重组人粒细胞刺激因子测活方法中几种常见显色方法的对比分析  

Comparison of Several Common Methods for Measuring the Biological Activity of Recombinant Human Granulocyte Colony Stimulating Factor

作　　者：史新昌[1] 刘兰[1] 秦玺[1] 王莹[1] 饶春明[1] 

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《中国药学杂志》2016年第13期1080-1084,共5页Chinese Pharmaceutical Journal

基　　金：十二五课题资助项目(2012ZX09304010);国家863计划课题资助项目(2012AA020805;2012AA02A304)

摘　　要：目的分析重组人粒细胞刺激因子生物学活性检测中,以NFS-60细胞为测活基础的几种常见显色/裂解/检测方法,所得检测结果是否一致。方法应用NFS-60细胞/MTT染色法、NFS-60细胞/MTS染色法、NFS-60细胞/CCK-8染色法、NFS-60细胞/化学发光法等检测重组人粒细胞刺激因子生物学活性,并对结果进行统计分析。统计分析了NFS-60细胞/MTT法双波长(570 nm检测,630 nm参比)、单波长(570 nm检测和630 nm检测)结果。分析比较了NFS-60细胞/MTS动态检测法、NFS-60细胞/CCK-8动态法检测结果。结果各生物学活性检测方法的检测结果差别不显著(P>0.05);NFS-60/MTT法双波长、单波长检测结果差别不显著(P>0.05)。NFS-60细胞/MTS动态检测法、NFS-60细胞/CCK-8动态法与NFS-60细胞/MTT染色法检测结果间差别不显著(P>0.05)。结论以NFS-60细胞为测活基础的几种常见的显色/裂解/检测方法,所得检测结果一致,为各实验室(应用不同显色/裂解/检测方法)给出的数据是否可以综合利用提供依据,为2015年版《中国药典》中重组人粒细胞刺激因子生物学活性检测方法的扩充提供支持,也为其他细胞因子生物学活性检测方法的扩充提供借鉴。OBJECTIVE To compare several common staining and detection methods using NFS-60 cells for biological activity test of recombinant human granulocyte colony stimulating factor （rhG-CSF）. METHODS The biological activity of rhG-CSF was detected using some common methods, named NFS-60 cells/MTT staining, NFS-60 cells/MTS staining, NFS-60 cells/CCK-8 staining, and NFS-60 cells/fluorescence staining. The biological activity was detected using the NFS-60 cells/MTF method using dual wave- length （570 nm detection, 630 nm reference） and single wavelength （570rim detection and 630rim detection） . The biological activity was detected using NFS-60 cells/MTS dynamic detection method and NFS-60 cell/CCK-8 dynamic method. Then, the results were ana- lyzed and compared. RESULTS The different methods were not significantly different （ P 〉 0. 05 ） ; the difference between the dual wavelength detection and single wavelength detection of NFS-60 cells/MTT was not significant （ P 〉 0. 05）. The results from NFS-60 cells/MTS dynamic detection method, NFS-60 cells/CCK-8 dynamic method and NFS-60 cells/MTF method had not significant difference （P 〉 0. 05）. CONCLUSION The biological activity determination results of the tested methods using NFS-60 cells are consist- ent. This study provides basis for utilization of results from different laboratories using different methods, support for expansion of the biological activity detection method of rhG-CSF in Chinese Pharmacopoeia, and reference for expansion of the biological activity detec- tion method of other cytokines.

关 键 词：重组人粒细胞刺激因子 生物学活性 NFS-60细胞 3-(4 5二甲基噻唑-2)-2 5-二苯基四氮唑溴盐 四氮唑蓝盐化合物MTS 2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2 4-二磺基苯)-2H-四唑单钠盐 双波长检测 单波长检测 

分 类 号：R917[医药卫生—药物分析学]