【作者】 杨倩； 刘润园； 于新新； 牛卫东；

【Author】 Qian Yang;Runyuan Liu;Xinxin Yu;Weidong Niu;Department of Stomatology, Dalian Medical University;

【机构】 大连医科大学口腔医学院；

【摘要】 目的:通过超速离心的方法分离牛乳小细胞外囊泡(small Extracellular vesicles,sEVs),鉴定牛乳sEVs形态特征及长期保存的稳定性,为进一步研究牛乳sEVs的生物学功能奠定基础。方法:取新鲜的脱脂牛乳,通过超速离心的方法从牛乳中分离sEVs,并通过透射电子显微镜、纳米颗粒追踪、蛋白免疫印迹对分离的新鲜牛乳sEVs及冻存6个月牛乳sEVs进行鉴定。结果:透射电子显微镜结果显示,牛乳sEVs形态较为规则,多呈圆形或椭圆形,具有典型的双层膜样结构,囊泡大小符合文献报道范围;冻存6个月牛乳sEVs仍具有典型的双层膜结构,其大小并未改变,镜下观察到少量团聚结构;纳米颗粒追踪分析结果显示,牛乳sEVs的中值粒径为114.6 nm,颗粒浓度为1.8E+13/mL;冻存6个月牛乳sEVs中值粒径为116.8nm,颗粒浓度为1.5E+13/mL;蛋白免疫印迹结果显示,牛乳sEVs阳性标记物CD63、CD81、Alix有表达,阴性标记物CD40无表达。结论:通过超速离心的方法可成功从新鲜脱脂牛乳中分离sEVs,且在-80℃下保存6个月并不会改变其大小、双层膜结构及颗粒浓度。更多还原

【Abstract】 Objectives:This research through the overspeed centrifugal method Separate milk small Extracellular vesicles(sEVs),Identificate milk sEVs morphological characteristics and long-term preservation of stability,and lay a foundation for further study on milk sEVs biology function.Methods:Take fresh skimmed milk,sEVs was isolated from milk by the overspeed centrifugal method and identificate fresh milk sEVs and sEVs cryopreserved six months by Transmission electron microscopy,nano particle tracking,Western Blot.Results:Transmission electron microscopy shows Milk sEVs form relatively rules,more assumes the circular or elliptic,with typical double membrane structure.vesicle size conform to the scope of literature reports;sEVs cryopreserved Six months still has the typical double-layer membrane structure.its size did not change.the microscopic observation has a small amount of aggregate structure;Nano particle tracking analysis shows Milk sEVs median diameter is 114.6 nm,particle concentration is 1.8 E+13/mL;sEVs cryopreserved six months median diameter is 116.8 nm,particle concentration is 1.5 E+13/mL;Western Blot shows Milk sEVs has positive marker CD63,CD81,Alix expression and negative marker CD40 expression.Conclusion:sEVs could be successfully separated from fresh skim milk,by using the method of overspeed centrifugal and it can keep 6 months under 80℃and this situation will not change its size,double layer membrane structure and particle concentration.更多还原