血清饥饿对结肠癌HCT116细胞外泌体分泌的影响  

Effect of serum starvation on secretion of exosomes in colorectal cancer HCT116 cells

作　　者：高佳佳[1] 李薇[1] 鞠强[1] 周兰萍[1] 赵晓航[1] 

机构地区：[1]北京协和医学院中国医学科学院肿瘤医院分子肿瘤学国家重点实验室,北京市100021

出　　处：《世界华人消化杂志》2016年第4期566-572,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.81572365;81372591;国家高技术发展计划("863"计划)基金资助项目;No.2012AA020206~~

摘　　要：目的:本研究尝试使用添加无外泌体血清(E x o s o m e-d e p l e t e d F B S)的培养基培养细胞并提取外泌体,比较无外泌体血清和传统的无血清两种不同的培养条件对结肠癌H C T116细胞外泌体分泌的影响,旨在优化外泌体提取条件,使之有利于后续功能研究.方法:采用提取前24 h无血清和添加无外泌体血清培养基培养结肠癌细胞,普通光学显微镜下观察两种培养条件下细胞的生长和形态,用超速离心法分步提取分泌到细胞培养上清中的外泌体,透射电子显微镜下观察外泌体的形态,蛋白免疫印迹法分析外泌体标志蛋白,以及纳米颗粒追踪计数分析外泌体大小和含量.结果:与无血清培养条件相比,添加无外泌体血清培养的HCT116细胞生长状态良好,细胞无明显死亡现象.两种培养条件分泌的外泌体形态相近,均呈圆形或椭圆形,由脂质双分子层包裹的小囊泡,直径为40-100 nm.无外泌体血清培养条件分泌的外泌体大约是无血清培养的6倍,二种培养条件下提取的外泌体直径均为90 nm左右,与电镜结果一致.两种培养条件提取的外泌体中均检测到HSP70和CD63外泌体标志蛋白.结论:添加无外泌体血清培养HCT116细胞分泌的外泌体与无血清培养相比无明显差异,但添加无外泌体血清培养的细胞状态比无血清培养的细胞状态好,外泌体的分泌可能更接近生理条件下的生长环境,分泌的外泌体数量更多,因此无外泌体血清培养条件较无血清培养更适合结肠癌来源外泌体的提取和研究.AIM： To investigate the influence of serum starvation on secretion of exosomes, to find a more conducive way to get more exosomes. METHODS： Fetal bovine serum （FBS）-free medium and medium containing exosome- depleted FBS were used to culture HCT116 cells. Ultracentrifugation was used to extract exosomes, and ordinary optical microscope was used to observe the physiological state of the cells. Exosome markers were detected by Western blot. Exosomes were observed by transmission electron microscopy and counted with Nanosight. RESULTS： Ordinary optical microscopy showed that cells cultured in medium containing exosome-depleted FBS grew in good state, and there was no obvious cell death compared with those cultured in FBS-free condition. Transmission electron microscopy showed that the exosomes secreted by cells had a diameter of 40-100 nm and were protected by a lipid bilayer. Western blot analysis showed that the exosomes were positive for CD63 and heat shock protein 70 in two kinds of culture conditions. The quantity of exosomes secreted by cells cultured in medium containing exosome-depleted FBS was about 6 times more than that by cells cultured in FBS-free medium.CONCLUSION： Exosomes secreted by HCT116 cells cultured in medium containing exosome- depleted FBS have no obvious differences from those by HCT116 cells cultured in FBS-free medium. However, the cells grow better and more exosomes can be obtained when cultured in medium containing exosome-depleted FBS.

关 键 词：外泌体 结肠癌 血清饥饿 无外泌体血清 

分 类 号：R735.35[医药卫生—肿瘤]