【摘要】：乳蛋白合成的機(jī)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(jìn)展。細(xì)胞核和細(xì)胞液之間如何應(yīng)答氨基酸信號(hào)從而調(diào)節(jié)乳蛋白的合成,仍是現(xiàn)今泌乳生物學(xué)領(lǐng)域尚待解決的重要科學(xué)問題。Ran作為一個(gè)重要的細(xì)胞增殖因子,在多種細(xì)胞活動(dòng)中發(fā)揮作用,包括DNA復(fù)制,細(xì)胞核膜重建,紡錘體的形成,物質(zhì)的核漿轉(zhuǎn)運(yùn)等。國內(nèi)外關(guān)于Ran基因與甘氨酰tRNA合成酶在奶牛乳腺發(fā)育過程中表達(dá)及相互作用的研究鮮有報(bào)道。本研究以泌乳期奶牛乳腺上皮細(xì)胞為模型。探索Ran基因與甘氨酰tRNA合成酶的相互作用,在奶牛乳腺中的調(diào)節(jié)作用,為動(dòng)物乳腺發(fā)育和泌乳調(diào)節(jié)機(jī)制的研究提供基礎(chǔ)資料,為人工調(diào)節(jié)乳品質(zhì)和乳產(chǎn)量提供理論依據(jù)。本研究以中國荷斯坦奶牛作為實(shí)驗(yàn)動(dòng)物,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(WB)和免疫熒光(IF)檢測細(xì)胞中角蛋白18(CK18)和酪蛋白(CSN2)的表達(dá)以鑒定細(xì)胞的純度和泌乳功能。實(shí)驗(yàn)通過在培養(yǎng)液中添加0.6 mmol/L的蛋氨酸,建立了細(xì)胞的泌乳模型。檢測添加蛋氨酸泌乳模型中,Ran過表達(dá)后GlyRS的表達(dá)顯著上升(p0.01)。以體外培養(yǎng)的泌乳期奶牛乳腺上皮細(xì)胞為研究對(duì)象,采用共定位技術(shù)初步檢測Ran與GlyRS是否相互作用,采用Western blotting技術(shù)探索在泌乳過程中Ran的與GlyRS成正相關(guān)。構(gòu)建重組質(zhì)粒PEX-7-Ran(Ran-RFP)和實(shí)驗(yàn)室構(gòu)建成功的GlyRS-GFP,對(duì)細(xì)胞進(jìn)行瞬時(shí)轉(zhuǎn)染,進(jìn)行熒光能量共振轉(zhuǎn)移實(shí)驗(yàn),用相關(guān)軟件進(jìn)行結(jié)果分析,證實(shí)Ran與GlyRS之間的相互作用。綜上所述,Ran與GlyRS在乳腺上皮細(xì)胞泌乳過程中相互作用,并且在泌乳過程中,呈正相關(guān)。
[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.
【學(xué)位授予單位】：哈爾濱師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823

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