乳源乳脂肪球膜蛋白组学分析及其对细胞凋亡的作用机制
发布时间：2018-07-21 18:03
【摘要】：乳脂肪球膜是包裹在乳脂肪液滴外的膜,主要由蛋白质、磷脂、氨基酸、多糖及多种微量元素构成,且具备多种生物学功能。近年来,从分子水平认识乳脂肪球膜蛋白质的生物学功能已经成为该领域的研究热点。本文基于对牛乳、山羊乳、水牛乳、牦牛乳和骆驼乳5种乳脂肪球膜的基本组成和含量的了解,采用同位素标记相对和绝对定量(iTRAQ)技术研究了5种乳脂肪球膜蛋白的蛋白组,并对特异性乳脂肪球膜蛋白质进行了生物信息学分析,并初步研究了5种乳脂肪球膜蛋白对HT-29结肠癌细胞和MCF-7乳腺癌细胞的抑制作用。首先对牛乳、山羊乳、水牛乳、牦牛乳和骆驼乳5种乳脂肪球膜的组分含量和各组分在不同乳源之间的差异进行了研究。结果表明,脂肪球的大小与脂肪含量呈正相关;乳脂肪球膜占脂肪球重量的2.07%~2.99%;5种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%;乳脂肪球膜中脂质含量在53.64%~66.34%,磷脂是极性脂质的主要成分,约占极性脂质的85%~98%。采用iTRAQ技术对5种乳脂肪球膜蛋白进行了相对定量鉴定,牛乳和山羊乳脂肪球膜的有效膜蛋白为335个,牦牛乳和骆驼乳为336个,水牛乳为334个;以牛乳脂肪球膜蛋白为基准,其他4种乳中丰度上升蛋白(1.5倍)的数量在58~72个;GO分析结果表明,乳脂肪球膜蛋白主要参与细胞的代谢、生物过程调节、应激反应和转运。聚类分析将乳脂肪球膜蛋白分为5类,其中高丰度蛋白(热点得分1)分别有28、13、12、9和18个。对聚类分析中的高丰度蛋白进行代谢通路分析,在高丰度蛋白所参与的通路中,超过50%的通路与癌症相关。根据主成分分析(因子累计贡献率85%),从5种乳脂肪球膜蛋白中提取出的特异性蛋白分别为牛乳2个、山羊乳8个、水牛乳6个、牦牛乳5个和骆驼乳9个。基于对5种乳脂肪球膜蛋白组的研究,选择乳凝集素(MFG-E8)、长链酯酰辅酶A合成酶(ACSL)、糖基化依赖细胞黏附分子1(GlyCAM1)和三磷酸腺苷结合转运蛋白G超家族成员2(ABCG2)4种高丰度的功能性蛋白进行了生物信息学分析。4种蛋白在物种间的差异较少,氨基酸序列相似度均达到了80%以上,二级结构都包括α-螺旋、无规则卷曲和延伸链,均属于不规则球状蛋白。MFG-E8蛋白有2个N-糖基化位点和2个保守结构域(表皮生长因子和F5/8型因子C端)。ACSL3蛋白包含ATP/AMP结合基序和脂肪酸结合基序2个重要的功能域。GlyCAM1蛋白有1个N-糖基化位点和4个O-糖基化位点,主要功能结构域位于20~152位氨基酸。ABCG2蛋白包括ABCG-EPDR和ABC-2type transporter结构域。研究了5种乳脂肪球膜蛋白对HT-29结肠癌细胞和MCF-7乳腺癌细胞的抑制作用。5种乳源乳脂肪球膜蛋白对两种细胞增殖抑制呈剂量和时间依赖。经牛乳、山羊乳和水牛乳脂肪球膜蛋白处理的HT-29细胞在S期发生阻滞,经山羊乳、牦牛乳和骆驼乳脂肪球膜蛋白处理的MCF-7细胞在S期发生阻滞。研究结果表明,乳脂肪球膜蛋白通过降低了2种细胞中线粒体的活性,破坏线粒体功能,诱导HT-29细胞和MCF-7细胞发生凋亡;乳脂肪球膜蛋白是通过上调Bax和Caspase-3的表达,同时下调Bcl-2的表达而达到的诱导细胞凋亡的目的。在5种乳脂肪球膜蛋白中,山羊乳和水牛乳脂肪球膜蛋白对HT-29细胞的作用效果最为显著,水牛乳脂肪球膜蛋白对MCF-7细胞的作用效果最明显。
[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.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS252.1
，
本文编号：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.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS252.1
，
本文编号：2136339
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