乳源乳脂肪球膜蛋白組學(xué)分析及其對細(xì)胞凋亡的作用機(jī)制
發(fā)布時間：2018-07-21 18:03
【摘要】：乳脂肪球膜是包裹在乳脂肪液滴外的膜,主要由蛋白質(zhì)、磷脂、氨基酸、多糖及多種微量元素構(gòu)成,且具備多種生物學(xué)功能。近年來,從分子水平認(rèn)識乳脂肪球膜蛋白質(zhì)的生物學(xué)功能已經(jīng)成為該領(lǐng)域的研究熱點。本文基于對牛乳、山羊乳、水牛乳、牦牛乳和駱駝乳5種乳脂肪球膜的基本組成和含量的了解,采用同位素標(biāo)記相對和絕對定量(iTRAQ)技術(shù)研究了5種乳脂肪球膜蛋白的蛋白組,并對特異性乳脂肪球膜蛋白質(zhì)進(jìn)行了生物信息學(xué)分析,并初步研究了5種乳脂肪球膜蛋白對HT-29結(jié)腸癌細(xì)胞和MCF-7乳腺癌細(xì)胞的抑制作用。首先對牛乳、山羊乳、水牛乳、牦牛乳和駱駝乳5種乳脂肪球膜的組分含量和各組分在不同乳源之間的差異進(jìn)行了研究。結(jié)果表明,脂肪球的大小與脂肪含量呈正相關(guān);乳脂肪球膜占脂肪球重量的2.07%~2.99%;5種乳脂肪球膜蛋白的含量在30.26%~42.51%,多糖的含量在2.96%~8.54%;乳脂肪球膜中脂質(zhì)含量在53.64%~66.34%,磷脂是極性脂質(zhì)的主要成分,約占極性脂質(zhì)的85%~98%。采用iTRAQ技術(shù)對5種乳脂肪球膜蛋白進(jìn)行了相對定量鑒定,牛乳和山羊乳脂肪球膜的有效膜蛋白為335個,牦牛乳和駱駝乳為336個,水牛乳為334個;以牛乳脂肪球膜蛋白為基準(zhǔn),其他4種乳中豐度上升蛋白(1.5倍)的數(shù)量在58~72個;GO分析結(jié)果表明,乳脂肪球膜蛋白主要參與細(xì)胞的代謝、生物過程調(diào)節(jié)、應(yīng)激反應(yīng)和轉(zhuǎn)運(yùn)。聚類分析將乳脂肪球膜蛋白分為5類,其中高豐度蛋白(熱點得分1)分別有28、13、12、9和18個。對聚類分析中的高豐度蛋白進(jìn)行代謝通路分析,在高豐度蛋白所參與的通路中,超過50%的通路與癌癥相關(guān)。根據(jù)主成分分析(因子累計貢獻(xiàn)率85%),從5種乳脂肪球膜蛋白中提取出的特異性蛋白分別為牛乳2個、山羊乳8個、水牛乳6個、牦牛乳5個和駱駝乳9個�；趯�5種乳脂肪球膜蛋白組的研究,選擇乳凝集素(MFG-E8)、長鏈酯酰輔酶A合成酶(ACSL)、糖基化依賴細(xì)胞黏附分子1(GlyCAM1)和三磷酸腺苷結(jié)合轉(zhuǎn)運(yùn)蛋白G超家族成員2(ABCG2)4種高豐度的功能性蛋白進(jìn)行了生物信息學(xué)分析。4種蛋白在物種間的差異較少,氨基酸序列相似度均達(dá)到了80%以上,二級結(jié)構(gòu)都包括α-螺旋、無規(guī)則卷曲和延伸鏈,均屬于不規(guī)則球狀蛋白。MFG-E8蛋白有2個N-糖基化位點和2個保守結(jié)構(gòu)域(表皮生長因子和F5/8型因子C端)。ACSL3蛋白包含ATP/AMP結(jié)合基序和脂肪酸結(jié)合基序2個重要的功能域。GlyCAM1蛋白有1個N-糖基化位點和4個O-糖基化位點,主要功能結(jié)構(gòu)域位于20~152位氨基酸。ABCG2蛋白包括ABCG-EPDR和ABC-2type transporter結(jié)構(gòu)域。研究了5種乳脂肪球膜蛋白對HT-29結(jié)腸癌細(xì)胞和MCF-7乳腺癌細(xì)胞的抑制作用。5種乳源乳脂肪球膜蛋白對兩種細(xì)胞增殖抑制呈劑量和時間依賴。經(jīng)牛乳、山羊乳和水牛乳脂肪球膜蛋白處理的HT-29細(xì)胞在S期發(fā)生阻滯,經(jīng)山羊乳、牦牛乳和駱駝乳脂肪球膜蛋白處理的MCF-7細(xì)胞在S期發(fā)生阻滯。研究結(jié)果表明,乳脂肪球膜蛋白通過降低了2種細(xì)胞中線粒體的活性,破壞線粒體功能,誘導(dǎo)HT-29細(xì)胞和MCF-7細(xì)胞發(fā)生凋亡;乳脂肪球膜蛋白是通過上調(diào)Bax和Caspase-3的表達(dá),同時下調(diào)Bcl-2的表達(dá)而達(dá)到的誘導(dǎo)細(xì)胞凋亡的目的。在5種乳脂肪球膜蛋白中,山羊乳和水牛乳脂肪球膜蛋白對HT-29細(xì)胞的作用效果最為顯著,水牛乳脂肪球膜蛋白對MCF-7細(xì)胞的作用效果最明顯。
[Abstract]:The milk fat globule membrane is a membrane encapsulated in the milk fat droplet, which is mainly composed of protein, phospholipid, amino acid, polysaccharide and a variety of trace elements, and has many biological functions. In recent years, it has become a hot topic in this field to understand the biological function of milk fat globule membrane protein from the molecular level. The basic composition and content of 5 kinds of milk fat spheres of milk, yak milk and camel milk were understood. The protein groups of 5 kinds of milk fat globules were studied by isotope labeling relative and absolute quantitative (iTRAQ) technology. The bioinformatics analysis of specific milk fat globule protein was carried out, and 5 kinds of milk fat globule membrane proteins were preliminarily studied. The inhibitory effects of HT-29 colon cancer cells and MCF-7 breast cancer cells. First, the content of 5 kinds of milk fat globules in milk, goat milk, buffalo milk, yak milk and camel milk were studied. The difference between the components in different Ruyuan were studied. The results showed that the size of the fat ball was positively correlated with the fat content; the milk fat globule membrane accounted for the fat ball weight. The content of the 5 kinds of milk fat globules is in the 30.26%~42.51%, the content of the polysaccharide is in the 2.96%~8.54%; the lipid content in the milk fat globule membrane is 53.64%~66.34%, the phospholipid is the main component of the polar lipid, and the 85%~98%. of the polar lipid is relative quantitative identification by iTRAQ technology to the milk fat globule membrane protein, milk and mountain. There are 335 effective membrane proteins in the fat globule membrane, 336 yak milk and camel milk, 334 buffalo milk, the milk fat globule protein as the base, and the number of