简化法与传统方法进行淋巴细胞胞内细胞因子染色效果的比较  

Staining of cytokines in lymphocytes by modified and traditional methods

作　　者：刘良忠[1] 胡建娥[1] 常世川[1] 熊德明[1] 朱川[1] 刘必宽[1] 陈睿[1] 谭建军[1] 李刚[1] 

机构地区：[1]重庆三峡中心医院肿瘤防治研究所,重庆404000

出　　处：《中国生物制品学杂志》2012年第5期633-637,共5页Chinese Journal of Biologicals

基　　金：重庆市医学重点研究室建设项目(2007-14);重庆市卫生局科研项目(2009-2-050)

摘　　要：目的分别采用简化法与传统方法进行淋巴细胞胞内细胞因子染色,通过流式细胞术检测比较两种方法的染色效果。方法取健康志愿者外周血,分离外周血单个核细胞(PBMC),分别经佛波酯(Phorbol myristate acetate,PMA)、离子霉素(Ionomycint,Ion)和莫能霉素(Monensin)刺激4 h,分别以传统法(细胞刺激后进行表面染色,再固定,破膜,行细胞内染色)和简化法(细胞刺激后直接固定,破膜,在不同时间段进行膜上膜内同时染色)进行细胞内染色。样本经抗体标记后,采用流式细胞术检测淋巴细胞的百分率。结果简化法染色检测Th1细胞的百分比与传统染色方法相比,差异无统计学意义(P>0.05);而简化法染色检测Th17细胞的百分比明显高于传统方法(P<0.05)。与传统法比较,应用简化法,细胞于破膜后固定0、48、96 h染色,检测Th2细胞的百分比差异无统计学意义(P>0.05)。结论简化法简化了操作流程,有利于流式细胞术的质控,可替代传统染色方法进行淋巴细胞胞内细胞因子染色。Objective To stain the cytokines in lymphocytes by modified and traditional methods, and compare the effectiveness by flow cytometry. Methods Peripheral blood samples of healthy volunteers were collected, from which PBMCs were isolated and subjected to intracellular staining by traditional and modified methods respectively. By the traditional method, the cells were stimulated, and stained on surface, then fixed and stained after the membranes were broken. However, by the modified method, the cells were directly fixed after stimulation, of which the membranes were broken, and the intramembrane and membrane surface staining were carried out simultaneously at various time points. The percentages of lymphocytes in the samples were determined by flow cytometry after the antibodies were labeled. Results The percentage of Thl cells stained by modified method showed no significant difference with that by traditional method （P 〉 0. 05 ）. However, the percentage of Th17 cells stained by modified method was significantly higher than that by traditional method（P 〈 0. 05）, while the percentages of Th2 cells 0, 48 and 96 h after the membranes were broken showed no significant difference （P 〉 0. 05）. Conclusion By the modified method, the procedure for test was simpli- fied. The modified method was beneficial to the quality control of flow cytometry, which might be used for staining of cytokines in lymphocytes instead of traditional method.

关 键 词：流式细胞术 细胞内染色 TH1细胞 TH2细胞 TH17细胞 

分 类 号：R392.33[医药卫生—免疫学] Q233[医药卫生—基础医学]