脂多糖刺激奶牛乳腺上皮细胞后外泌体的分离及鉴定

Isolation and identification of exosomes produced by stimulating mammary epithelial cells of dairy cows with lipopolysaccharide

目的分离并鉴定脂多糖(lipopolysaccharide,LPS)刺激奶牛乳腺上皮细胞产生的外泌体。方法培养奶牛乳腺上皮细胞,经20μg/mL的LPS刺激后,采用超速离心法提取细胞上清中的外泌体,通过透射电镜及Western blot法对分离物进行鉴定,同时以正常奶牛乳腺上皮细胞为对照组。结果奶牛乳腺上皮细胞产生的外泌体形态为40~120 nm的膜泡盘样结构;外泌体中含有CD63、HSP90和HSP70 3种特异性标志分子,且表达均低于对照组。结论成功分离了奶牛乳腺上皮细胞产生的外泌体,LPS刺激可影响外泌体表面标志分子的表达。

Objective To isolate and identify the exosomes produced by stimulating the mammary epithelial cells of dairy cows with lipopolysaccharide(LPS). Methods The mammary epithelial cells of dairy cows were cultured and stimulated with 20 μg/mL LPS. The exosomes in cell culture supernatant were extracted by ultracentrifugation and identified by transmission electron microscopy(TEM)and Western blot,using that derived from normal cow mammary epithelial cells as control. Results The exosomes derived from mammary epithelial cells of dairy cows were in shapes of round or oval saucers at sizes of 40 ~ 120 nm. Specific markers CD 63,HSP90 and HSP70 were expressed in the exosomes,of which the levels were lower than those in control group. Conclusion The exosomes produced by stimulation of cow mammary epithelial cells with LPS were successfully isolated,and LPS stimulation affected the expression of surface markers of exosmoes.