other 4 kinds of milk (1.5 times) in the other 4 kinds of milk (1.5 times). The results of GO analysis show that the milk fat globule protein is mainly involved in cell metabolism, biological process regulation, stress response. Cluster analysis divided milk fat globule membrane proteins into 5 categories, with high abundance proteins (hot spots 1) with 28,13,12,9 and 18. Metabolic pathways were analyzed for high abundance proteins in cluster analysis, and more than 50% of the pathways involved in high abundance proteins were associated with cancer. According to principal component analysis (cumulative contribution rate of factors) 85%) the specific proteins extracted from 5 kinds of milk fat globules were milk 2, 8 goat milk, 6 buffalo milk, 5 yak milk and 9 camel milk. Based on the study of 5 milk fat globules group, MFG-E8, long chain ester acyl coenzyme A synthase (ACSL), glycosylation dependent cell adhesion molecules 1 (GlyCAM1) and three Adenosine phosphate binding transporter G superfamily member 2 (ABCG2) 2 (ABCG2) 4 kinds of high abundance functional proteins, bioinformatics analysis of.4 species with less difference among species, the similarity of amino acid sequences reached more than 80%, and the two structure consisted of alpha helix, irregular curl and extension chain, all of which belonged to irregular globular protein.MFG-E8 eggs There are 2 N- glycosylation sites and 2 conservative domains (epidermal growth factor and F5/8 type factor C terminal).ACSL3 protein containing 2 important functional domains, ATP/AMP binding and fatty acid binding, 1 N- glycosylation sites and 4 O- glycosylation sites, and the main functional domains are 20~152 bit amino acid.ABCG2 protein including A BCG-EPDR and ABC-2type transporter domains. The inhibitory effects of 5 kinds of milk fat globules on HT-29 colon cancer cells and MCF-7 breast cancer cells.5 species,.5 species, Ruyuan milk fat globule protein, were dosed and time dependent on the inhibition of the proliferation of two cells. The HT-29 cells treated by milk, goat milk and milk fat globule protein were in S phase MCF-7 cells treated with goat milk, yak milk and camel milk fat globules were blocked in the S phase. The results showed that milk fat globule membrane protein could destroy mitochondrial function in 2 cells, destroy mitochondrial function and induce apoptosis of HT-29 cells and MCF-7 cells; milk fat globule membrane protein is through up regulation of Bax In 5 kinds of milk fat globules, the effect of goat milk and buffalo milk fat globule membrane protein on HT-29 cells is the most significant in 5 kinds of milk fat globule membrane proteins. The effect of buffalo milk fat globule protein on MCF-7 cells is the most obvious.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS252.1
，
本文編號：2136339
[Abstract]:The milk fat globule membrane is a membrane encapsulated in the milk fat droplet, which is mainly composed of protein, phospholipid, amino acid, polysaccharide and a variety of trace elements, and has many biological functions. In recent years, it has become a hot topic in this field to understand the biological function of milk fat globule membrane protein from the molecular level. The basic composition and content of 5 kinds of milk fat spheres of milk, yak milk and camel milk were understood. The protein groups of 5 kinds of milk fat globules were studied by isotope labeling relative and absolute quantitative (iTRAQ) technology. The bioinformatics analysis of specific milk fat globule protein was carried out, and 5 kinds of milk fat globule membrane proteins were preliminarily studied. The inhibitory effects of HT-29 colon cancer cells and MCF-7 breast cancer cells. First, the content of 5 kinds of milk fat globules in