荧光素标记检测乙型肝炎病毒表面抗原特异性杀伤性T淋巴细胞方法的探讨  

A Sensitive and Convenient Fluorescence Assay for the Determination of Cytotoxic T Lymphocyte Activity Primed by HBsAg

作　　者：吴婷[1] 陈敏[1] 欧山海[1] 何水珍[1] 程通[1] 张军[1] 夏宁邵[1] 

机构地区：[1]厦门大学福建省医学分子病毒学研究中心,福建厦门361005

出　　处：《病毒学报》2005年第2期113-117,共5页Chinese Journal of Virology

基　　金：教育部跨世纪人才优秀培养计划

摘　　要：用绿色荧光前体化合物CalceinAM标记靶细胞,经过与杀伤性T淋巴细胞(CTL)效应细胞数小时的孵育,通过分析培养上清中的荧光强度检测CTL效应。通过对一系列标记条件的研究:不同浓度CalceinAM标记靶细胞、不同洗涤缓冲液、不同标记细胞浓度、不同洗涤次数、不同裂解液配方,确定CalceinAM荧光素标记法的最佳条件。用该方法检测乙型肝炎病毒表面抗原的DNA疫苗免疫Balb/c小鼠对P815S细胞的CTL效应,E/T比为10时,杀伤效率接近饱和,达到65%。通过荧光显微镜直接观察杀伤后的P815S细胞,细胞破裂程度与计算出的杀伤效率有一定相关性。The use of chromium-release assay to determine the cytotoxicity of effector against target cells has various limitations mostly due to the inherent properties of the radioactive substance. Alternative methods to detect CD8+ activity including ELISPOT,tetramer staining,or flow cytometry are indirect. or time consuming detection . A non-fluorescent substance Calcein AM has been used to stain the target cells and then intracellularly converted to green fluorescent calcein by esterase activity,after several hours' coculturing with effector,intensity of fluorescence in supernatant was assayed to determine the specific lysis ratio of cytotoxic T lymphocyte (CTL) .This method is convenient and nontoxic,but has high background which limits its large-scale use.In this report, several labeling conditions were tried to decrease its background and increase its sensitivity which included: labeled the target cells with μmol/L,10μmol/L or 25μmol/L Calcein AM;labeled the target cells at 1 × 106,2 × 106 or 4 × 106 cells/200μ labeling buffer; washed the labeled cells with PBS or PBS containing 10% FCS 1 -3 times to remove extracellular calcein; total lysis of the labeled target cells with different concentrations of SDS or Triton, and at last the optimal labeling and assay conditions were determined that were to label the target cells with 25μmol/L Calcein AM in 37 ℃ CO2 incubator for 30min;the labelling concentration of the target cells was 1 × 106-2 × 106 cells/200μ l labeling buffer; the labeled cells were washed with PBS containing 10% FCS for 3 times;0.025% SDS was used for total lysis of the labeled target cells to determine the specific lysis ratio.This improved fluorescence assay was used to detect CTL activity of Balb/c mice primed by HBsAg DNA vaccine pcD-NA3.1-S and boosted by recombinant vaccinia virus WR-S which contains HBsAg coding gene.The DNA vaccine vector pcDNA3. 1 was used as negative control. One week later, the splenocytes were separated and in vitro stimulated by mitomycin C treated P815S for 5

关 键 词：CTL 乙型肝炎病毒表面抗原 T淋巴细胞 特异性杀伤 靶细胞 荧光素标记 培养上清 浓度 孵育 洗涤次数 

分 类 号：Q783.1[生物学—分子生物学] R735.7[医药卫生—肿瘤]