螺旋藻蛋白源生物活性肽的制备及其抗皮肤光老化机理研究

发布时间：2018-02-11 19:54

  本文关键词： 螺旋藻 生物活性肽 抗氧化 抗癌 抗光老化　出处：《华南理工大学》2016年博士论文　论文类型：学位论文

【摘要】：本文以螺旋藻粉末为原料,采用低温超高压连续流法提取螺旋藻蛋白,利用生物酶解法结合柱层析纯化技术制备具有体外抗氧化和抗光老化活性的多肽组分,采用基质辅助激光解离质谱(MALDI-TOF-MS/MS)和PEAKS软件等手段鉴定多肽的纯度并得到6个多肽序列,随后,固相合成多肽。采用MTT法、流式细胞凋亡分析法和猩红苦味酸染色法分别测定细胞存活率、细胞凋亡率和胶原蛋白产量,研究多肽组分对中波紫外(UVB)老化的人永生化表皮细胞(Hacat)和人皮肤成纤维细胞(HSF)存活率的影响,筛选出具有抗皮肤光老化活性的多肽。并且,通过动物模型对得到的活性多肽进行抗皮肤光老化活性研究,结合同位素标记相对和绝对定量(iTRAQ)蛋白质组学技术揭示其作用机理。制备获得的胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa组分清除ABTS自由基的半抑制浓度(IC_(50)值)分别为76.16±3.33μg/mL、756.36±11.42μg/mL和555.99±11.96μg/mL。将UVB老化组中HSF细胞增殖率和胶原蛋白产量设为100,阳性对照五胜肽、胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa组分作用后HSF增殖率分别为100.80±7.40、129.11±1.85、121.97±11.25和120.86±11.32;胶原蛋白产量分别为156.68±10.87、122.87±8.16、102.19±2.36和151.04±8.16。以上结果表明,胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa组分的抗氧化效果均较强,且能通过促进UVB老化的HSF增殖或者胶原蛋白产量的提升来实现其体外抗光老化效果。此外,分别从胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa组分中筛选出了抗氧化和抗光老化活性均比较强的组分T_3、P_3和A_6,并从中鉴定得到六条多肽,其序列分别为:(Ⅰ)GMCCSR;(Ⅱ)FFEFF;(Ⅲ)EYFDALA;(Ⅳ)VTAPAASVAL;(Ⅴ)ANAAFRPR;(Ⅵ)WVAGLGYFTKNGGPK。生物活性研究结果表明,多肽Ⅰ号、Ⅲ号和Ⅵ号抗氧化活性较强,其中多肽Ⅰ号保护人红细胞的效果最佳,浓度达到100μg/mL时,样品的溶血抑制率与正常组没有显著性差异,且多肽Ⅰ号能显著促进UVB老化的HSF增殖和胶原蛋白的产生。基于多肽Ⅰ号、Ⅲ号和Ⅵ号的皮肤渗透性较差,对多肽序列进行乙酰酰胺化修饰和棕榈酰化修饰。结果表明,相比于未修饰之前,乙酰酰胺化、棕榈酰化多肽Ⅰ号和Ⅲ号中,α-螺旋和无规卷曲的比例逐渐增多,β-折叠的比例逐渐降低,且棕榈酰化多肽Ⅰ号和Ⅲ号中的上述二级结构均已显著不同于未修饰多肽。在生物活性方面,棕榈酰化多肽Ⅰ号和Ⅲ号抗癌活性显著增加,且对皮肤癌细胞A375的增殖抑制作用显著强于阳性对照五胜肽。相比于多肽Ⅲ号,同种修饰的多肽Ⅰ号对Hacat的毒性较低。乙酰酰胺化多肽Ⅰ号能显著促进UVB老化的Hacat细胞增殖,降低其凋亡峰比例,有效拮抗UVB导致的S期阻滞的形成,降低模型组中S期细胞比例,增加G_1期细胞比例,从而起到保护UVB老化的Hacat细胞的作用。小鼠体内抗光老化实验结果表明,乙酰酰胺化多肽Ⅰ号(P_1)能通过降低皮肤组织中丙二醛(MDA)含量,细胞质基质中金属基质蛋白酶(MMP-1和MMP-3)的表达量,增加胶原蛋白含量和组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)以及过氧化氢酶(CAT)等抗氧化酶的活力来实现其体内抗光老化效果。棕榈酰化多肽Ⅰ号(P_2)主要通过增加胶原蛋白含量来发挥其体内抗光老化效果。iTRAQ差异蛋白质组学全面鉴定分析了小鼠皮肤光老化以及P_1和P_2抗光老化相关的GO功能和KEGG代谢通路。P_1具有较强的抗氧化活性,可以调节线粒体电子传递功能。P_2具有较强的抗癌活性,可以调节p53控制的内在凋亡信号通路。在光老化的基础上,P_1和P_2分别作用后,检测到的差异蛋白在信号通路帕金森氏病和阿尔茨海默氏病中的数目最多,与P_1和P_2抗光老化活性显著相关的信号通路是两组分系统通路。
[Abstract]:In this paper, spirulina powder as raw materials, using low temperature high pressure continuous extraction of Spirulina protein purification method, preparation technology has in vitro antioxidant and anti aging activity of peptides combined with column chromatography using bio enzymatic method, using matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS/MS) and PEAKS software and other means to identify the purity of peptides and 6 polypeptides then, the sequence, the solid phase peptide synthesis. By MTT method, respectively. Cell viability was measured by FACS analysis and scarlet picric acid staining, apoptosis rate and collagen production of polypeptide components of the medium wave ultraviolet (UVB) aging immortalized human epidermal cells (Hacat) and human skin fibroblasts (the influence of the survival rate, HSF) were screened with anti photoaging peptides. And, to get the anti peptide activity of skin photoaging by animal model. A contract labeled relative and absolute quantification (iTRAQ) proteomics reveals the mechanism. The obtained 3 kDa of trypsin, pepsin and papain 3 kDa 3 kDa component of ABTS free radical scavenging half inhibitory concentration (IC_ (50) value) were 76.16 + 3.33 g/ mL, 756.36 g/mL + 11.42 and 555.99 + 11.96 g/mL. UVB group in aging HSF cell proliferation and collagen production is set to 100, five positive control peptide, 3 kDa trypsin, pepsin and papain 3 kDa 3 kDa component after proliferation rate of HSF was 100.80 + 7.40129.11 + 1.85121.97 + 11.25 and 120.86 + 