肝癌细胞外泌体的分离与鉴定

Isolation and identification of exosomes of hepatocellular carcinoma cells

目的:针对目前尚缺理想的肿瘤细胞提取方案,探寻分离纯化肝癌细胞培养液中外泌体的新方法.方法:连续适应无血清培养肝癌SMMC-7721细胞,收集细胞上清液,采用改良超速离心法分离、纯化培养上清液中肝癌细胞外泌体;透射电镜观察其形态,Nanosight技术分析粒径;Western blot分析其特异蛋白Alix、CD63、CD9的表达并利用SDS-PAGE电泳分析肝癌细胞和外泌体的蛋白谱.结果:透射电镜观察到的肝癌细胞外泌体,外形呈圆形或椭圆形;Nanosight技术分析颗粒直径峰值为122 nm,直径30-150 nm之间颗粒占72.16%;Western blot证实在细胞培养上清液和外泌体提取液中均能检测到Alix、CD63、CD9的蛋白表达;SDS-PAGE电泳清晰显示外泌体中高丰度大分子结构蛋白明显少于肝癌细胞.结论:利用连续适应无血清细胞培养结合改良超速离心法能提取高纯度的外泌体,为肝癌细胞外泌体标志物研究奠定基础.

AIM：To seek a new method for isolating and purifying exosomes from the supernatants of cultured human hepato cellular carcinoma cells.METHODS：Human hepatocellular carcinoma cells SMMC-7721 were cultured to adapt to continuous serum-free culture,and the culture supernatants were collected.Exosomes derived from SMMC-7721 were isolated and purified from the supernatants by improved ultracentrifugation.Transmission electron microscopy（TEM） was used to observe the morphology of isolated exosomes.Particle size of exosomes was detected with Nanosight technology.Western blot analysis was used to examine CD63,CD9,and Alix exosomal protein expression and SDS-PAGE was used for exosomal protein analysis.RESULTS：The isolated exosomes were round or oval in shape under TEM.The peak diameter of exosomes was 122 nm,and particles with a diameter between 30-150 nm accounted for 72.16%.The expression of Alix,CD63 and CD9 could be detected in the supernatants of cultured cells and isolated exosomes.SDS-PAGE showed that the abundant macromolecular structure proteins were significantly less in exosomes than in SMMC-7721 cells.CONCLUSION：Continuous adaptation to serum-free culture combined with improved ultracentrifugation can be used to isolate and purify exosomes efficiently.This is helpful for further study of exosome markers of hepatocellular carcinoma cells.