relevant software to confirm the interaction between Ran and GlyRS. In conclusion, ran and GlyRS interact with each other during lactation of mammary epithelial cells, and there is a positive correlation during lactation.
【学位授予单位】：哈尔滨师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
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