摘要
目的优化B细胞亚群及其组织分布的流式细胞分析的荧光染色方案,以用于免疫调控药物的活性评价。方法取小鼠外周血、腹膜腔细胞、脾细胞、淋巴结细胞和骨髓细胞,分别通过3种荧光抗体染色方案对各种细胞进行染色:①抗B220,CD5和CD11b的荧光抗体及7-氨基放线菌素D(7-AAD)染色;②抗B220,CD43,CD93,CD23和IgM的荧光抗体及7-AAD染色;③抗B220,CD27和CD138的荧光抗体及7-AAD染色。然后利用流式细胞术分析B细胞亚群,通过优化的表面标志物组合确定各种B细胞亚群,再统计各种B细胞亚群在多种组织中的分布。结果第①种染色方案可区分B1a,B1b和B2细胞;第②种染色方案可区分原始B细胞、不成熟和成熟B细胞;第③种染色方案可区分记忆性B细胞和浆细胞。外周血中主要包含成熟B细胞和一种特殊的不成熟B细胞;腹膜腔中主要包含B1a细胞和成熟B细胞;脾中主要包含成熟B细胞、边缘区B细胞及T1和T2期不成熟B细胞;淋巴结中主要包含成熟B细胞;骨髓中包含各种发育阶段B细胞,主要包含前B细胞(pre-B细胞)和T1期不成熟B细胞。记忆性B细胞和浆细胞在各种组织中都只占B细胞的较小比例。结论本研究采用的荧光染色方案可特异准确地分析B细胞亚群及其组织分布,可用于免疫调控药物的活性评价。
OBJECTIVE To optimize the analysis of B-cell subgroups and their tissue distribution with flow cytometry.METHODS The peripheral blood,peritoneal cavity cells,splenocytes,lymph node cells and bone marrow cells of mice were prepared and stained with three combinations of fluorescent antibodies and dead cell dye:①combination of anti-B220,anti-CD5,anti-CD11b,and 7-aminoactinomycin D(7-AAD);②combination of anti-B220,anti-CD43,anti-CD93,anti-CD23,and 7-AAD;③combination of anti-B220,anti-CD27,anti-CD138,and 7-AAD.These cells were analyzed by flow cytometry and Bcell subgroups were identified with optimized biomarkers on cell surface.Finally,tissue distributions of B-cell groups were analyzed.RESULTS B1a,B1b and B2 cells were identified by the first staining combination of fluorescent antibodies and dead cell dye.Original B cells,immature B cells and mature B cells were clearly distinguished by the second staining combination.Memory B cells and plasma cells were identified by the third staining combination.In peripheral blood,there were mostly mature B cells and a specific immature B cells.The peritoneal cavity contained B1a and mature B cells.The spleen had multiple B-cell subgroups,including mature B cells,marginal zone B cells,immature B cells in T1 and T2 stages.In lymph nodes,there were mainly mature B cells.Bone marrow contained almost each of B-cell subgroups,including pre-B cells and immature B cells in T1 stage.Memory B cells and plasma cells made up small proportions of B cells in all tissues.CONCLUSION B-cell subgroups and their tissue distributions can be specifically and accurately analyzed with this optimized flow cytometry,which is applicable to active evaluation of immunomodulatory drugs.
作者
郭冉
李芸芊
屠鹏飞
朱枝祥
GUO Ran;LI Yun-qian;TU Peng-fei;ZHU Zhi-xiang(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2020年第4期302-310,共9页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(81603361)
北京中医药大学研究生自主课题(2019-JYB-XS-053)。
关键词
B细胞亚群
标志物
组织分布
流式细胞术
B-cell subgroups
biomarker
tissue distribution
flow cytometry
