牛奶外泌体对皮肤屏障的影响研究

Study on the effect of bovine milk exosomes on skin barrier

通过密度梯度离心的方法纯化得到了牛奶外泌体,并研究了牛奶外泌体对皮肤屏障的影响。结果表明,制备的牛奶外泌体呈现出典型的外泌体形态、粒径和标志物特征;分析牛奶外泌体的蛋白组数据,聚类显示,它对细胞间粘附的生物学过程的影响是显著的,提示了牛奶外泌体可能具有修护皮肤屏障的潜能。进一步分析牛奶外泌体对人永生化角质形成细胞(HaCat)的蛋白表达的影响,蛋白质谱数据显示,细胞的紧密联接蛋白Claudin-1和Claudin-6表达上调,紧密联接通路相关的负调控因子PPP2R1A和PPP2R2A下调。细胞实验发现,牛奶外泌体上调HaCat中Claudin-1基因的转录水平和翻译水平。动物实验进一步验证了牛奶外泌体对小鼠皮肤中Claudin-1的表达影响,发现48 h后,Claudin-1的表达显著提升。

Bovine milk exosomes were purified by density centrifugation,and their effect on skin barrier was studied.TEM,nanoFCM,Western-blot,LC-MS and BCA test showed that,the obtained bovine milk exosomes were highly pure exosome product whose appearance,particle size and markers were consistent with the typical characteristics of exosomes.Proteomics was then employed.The results showed that,the bovine milk exosomes contributed to cell-cell adhesion,indicative of an effect of skin barrier repair.HaCat cells were then incubated with bovine milk exosomes,and the cell proteomics analysis proved that tight junction proteins such as Claudin-1 and Claudin-6 were upregulated by bovine milk exosomes at all dosages,while some negative regulatory factors like PPP2R1A and PPP2R2A were downregulated.Molecular analysis identified with the proteomics results showed that Claudin-1 was upregulated at both transcription and translation levels.The bovine milk exosomes were then labeled with fluorescent dye and topically applied on mouse skin.Trans-epidermal trace of labeled bovine milk exosomes was demonstrated by fluorescence confocal microscopy.Immunohistochemical detection further verified that Claudin-1 was enhanced after 48 hours’topical application on mouse skin.