11.32; collagen production was 156.68 + 10.87122.87 + 8.16102.19 + 2.36 and 151.04 + 8.16. the above results showed that 3 kDa of trypsin, the antioxidant effect of pepsin and papain 3 kDa 3 kDa were strong, and can promote the aging of UVB H The proliferation of SF or collagen yield increase to achieve its in vitro anti photoaging effects. In addition, 3 kDa respectively from trypsin, pepsin and papain 3 kDa 3 kDa components were screened from antioxidant and anti-aging activity were relatively strong components T_3, P_3 and A_6, and obtained six peptides from the sequence identification, respectively: (I) GMCCSR; (II) FFEFF; (III) EYFDALA; (IV) VTAPAASVAL; (V) ANAAFRPR; (VI) WVAGLGYFTKNGGPK. biological activity results showed that polypeptide 1, III and VI strong antioxidant activity, the polypeptide 1 protection of human red blood cells the best effect, the concentration reached 100 g/mL, the sample hemolysis inhibition rate compared with the normal group had no significant difference, and the polypeptide 1 can significantly promote the proliferation of HSF and collagen UVB aging. The polypeptide 1 based on low permeability III and VI skin of polypeptide sequence For acetylation amidation and palmitoylation modification. The results show that, compared to the unmodified before acetylation amidation, palmitoylated peptides I and III, alpha helix and random coil ratio gradually increased, the proportion of p-sheet decreased gradually, and palmitoylated peptides I and III in the two stage structure are significantly different from unmodified peptides. In biological activity, palmitoylated peptide I and III anticancer activity increased significantly, and the skin cancer A375 cell proliferation inhibition was significantly stronger than the positive control five peptides. Compared to peptide III, toxic polypeptide 1 the same modification of Hacat is low. Acetyl amidated polypeptide 1 can significantly promote the proliferation of Hacat cells UVB of aging, reduce the apoptosis peak ratio, form an effective antagonist of UVB induced S arrest, reduce the proportion of cells in the model group increased in S phase, the cell proportion of G_1 phase, from And to protect the UVB aging Hacat cells. The mice anti light aging experimental results show that the acetyl amidated polypeptide 1 (P_1) by reduction of malondialdehyde (MDA) content in skin tissue, metal matrix orcytomatrix protease (MMP-1 and MMP-3) expression, increase the content of collagen and tissue superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) and antioxidant enzyme activity in vivo to achieve its anti-aging effect. Palmitoylated polypeptide 1 (P_2) mainly by increasing the content of collagen exerts its anti-aging effect of.ITRAQ in vivo proteomic analysis of mouse skin light aging as well as P_1 and P_2 anti light aging related function of GO and KEGG.P_1 pathway has strong antioxidant activity comprehensive identification, can regulate mitochondrial electron transfer function.P_2 has strong Anticancer activity can modulate the intrinsic apoptosis pathway of p53 control. Based on light aging, P_1 and P_2 respectively, the number of proteins detected in the signal pathway of Parkinson's disease and Alzheimer's disease in the most, with P_1 and P_2 anti photoaging activity significantly related signaling pathways is two component system pathway.

【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：O629.7;R96
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本文编号：1503867