成骨细胞源性外泌体的分离提取和鉴定

Isolation,extraction and identification of exosomes derived from osteoblasts

目的分离成骨细胞来源的外泌体,并对其进行鉴定。方法体外培养成骨细胞,用差速离心法分离、提取成骨细胞分泌的外泌体,经透射电子显微镜观察其形态及大小,采用蛋白质印迹法(WB)检测外泌体跨膜蛋白CD9和成骨细胞特异性碱性磷酸酶(AKP)的表达。结果成骨细胞源性外泌体为直径30~100 nm的圆形或椭圆形结构,高表达CD9和AKP蛋白。结论差速离心法提取的成骨细胞源性外泌体,具有外泌体的一般特性,并高表达成骨细胞特异性蛋白AKP。

Objective The exosomes from osteoblasts were isolated and identified.Methods Osteoblasts were cultured in vitro.The exosomes secreted from osteoblasts were separated and extracted by differential centrifugation.The morphology and size of exosomes were observed by transmission electron microscopy.The expression of CD9 and alkaline phosphatase in osteoblasts were detected by Western blotting.Results The exosomes of osteoblasts were round or oval structures with a diameter of 30~100 nm,which highly expressed CD9 and AKP protein.Conclusion The osteoblast derived from exosomes have the general characteristics of exosomes and highly express the osteoblast specific protein AKP.