【摘要】：食源性肽是以食用蛋白為原料,采用蛋白酶酶解、分離純化等工藝研制而成小分子肽段混合物。食源性肽來源廣泛,根據(jù)蛋白質(zhì)原料來源的不同,主要分為動(dòng)物源蛋白肽和植物源蛋白肽兩大類。近年來,對(duì)食源性肽的活性研究主要集中于抗氧化、降血壓和抑菌等生物活性方面,而對(duì)其在細(xì)胞遷移侵襲方面的研究較少。因此,在本研究中,分別以大豆和蛋清為來源,考察兩種不同來源的功能肽對(duì)細(xì)胞遷移調(diào)控作用。我們首先用計(jì)算機(jī)模擬胰蛋白酶/胃蛋白酶對(duì)大豆蛋白進(jìn)行水解,產(chǎn)生一系列的多肽片段,再依據(jù)與Hsp90對(duì)接消耗的自由能的大小排序,得到消耗自由能最低的5個(gè)大豆蛋白肽序列。我們采用劃痕愈傷實(shí)驗(yàn)考察5種大豆蛋白肽對(duì)胰腺癌細(xì)胞MIAPaCa-2遷移能力影響,結(jié)果顯示七肽Leu-Val-Thr-Val-Pro-Gln-Phe(LVTVPQF)對(duì)MIAPaCa-2細(xì)胞遷移具有抑制作用。Transwell實(shí)驗(yàn)結(jié)果進(jìn)一步證實(shí)了LVTVPQF對(duì)MIAPaCa-2細(xì)胞遷移的抑制效果,同時(shí),我們通過細(xì)胞增殖試驗(yàn)證實(shí)LVTVPQF對(duì)細(xì)胞的生長(zhǎng)增殖沒有影響,即沒有細(xì)胞毒性。此外,利用Western-Blot檢測(cè)蛋白水平的變化,證明LVTVPQF以濃度依賴的方式抑制細(xì)胞外分泌型Hsp90α及其下游MMP-9水平,而對(duì)細(xì)胞內(nèi)的Hsp90及其客戶蛋白AKT,CDK4均無(wú)抑制作用。以上結(jié)果表明大豆蛋白衍生肽LVTVPQF通過抑制分泌型Hsp90α和下游基質(zhì)MMP-9的表達(dá),起到抑制胰腺癌細(xì)胞遷移的作用。接下來我們以蛋清濃縮蛋白粉為原料,采用Alcalase堿性蛋白酶和風(fēng)味蛋白酶酶解蛋清濃縮蛋白粉,酶解液經(jīng)過超濾膜截留,通過MTS實(shí)驗(yàn)可知混合組分EWPH1和EWPH1(超濾)組分均對(duì)HaCaT細(xì)胞沒有毒性,經(jīng)過劃痕愈傷實(shí)驗(yàn)和Transwell實(shí)驗(yàn)的篩選,我們選擇較優(yōu)組分EWPH1(超濾)進(jìn)行后續(xù)的純化以及促細(xì)胞遷移研究。將EWPH1(超濾)組分經(jīng)過葡聚糖凝膠G-15進(jìn)行分離純化,得到EWP1,EWP2,EWP3,EWP4四個(gè)組分,通過MTS實(shí)驗(yàn)、愈傷實(shí)驗(yàn)和Transwell實(shí)驗(yàn),我們選擇較優(yōu)組分EWP2進(jìn)行后續(xù)的純化以及促細(xì)胞遷移研究。EWP2經(jīng)過LC-MS分析,得到EWP2中含有的多肽的氨基酸序列,并合成了九肽Lys-Pro-His-Ala-Glu-Val-Val-Leu-Arg(KPHAEVVLR)。MTS實(shí)驗(yàn)可知KPHAEVVLR對(duì)皮膚成纖維細(xì)胞HSF的增殖有促進(jìn)的作用,同時(shí)Transwell實(shí)驗(yàn)結(jié)果證實(shí)KPHAEVVLR能夠促進(jìn)HSF細(xì)胞遷移。在本研究中,我們選擇了大豆肽和蛋清肽兩種食源性肽,分別從抑制胰腺癌細(xì)胞遷移和促進(jìn)皮膚傷口愈合作用兩方面進(jìn)行了研究。一方面我們發(fā)現(xiàn)大豆肽通過影響分泌型Hsp90α和下游MMP-9等蛋白的表達(dá),進(jìn)而抑制胰腺癌細(xì)胞的遷移。另一方面,我們從蛋清肽酶解物中分離出了九肽KPHAEVVLR,并通過實(shí)驗(yàn)證明其可促進(jìn)皮膚成纖維細(xì)胞HSF的增殖和遷移。這些實(shí)驗(yàn)結(jié)果表明食源性肽在調(diào)控細(xì)胞遷移方面具有很高的研究潛力和研究?jī)r(jià)值,同時(shí)為開發(fā)新型調(diào)控細(xì)胞遷移類食源肽產(chǎn)品提供理論依據(jù)及參考價(jià)值。
[Abstract]:The food-derived peptide is a small-molecular-peptide mixture which is prepared by using an edible protein as a raw material, using a process such as protease enzymolysis, separation and purification, and the like. The source of the food-derived peptide is wide, and is mainly divided into the animal source protein peptide and the plant source protein peptide according to the difference of the source of the protein raw material. In recent years, the research on the activity of food-derived peptides mainly focuses on the biological activities such as anti-oxidation, blood pressure lowering and bacteriostasis, and has little research on cell migration and invasion. Therefore, in this study, the effect of functional peptides from two different sources on cell migration was investigated by using soybean and egg white as a source. We first use the computer to simulate the hydrolysis of the soybean protein with the trypsin/ pepsin, produce a series of polypeptide fragments, and then, according to the size of the free energy consumed by the Hsp90, the five soybean protein peptide sequences with the lowest consumption can be obtained. The effect of five soybean protein peptides on the migration of MIAPaCa-2 in pancreatic cancer cells was investigated