高浓度草酸作用下HK-2细胞系中外泌体的提取与鉴定  

Isolation and identification of exosomes secreted from HK-2 cells exposed to high oxalate

作　　者：何子奇[1] 关晓峰[1] 刘权[1] 刘云龙[1] 邓耀良[1] He Ziqi Guan Xiaofeng Liu Quan Liu Yunlong Deng Yaoliang(Department of Urology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China)

机构地区：[1]广西医科大学第一附属医院泌尿外科,530021

出　　处：《中华泌尿外科杂志》2017年第9期712-716,共5页Chinese Journal of Urology

基　　金：国家自然科学基金（81360113）;广西卫计委自筹经费科研课题（Z2016311）

摘　　要：目的 探讨高浓度草酸作用下HK-2细胞系中外泌体提取与鉴定的方法.方法 2016年6月至2017年1月进行实验.配制含草酸浓度分别为0、0.25、0.50、1.00、2.00、4.00、5.00、8.00、10.00mmoL/L的DMEM/F12无血清培养基,分别对HK-2细胞干预48 h,然后用CCK-8法检测不同浓度草酸对HK-2细胞生存率的影响.结合倒置显微镜下观察细胞的形态学变化,选择2.00 mmol/L的草酸作为实验浓度.用2.00 mmol/L草酸对HK-2细胞系干预48 h,收集干预后的HK-2细胞系的上清液,采用超速离心法提取外泌体;透射电镜观察外泌体的形态、大小;Nanosight系统检测外泌体的粒径与分布;蛋白质印迹法检测外泌体特异性蛋白HSP70、CD63的表达.结果 CCK-8法检测结果显示0、0.25、0.50、1.00、2.00、4.00、5.00、8.00、10.00 mmol/L组细胞的生存率分别为（100.0±4.0）％、（97.7±1.5）％、（97.3±2.1）％、（87.7±2.1）％、（76.0±1.0）％、（58.1±2.6）％、（52.7±1.5）％、（37.7±3.2）％、（31.3±2.0）％.透射电镜下外泌体呈杯口状,包膜为典型的双层膜结构.Nanosight系统显示2.00mmoL/L组外泌体峰值粒径为56 nm,整体平均粒径为87 nm,粒径为30～150 nm的比例为91.2％.蛋白质印迹法检测结果显示外泌体中明显表达HSP70和CD63蛋白,提取外泌体后的HK-2细胞表达HSP70蛋白,不表达CD63蛋白.结论 2.00 mmol/L草酸作用HK-2细胞48 h后,可以通过超速离心法提取丰富的、较高纯度的外泌体,这将为后续深入研究高浓度草酸背景下外泌体的信号转导奠定基础.Objective To investigate the method for isolating and identification exosomes from the HK-2 cells exposed to high oxalate.Methods HK-2 cells were cultured to serum-free culture medium and treated with oxalate at the concentration from 0 mmol/L to 10.00 mmol/L for 48 h.CCK-8 assays were performed to measure the cells proliferation.Combined the cell morphology,observed under inverted microscope with statistical analysis,we finally 2.00 mmol/L oxalic acid as the experimental concentration.The HK-2 cell was exposed to 2.00 mmol/L oxalate for 48 h.The supernatants were collected.Exosomes were isolated and purified from the supernatants by ultracentrifugation.Transmission electron microscopy （TEM） was used to observe the morphology of isolated exosomes.Particle size of exosomes were detected with Nanosight technology.Western blot analysis was used to examine the experession of HSP70,CD63.Results CCK-8 assay showed that the cell viability in each group,including （100.0 ± 4.0） % in 0 mmol/L group;（97.7 ± 1.5）% in the 0.25 mmol/L group;（97.3 ±2.1）% in the 0.50 mmol/L group;（87.7 ± 2.1） % in the 1.00 mmol/L group;（76.0 ± 1.0） % in the 2.00 mmol/L group;（58.1 ± 2.6） % in the 4.00 mmol/L group;（52.7 ± 1.5） % in the 5.00 mmol/L group;（37.7 ± 3.2） % in the 8.00 mmol/L group;（31.3 ±2.0）% in the 10.00 mmol/L group.The isolated exosomes demonstrated round or oval shape under TEM.The peak particle size was 56 nm,which the overall mean particle size was 87 nm.Those and particles with a diameter between 30-150 nm accounted for 91.2%.In this experiment,The expression of HSP70,CD63 could be detected in the isolated exosomes.However,only the expression of HSP70 could be detected in the HK-2 cells.Conclusions Under the treatment of 2 mmol/l oxalate for 48 hours,Ultracentrifugation can be used to isolate and purify exosomes efficiently from the HK-2 cells.This is helpful for further study of exosome as mediator of cell-to-cell communication.

关 键 词：外泌体 肾结石 草酸 

分 类 号：R692.4[医药卫生—泌尿科学]