为促进海参主要活性成分多糖和多肽的协同开发利用,选取浙江的东海乌参工业酶解生产的超滤截留部分的蛋白聚糖为原料,进行进一步酶解和纯化制备多肽,采用化学和波谱分析等方法对其进行结构解析,细胞划痕法等方法研究多肽对肿瘤细胞迁移的抑制作用,并且通过转录组学分析活性多肽抑制细胞迁移作用的可能途径并利用Western Blot进行验证.结果表明,以蛋白聚糖为原料酶解获得的多肽属于胶原蛋白肽的范畴,具有细胞迁移抑制活性的多肽GT2-0主要集中在8-10肽.转录组学分析表明乌参活性肽GT2-0在抑制HCT116结肠癌细胞增殖及迁移时与ERBB以及NF-κB多条通路相关.利用 Western Blot 检测总β-catenin,P-β-catenin,Cyclin-D1,C-myc 蛋白的表达,来探究 GT2-0 在 Wnt/β-catenin 信号通路中蛋白的表达情况,结果表明GT2-0通过上调P-β-catenin和下调β-catenin蛋白表达,并激活的下游双转录因子引起的Cyclin-D1,Cmyc蛋白表达从而发挥细胞迁移抑制作用最终起到良好抗肿瘤效果.本研究对乌参活性肽的结构和抗肿瘤活性做了系统的研究和讨论,为浙江优势海参资源东海乌参生物活性成分的研究和发展提供了重要基础和参考.
Study on Preparation of Active Peptide from Acaudina leucoprocta and Its Inhibitory Activity on Cell Migration
In order to promote the collaborative development and utilization of polysaccharides and pep-tides in sea cucumber.The proteoglycans of ultrafiltration intercepted part by industrial enzymatic hydrolysis of Acaudina leucoprocta from East China Sea were used as raw materials.The peptides were prepared by further enzymatic hydrolysis and purification,and their structure was analyzed by chemical and spectral analysis methods.The inhibitory effect of the peptides on the migration of tumor cells was studied by cell scratches method and other methods.Besides,the possible pathway of inhibitory effect of active peptides on the migra-tion of tumor cells was analyzed by transcriptomics and verified by Western Blot.The results showed that polypeptides obtained from proteoglycans belonged to the collagen peptides,and the polypeptide GT2-0 with cell migration inhibition activity mainly concentrated in octapeptide-decapeptide.It was verified that the activ-ityof peptide GT2-0 was associated with multiple pathways of ERBB and NF-κB in inhibiting the proliferation and migration of HCT116 colon cancer cells.Finally,the expressions of total β-caten in,P-β-catenin,Cyclin-D1 and C-myc were detected by Western Blot.The results showed that GT2-0 up-regulated P-β-catenin pro-tein expression and down-regulated β-catenin protein expression,resulting in down-regulated Cyclin-D1 in-duced by activated downstream dual transcription factors.The expression of Cmyc protein plays an inhibitory role in cell migration and finally has a good anti-tumor effect.This study systematically studied and discussed the structure and antitumor activity of the active peptide of A.leucoprocta,which provided an important basis and reference for future research and development of sea cucumber in East China Sea.
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