milk, goat milk, buffalo milk, yak milk and camel milk were studied. The difference between the components in different Ruyuan were studied. The results showed that the size of the fat ball was positively correlated with the fat content; the milk fat globule membrane accounted for the fat ball weight. The content of the 5 kinds of milk fat globules is in the 30.26%~42.51%, the content of the polysaccharide is in the 2.96%~8.54%; the lipid content in the milk fat globule membrane is 53.64%~66.34%, the phospholipid is the main component of the polar lipid, and the 85%~98%. of the polar lipid is relative quantitative identification by iTRAQ technology to the milk fat globule membrane protein, milk and mountain. There are 335 effective membrane proteins in the fat globule membrane, 336 yak milk and camel milk, 334 buffalo milk, the milk fat globule protein as the base, and the number of other 4 kinds of milk (1.5 times) in the other 4 kinds of milk (1.5 times). The results of GO analysis show that the milk fat globule protein is mainly involved in cell metabolism, biological process regulation, stress response. Cluster analysis divided milk fat globule membrane proteins into 5 categories, with high abundance proteins (hot spots 1) with 28,13,12,9 and 18. Metabolic pathways were analyzed for high abundance proteins in cluster analysis, and more than 50% of the pathways involved in high abundance proteins were associated with cancer. According to principal component analysis (cumulative contribution rate of factors) 85%) the specific proteins extracted from 5 kinds of milk fat globules were milk 2, 8 goat milk, 6 buffalo milk, 5 yak milk and 9 camel milk. Based on the study of 5 milk fat globules group, MFG-E8, long chain ester acyl coenzyme A synthase (ACSL), glycosylation dependent cell adhesion molecules 1 (GlyCAM1) and three Adenosine phosphate binding transporter G superfamily member 2 (ABCG2) 2 (ABCG2) 4 kinds of high abundance functional proteins, bioinformatics analysis of.4 species with less difference among species, the similarity of amino acid sequences reached more than 80%, and the two structure consisted of alpha helix, irregular curl and extension chain, all of which belonged to irregular globular protein.MFG-E8 eggs There are 2 N- glycosylation sites and 2 conservative domains (epidermal growth factor and F5/8 type factor C terminal).ACSL3 protein containing 2 important functional domains, ATP/AMP binding and fatty acid binding, 1 N- glycosylation sites and 4 O- glycosylation sites, and the main functional domains are 20~152 bit amino acid.ABCG2 protein including A BCG-EPDR and ABC-2type transporter domains. The inhibitory effects of 5 kinds of milk fat globules on HT-29 colon cancer cells and MCF-7 breast cancer cells.5 species,.5 species, Ruyuan milk fat globule protein, were dosed and time dependent on the inhibition of the proliferation of two cells. The HT-29 cells treated by milk, goat milk and milk fat globule protein were in S phase MCF-7 cells treated with goat milk, yak milk and camel milk fat globules were blocked in the S phase. The results showed that milk fat globule membrane protein could destroy mitochondrial function in 2 cells, destroy mitochondrial function and induce apoptosis of HT-29 cells and MCF-7 cells; milk fat globule membrane protein is through up regulation of Bax In 5 kinds of milk fat globules, the effect of goat milk and buffalo milk fat globule membrane protein on HT-29 cells is the most significant in 5 kinds of milk fat globule membrane proteins. The effect of buffalo milk fat globule protein on MCF-7 cells is the most obvious.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS252.1
，
本文編號：2136339
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