运用磁珠分选法建立免疫细胞共培养体系

目的以相互作用的几种免疫细胞上共同表达的抗原物质为标志,运用磁珠分选技术,从慢性乙型肝炎患者外周血单个核细胞中同时获得并建立体外多细胞共培养体系,体外观察细胞之间的相互作用.方法采用梯度离心法分离30例慢性乙肝病毒感染者外周血单个核细胞(PBMC),采用免疫磁珠分选法分离获得 CXCR5+细胞,通过流式细胞仪检测 CXCR5+细胞纯度和组成,并将 CXCR5+细胞进行体外培养.在分离得到的 CXCR5+细胞的基础上,将 CXCR5+细胞分为3组,分别为对照组:不加任何刺激;实验组一:加入 IL 21刺激培养,实验组二:与人肝癌细胞系(HepG2)2215细胞混合培养.体外培养一周后,流式细胞仪检测实验组与对照组细胞 B细胞数量变化,ELISA测定培养体系中上清液 HBe抗体的含量.结果1)用流式细胞仪鉴定 CXCR5+细胞的纯度和构成:CXCR5+细胞纯度为85.5±5.8%,其中 Tfh细胞占23.8±7.4%,B细胞占35.6±7.6%;2)培养7天后,IL 21刺激组 B细胞百分率为47.2±1.8%,与(HepG2)2215混合培养组 B细胞百分率为40.2±3.5%,空白对照组 B细胞百分率为36.6±7.5%,IL 21刺激组与对照组,(HepG2)2215混合培养组与对照组 B细胞百分率均有显著差异(p<0.05);3)ELISA检测培养液上清 HBeAb定量,IL 21刺激组为0.668±0.094pg/ml,空白对照组为0.378±0.088pg/ml,两组有显著差异(p<0.05).结论使用间接免疫磁珠分离法可成功建立 CXCR5+B淋巴细胞和 Tfh细胞的共培养体系,为进一步体外研究细胞之间相互关系奠定基础

Establishment of Co-culture System of Immunal Cell Using Bead Separation

Objective To establish a co-culture system of several immune cells in vitro ,which express the same antigens as a symbol and observe the interaction among those cells ,the immunomagnetic bead is used to isolate them from peripheral blood mononuclear cells in patients with chronic hepatitis B . Materails and Methods Mononuclear cells were isolated from fresh peripheral blood of 30 cases of＇ CHB patients by Ficoll-Hypaque density gradient eentrifugation. Isolate the CXCR5 ＋ cells from PBMC by magnetic beads methods and then detection the purity and composition of CXCR5 ＋ cells by FCM. This CXCR5 ＋ cells were divided into 3 groups：the control group：without any stimulation, the experimental group one：cuhured with the stimulus of IL-21 alone for one week, the experimental group two：co-cultured with human hepa- toma cell line （ HepG2 ） 2215 ceils alone for one week. Flow cytometry was used to detect the propotion of B ceils after 7 days culture. ELISA was used to detect the level of secreted HBeAb in culture supernant. Results 1 ） Detection the purity and composition of CXCR5 ＋ cells by FCM,The purity of CXCR5 ＋ ceils was 85.5 ＋5.8% which were composed of 23.8 ＋7.4% Tfh ceils and 35.6 ＋7.6% B ceils. 2） After 7 days culture,the percent of B ceils which stimulated by IL-21 （47.2 ± 1.8% ） was.higher than blank controls （36. 6 ±7.5% ） ,and which mixed culture with （ HepG2）2215 cells（40.2 ±3.5% ） was higher than blank controls （36.6 ±7.5% ） （ P 〈0.05）. 3） Detection the quantity of HBeAb in Supernatant by ELISA , The group stimulated by IL-21 （0. 668 ＋ 0. 094pg/ml） were significantly different from blank controls（0. 378 ＋ 0. 088 pg/ml） （ P 〈 0. 05 ）. Conclusion The co-cuhure system of CXCR5 ＋ Tfh cells and B lymphocytes can be successfully established by use of indirect immunomagnetic bead separation method, which will benefit the study On the interaction of between different ceils in vitro.