人类B细胞转化为抗体分泌浆细胞的体外研究  

Monitoring the generation of antibody-secreting plasma cells from human B lymphocytes in vitro

作　　者：廖爱华[1] 王燕[1] 张玲[1] 

机构地区：[1]华中科技大学同济医学院生殖医学中心,武汉430030

出　　处：《中华微生物学和免疫学杂志》2007年第7期647-650,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(30571710);教育部留学回国人员科研启动基金(2006331)

摘　　要：目的体外示踪不同刺激条件下人类B细胞转化为抗体分泌浆细胞的能力。方法对5名健康自愿者采用B细胞阴性选择磁珠分离法分离和体外培养B细胞,观察4种培养条件下(培养液对照组、PWM组、细胞因子组及PWM+细胞因子组)、不同时间点B细胞转化为抗体分泌浆细胞的能力。采用双荧光标记(FITC-CD27、PE-CD38)流式细胞技术监测B细胞4种表面标志物的动态变化;采用酶联免疫法(ELISA)测定不同时间点培养上清液中Ig浓度;采用酶联免疫斑点法(ELISPOT)测定不同时间点抗体分泌B细胞数。结果B细胞产生抗体浓度明显与培养条件有关。PWM刺激的B细胞培养上清液中Ig浓度最高(P=0.003);PWM+细胞因子组次之;细胞因子组与对照组差异无统计学意义。流式细胞分析结果显示,培养第7天浆细胞前体达峰,第10天浆细胞百分率达峰。活细胞数以细胞因子组最多,培养10d后仍呈增殖状态;而PWM组很快下降。ELISPOT法测定的lg分泌B细胞数也以PWM组最佳,PWM+细胞因子组次之。结论从Ig产生能力看,以PWM组最佳;从细胞增殖和浆细胞的存活来看,以细胞因子组最佳。表明体外诱导B细胞产生抗体不仅需要有效的刺激原,同时还需要必需的细胞因子信号促使细胞增殖和维持细胞的存活。Objective To monitor the kinetic profile of generation of plasma cells from human B cells in vitro under different stimulating conditions. Methods Human B cells from 5 healthy volunteers were isolated by B cell separation kit. The enriched B cells were cultured under four different conditions [ medium only, medium ＋ PWM, medium＋ cytokines（IL-2, IL-10, CD4OL） and medium＋ PWM＋ cytokines], to explore the efficiency of human B cells differentiation into Ig-secreting plasma cells at different time points by 5, 7, 10 and 12 days. The following three assays were to be detected： 1. levels of Ig in culture supernatants by ELISA; 2. kinetic changes of four different B cell phenotypes measured by flow cytometry; 3. Ig secreting B cells by ELISPOT assay. Results IgG production greatly differed depending on the culture conditions. IgG concentrations in supernatants from PWM- stimulated B cells were highest among those conditions（P =0.003）. By FACS analysis on day 7 and day 10, the gated percentage of precursors of plasma cells and plasma cells arrived at peak level, respectively. That of memory B cells decreased obviously. Events of plasma cells decreased obviously apart from cytokines-stimulated B cells. Conclusion PWM is the optimal stimulus in view of IgG production; Cytokines as IL-2, IL-10, CD40L are the optimal stimuli in view of long survival of plasma cells. To obtain optimal Ig production from B cells, both powerful stimuli and essential cytokine signals are required.

关 键 词：B淋巴细胞 浆细胞 免疫球蛋白 流式细胞技术 酶联免疫斑点法 

分 类 号：R392[医药卫生—免疫学]