by means of scratch. The results showed that the seven-peptide Leu-Val-Thr-Val-Pro-Gln-Phe (LVTVPQF) had an inhibitory effect on the migration of MIAPaCa-2 cells. Transwell's experimental results further confirmed the inhibitory effect of LVTVPQF on the migration of MIAPaCa-2 cells. At the same time, we confirmed that LVTVPQF had no effect on the growth and proliferation of cells by cell proliferation assay, i.e. no cytotoxicity. In addition, by using Western-Blot to detect the change of protein level, it was shown that LVTVPQF inhibited the level of extracellular secretion type Hsp90 and its downstream MMP-9 level in a concentration-dependent manner, and there was no inhibition of Hsp90 and its client protein AKT and CDK4 in the cells. The above results show that the protein-derived peptide LVTVPQF plays a role in inhibiting the cell migration of pancreatic cancer by inhibiting the expression of the secreted Hsp90 antigen and the downstream matrix MMP-9. In that follow, the egg white concentrated protein powder is taken as a raw material, an Alcalase alkaline protease and a flavor protease are adopted to solve the egg white concentration protein powder, the enzymolysis liquid is intercepted by the ultrafiltration membrane, and the mixed component EWPH1 and the EWPH1 (ultrafiltration) component have no toxicity to the HaCaT cell through the MTS experiment. The better component EWPH1 (ultrafiltration) was selected for subsequent purification and cell migration through the screening of the scratch callus and the Transwell test. The components of EWP1, EWP2, EWP3 and EWP4 were separated and purified by the sephadex G-15 to obtain the four components of EWP1, EWP2, EWP3 and EWP4. EWP2 was analyzed by LC-MS to obtain the amino acid sequence of the polypeptide contained in EWP2, and the nine-peptide Lys-Pro-His-Ala-Glu-Val-Val-Leu-Arg (KPHAEVVLR) was synthesized. The MTS experiment shows that the KPHAEEVVLR has the effect of promoting the proliferation of the skin fibroblast HSF, and the Transwell experimental results confirm that the KPHAVEVVLR can promote the migration of the HSF cells. In this study, we selected two kinds of food-derived peptides of soybean peptide and egg white peptide to study the effects of inhibiting the migration of pancreatic cancer cells and promoting the healing of skin wound, respectively. On the one hand, we found that soybean peptides inhibit the migration of pancreatic cancer cells by affecting the expression of secreted Hsp90 and downstream MMP-9. On the other hand, we isolated the 9-peptide KPHAGEVVLR from the egg white peptidase solution and demonstrated that it can promote the proliferation and migration of the skin fibroblasts HSF. The results of these experiments show that the food-derived peptide has high research potential and research value in regulating cell migration, and also provides a theoretical basis and reference value for developing a new type of regulatory cell migration type food source peptide product.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R151

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