分离、纯化及鉴定肝细胞和免疫细胞分泌的HMGB1  

Purification and identification of HMGB1 secreted by liver cells and immune cells

作　　者：萧梅芳[1] 戴霞红[1] 周蓉蓉[1] 刘建平[2] 章保新[1] 赵树山[1] 范学工[1] 

机构地区：[1]中南大学湘雅医院传染科,长沙410008 [2]中南大学现代分析测试中心,长沙410078

出　　处：《中南大学学报（医学版）》2011年第11期1065-1070,共6页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(30972621);湖南省自然科学基金(11JJ4074);湖南省科技厅计划项目(2011TT2060);中南大学大学生创新实验计划(YD10145);中南大学湘雅医学院优秀博士生选题计划~~

摘　　要：目的:分离纯化肝细胞HepG2与免疫细胞U937分泌的高迁移率族蛋白1(high mobility group box-1 protein,HMGB1),并加以鉴定。方法:体外培养人肝细胞HepG2与免疫细胞U937,采用400 ng/mL的脂多糖(lipopolysaccharide,LPS)刺激20 h后收集细胞培养液上清。采用超滤浓缩,阳离子交换层析,阴离子交换层析,Sephadex G75凝胶过滤层析结合免疫沉淀的方法,进行分离纯化;聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western印迹进行蛋白分子质量及性质鉴定。结果:2株细胞分离纯化得到的蛋白经SDS-PAGE鉴定其纯度达90%以上,分子质量约为26 kD,Western印迹鉴定为HMGB1。结论:该纯化方法可获得纯度较高的HMGB1。Objective To purify and identify HMGB1 secreted by liver cells HepG2 and immune cells U937.Methods We cultured the liver cell lines HepG2 and immune cell lines U937,and stimulated them with HMGB1（400 ng/mL） for 20 h.Then the supernatant was collected.Ultrafiltration centrifugation,CM-Sepharose cation,DEAE-Sepharose anion exchange chromatography,Sephadex G75-gel filtration chromatography,and immunoprecipitation were used for purification.The molecular weight and identity of HMGB1 was confirmed by SDS-PAGE and Western blot.Results A sharp stained protein band with a molecular weight of about 26 kD was obtained by SDS-PAGE analysis and shown to be HMGB1 confirmed by Western blot.Conclusion High purified HMGB1 can be separated from these two cell lines.

关 键 词：高迁移率族蛋白1 分离纯化 HEPG2细胞 U937细胞 

分 类 号：R346[医药卫生—基础医学]