食源性多肽对细胞迁移作用机制研究

发布时间：2019-06-03 15:13

【摘要】：食源性肽是以食用蛋白为原料,采用蛋白酶酶解、分离纯化等工艺研制而成小分子肽段混合物。食源性肽来源广泛,根据蛋白质原料来源的不同,主要分为动物源蛋白肽和植物源蛋白肽两大类。近年来,对食源性肽的活性研究主要集中于抗氧化、降血压和抑菌等生物活性方面,而对其在细胞迁移侵袭方面的研究较少。因此,在本研究中,分别以大豆和蛋清为来源,考察两种不同来源的功能肽对细胞迁移调控作用。我们首先用计算机模拟胰蛋白酶/胃蛋白酶对大豆蛋白进行水解,产生一系列的多肽片段,再依据与Hsp90对接消耗的自由能的大小排序,得到消耗自由能最低的5个大豆蛋白肽序列。我们采用划痕愈伤实验考察5种大豆蛋白肽对胰腺癌细胞MIAPaCa-2迁移能力影响,结果显示七肽Leu-Val-Thr-Val-Pro-Gln-Phe(LVTVPQF)对MIAPaCa-2细胞迁移具有抑制作用。Transwell实验结果进一步证实了LVTVPQF对MIAPaCa-2细胞迁移的抑制效果,同时,我们通过细胞增殖试验证实LVTVPQF对细胞的生长增殖没有影响,即没有细胞毒性。此外,利用Western-Blot检测蛋白水平的变化,证明LVTVPQF以浓度依赖的方式抑制细胞外分泌型Hsp90α及其下游MMP-9水平,而对细胞内的Hsp90及其客户蛋白AKT,CDK4均无抑制作用。以上结果表明大豆蛋白衍生肽LVTVPQF通过抑制分泌型Hsp90α和下游基质MMP-9的表达,起到抑制胰腺癌细胞迁移的作用。接下来我们以蛋清浓缩蛋白粉为原料,采用Alcalase碱性蛋白酶和风味蛋白酶酶解蛋清浓缩蛋白粉,酶解液经过超滤膜截留,通过MTS实验可知混合组分EWPH1和EWPH1(超滤)组分均对HaCaT细胞没有毒性,经过划痕愈伤实验和Transwell实验的筛选,我们选择较优组分EWPH1(超滤)进行后续的纯化以及促细胞迁移研究。将EWPH1(超滤)组分经过葡聚糖凝胶G-15进行分离纯化,得到EWP1,EWP2,EWP3,EWP4四个组分,通过MTS实验、愈伤实验和Transwell实验,我们选择较优组分EWP2进行后续的纯化以及促细胞迁移研究。EWP2经过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实验可知KPHAEVVLR对皮肤成纤维细胞HSF的增殖有促进的作用,同时Transwell实验结果证实KPHAEVVLR能够促进HSF细胞迁移。在本研究中,我们选择了大豆肽和蛋清肽两种食源性肽,分别从抑制胰腺癌细胞迁移和促进皮肤伤口愈合作用两方面进行了研究。一方面我们发现大豆肽通过影响分泌型Hsp90α和下游MMP-9等蛋白的表达,进而抑制胰腺癌细胞的迁移。另一方面,我们从蛋清肽酶解物中分离出了九肽KPHAEVVLR,并通过实验证明其可促进皮肤成纤维细胞HSF的增殖和迁移。这些实验结果表明食源性肽在调控细胞迁移方面具有很高的研究潜力和研究价值,同时为开发新型调控细胞迁移类食源肽产品提供理论依据及参考价值。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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