發(fā)布時間：2018-02-11 19:54

  本文關(guān)鍵詞： 螺旋藻 生物活性肽 抗氧化 抗癌 抗光老化　出處：《華南理工大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：本文以螺旋藻粉末為原料,采用低溫超高壓連續(xù)流法提取螺旋藻蛋白,利用生物酶解法結(jié)合柱層析純化技術(shù)制備具有體外抗氧化和抗光老化活性的多肽組分,采用基質(zhì)輔助激光解離質(zhì)譜(MALDI-TOF-MS/MS)和PEAKS軟件等手段鑒定多肽的純度并得到6個多肽序列,隨后,固相合成多肽。采用MTT法、流式細胞凋亡分析法和猩紅苦味酸染色法分別測定細胞存活率、細胞凋亡率和膠原蛋白產(chǎn)量,研究多肽組分對中波紫外(UVB)老化的人永生化表皮細胞(Hacat)和人皮膚成纖維細胞(HSF)存活率的影響,篩選出具有抗皮膚光老化活性的多肽。并且,通過動物模型對得到的活性多肽進行抗皮膚光老化活性研究,結(jié)合同位素標(biāo)記相對和絕對定量(iTRAQ)蛋白質(zhì)組學(xué)技術(shù)揭示其作用機理。制備獲得的胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa組分清除ABTS自由基的半抑制濃度(IC_(50)值)分別為76.16±3.33μg/mL、756.36±11.42μg/mL和555.99±11.96μg/mL。將UVB老化組中HSF細胞增殖率和膠原蛋白產(chǎn)量設(shè)為100,陽性對照五勝肽、胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa組分作用后HSF增殖率分別為100.80±7.40、129.11±1.85、121.97±11.25和120.86±11.32;膠原蛋白產(chǎn)量分別為156.68±10.87、122.87±8.16、102.19±2.36和151.04±8.16。以上結(jié)果表明,胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa組分的抗氧化效果均較強,且能通過促進UVB老化的HSF增殖或者膠原蛋白產(chǎn)量的提升來實現(xiàn)其體外抗光老化效果。此外,分別從胰蛋白酶3 kDa、胃蛋白酶3 kDa和木瓜蛋白酶3 kDa組分中篩選出了抗氧化和抗光老化活性均比較強的組分T_3、P_3和A_6,并從中鑒定得到六條多肽,其序列分別為:(Ⅰ)GMCCSR;(Ⅱ)FFEFF;(Ⅲ)EYFDALA;(Ⅳ)VTAPAASVAL;(Ⅴ)ANAAFRPR;(Ⅵ)WVAGLGYFTKNGGPK。生物活性研究結(jié)果表明,多肽Ⅰ號、Ⅲ號和Ⅵ號抗氧化活性較強,其中多肽Ⅰ號保護人紅細胞的效果最佳,濃度達到100μg/mL時,樣品的溶血抑制率與正常組沒有顯著性差異,且多肽Ⅰ號能顯著促進UVB老化的HSF增殖和膠原蛋白的產(chǎn)生�；诙嚯蘑裉枴ⅱ筇柡廷鎏柕钠つw滲透性較差,對多肽序列進行乙酰酰胺化修飾和棕櫚�；揎棥＝Y(jié)果表明,相比于未修飾之前,乙酰酰胺化、棕櫚酰化多肽Ⅰ號和Ⅲ號中,α-螺旋和無規(guī)卷曲的比例逐漸增多,β-折疊的比例逐漸降低,且棕櫚�；嚯蘑裉柡廷筇栔械纳鲜龆壗Y(jié)構(gòu)均已顯著不同于未修飾多肽。在生物活性方面,棕櫚酰化多肽Ⅰ號和Ⅲ號抗癌活性顯著增加,且對皮膚癌細胞A375的增殖抑制作用顯著強于陽性對照五勝肽。相比于多肽Ⅲ號,同種修飾的多肽Ⅰ號對Hacat的毒性較低。乙酰酰胺化多肽Ⅰ號能顯著促進UVB老化的Hacat細胞增殖,降低其凋亡峰比例,有效拮抗UVB導(dǎo)致的S期阻滯的形成,降低模型組中S期細胞比例,增加G_1期細胞比例,從而起到保護UVB老化的Hacat細胞的作用。小鼠體內(nèi)抗光老化實驗結(jié)果表明,乙酰酰胺化多肽Ⅰ號(P_1)能通過降低皮膚組織中丙二醛(MDA)含量,細胞質(zhì)基質(zhì)中金屬基質(zhì)蛋白酶(MMP-1和MMP-3)的表達量,增加膠原蛋白含量和組織中超氧化物歧化酶(SOD)、谷胱甘肽過氧化物酶(GSH-Px)以及過氧化氫酶(CAT)等抗氧化酶的活力來實現(xiàn)其體內(nèi)抗光老化效果。棕櫚酰化多肽Ⅰ號(P_2)主要通過增加膠原蛋白含量來發(fā)揮其體內(nèi)抗光老化效果。iTRAQ差異蛋白質(zhì)組學(xué)全面鑒定分析了小鼠皮膚光老化以及P_1和P_2抗光老化相關(guān)的GO功能和KEGG代謝通路。P_1具有較強的抗氧化活性,可以調(diào)節(jié)線粒體電子傳遞功能。P_2具有較強的抗癌活性,可以調(diào)節(jié)p53控制的內(nèi)在凋亡信號通路。在光老化的基礎(chǔ)上,P_1和P_2分別作用后,檢測到的差異蛋白在信號通路帕金森氏病和阿爾茨海默氏病中的數(shù)目最多,與P_1和P_2抗光老化活性顯著相關(guān)的信號通路是兩組分系統(tǒng)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.

【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：O629.7;R96
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本文編號：1503867