免疫沉淀分离外泌体对PC12细胞上清中α-syn含量的影响

EFFECT OF EXOSOME ISOLATION BY IMMUNOPRECIPITATION ONΑ-SYNUCLEIN LEVEL IN THE SUPERNATANT OF PC12 CELLS

目的探讨采用免疫沉淀方法分离PC12多巴胺能细胞上清中外泌体对细胞上清中α-突触核蛋白(α-syn)含量的影响。方法利用多西环素诱导PC12细胞高表达α-syn,培养24 h后收集细胞上清液,采用免疫沉淀方法进行外泌体的分离纯化,采用酶联免疫吸附测定方法测定外泌体分离前后细胞上清中α-syn的含量。结果采用免疫沉淀方法可以分离出细胞上清中的外泌体,分离出的外泌体表达特异性标记物alix和CD63。外泌体分离后,上清中α-syn的含量从(2999.0±193.0)ng/L下降为(2276.0±81.8)ng/L,降低了(723.0±111.2)ng/L(t=4.580,P<0.05)。结论免疫沉淀分离外泌体能够降低PC12细胞上清中α-syn的水平。

Objective To investigate the effect of isolation of exosomes from the supernatant of PC12 dopaminergic cells by immunoprecipitation on theα-synuclein(α-syn)level in cell supernatant.Methods Doxycycline was used to induceα-syn over-expression in PC12 cells.After 24 h of culture,the supernatant was collected,and the exosomes were isolated and purified by immunoprecipitation.The level ofα-syn in the supernatant before and after exosome isolation was determined by enzyme-linked immunosorbent assay.Results Exosomes in cell supernatant could be isolated by immunoprecipitation,and specific markers alix and CD63 were expressed in the isolated exosomes.After exosome isolation,the level ofα-syn in the supernatant decreased from(2999.0±193.0)ng/L to(2276.0±81.8)ng/L by(723.0±111.2)ng/L(t=4.580,P<0.05).Conclusion Exosome isolation by immunoprecipitation can reduce the level ofα-syn in the supernatant of PC12 cells.