SD大鼠星形胶质细胞来源外泌体的提取及鉴定

Extraction and Identification of Exosomes Derived from Sprague-Dawley Cerebral Cortex Astrocytes

对原代培养的SD大鼠星形胶质细胞分泌的外泌体进行提取并鉴定。首先原代培养大鼠星形胶质细胞,免疫荧光鉴定星形胶质细胞标记性蛋白(胶质纤维酸性蛋白:glial fibrillary acidic protein,GFAP)以鉴定星形胶质细胞的纯度,然后采用超速离心法对大鼠原代星形胶质细胞培养上清液中的外泌体进行分离和提取;外泌体纳米微粒追踪技术检测所提取外泌体的粒径大小和浓度,透射电镜观察其形态特征,免疫印迹检测外泌体特异性标记物跨膜蛋白CD9、肿瘤易感蛋白(tumour-susceptibility protein,TSG101)、热休克蛋白70(heat shock proteins 70,Hsp70)、ALG-2相互作用蛋白X(ALG-2-interacting protein X,ALIX)蛋白的表达等方法对所提取的外泌体进行鉴定。纳米微粒追踪技术显示提取的外泌体平均大小为(107.6±44)nm,占比为99.1%,符合外泌体大小的标准,并且浓度较高;透射电镜观察到细胞培养上清液提取物中有典型呈杯托状的外泌体,其胞膜完整,含有低电子密度的物质;免疫印迹检测显示所提取物中有外泌体特异性蛋白CD9、TSG101、HSP70、ALIX的表达。实验结果表明,采用超速离心法能从大鼠原代星形胶质细胞培养上清液中提取得到高纯度的外泌体,为后续研究星形胶质细胞来源外泌体的功能提供了良好的实验基础。

The aim of this study is to isolate and identify exosomes from primary cultured astrocytes of SD rats.Firstly,the astrocytes of SD rats were cultured,and the astrocytes marker protein(glial fibrillary acidic protei n,GFAP)was identified by immunofluorescence to identify the purity of astrocytes.Then the exosomes in the culture supernatant of primary astrocytes of SD rats were isolated and extracted by ultracentrifugation.The particle size and concentration of the exosomes were detected by nanoparticle tracking analysis,the morphological characteristics of the exosomes were observed by transmission electron microscope,and the exosomes were identified by Western blotting to detect the expression of CD9 of Tetraspanins,tumour-susceptibility protein,heat shock proteins 70 and ALG-2-interacting protein X proteins.Nanoparticle tracking analysis showed that the average size of the isolated exosomes was(107.6±44)nm,accounting for 99.1%,which conformed to the standard of exosomes size,and the concentration was high;transmission electron microscope showed that there were exosomes with typical oval cup shape in the supernatant of cell culture,the membrane of exosomes was intact and contained low electron density materials;Western blotting showed that the exosomes specific proteins CD9,TSG101,HSP70 and ALIX were expressed in the extract.The experimental results show that the exosomes with high purity can be extracted from the culture supernatant of SD rats primary astrocytes by ultracentrifugation,which provides a good experimental basis for the follow-up study of the function of exosomes derived fromastrocytes.