检验医学 ›› 2013, Vol. 28 ›› Issue (1): 61-66.DOI: 10.3969/j.issn.1673-8640.2013.01.015

• 技术研究与评价.论著 • 上一篇    下一篇

流式细胞术检测人外周血产生IL-22和/或IL-17的淋巴细胞亚群及其在结核病患者中的应用

梁华1,梁友宝2,贺文欣3,杨军平1,孙国宏1,杨小军1,李柏青4   

  1. 1. 江西中医学院附属医院检验科,江西 南昌 330006;2.蚌埠市第一医院检验科,安徽 蚌埠 233030;3.蚌埠医学院免疫学教研室 安徽省感染与免疫重点实验室,安徽 蚌埠 233030
  • 收稿日期:2012-11-26 修回日期:2012-08-24 出版日期:2013-01-30 发布日期:2013-01-10
  • 通讯作者: 李柏青,联系电话:0552-3171086。
  • 作者简介:梁华,男,1975年生,硕士,主管技师,主要从事细胞免疫学研究。
  • 基金资助:

    国家科技重大专项基金资助项目(2008ZX10003-011)

Detection of interleukin-22/17-producing lymphocytes subpopulations in peripheral blood of human by flow cytometry and application in patients with tuberculosis

  1. 1. Department of Clinical Laboratory,the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine,Jiangxi Nanchang 330006,China;2. Department of Clinical Laboratory,the First Hospital of Bengbu,Anhui Bengbu 233030,China;3. Department of Immunology,Bengbu Medical College,Anhui Key Laboratory of Infection and Immunity,Anhui Bengbu 233030,China
  • Received:2012-11-26 Revised:2012-08-24 Online:2013-01-30 Published:2013-01-10

摘要: 目的 建立用流式细胞术(FCM)检测人外周血产白细胞介素22(IL-22)的不同淋巴细胞亚群的方法,并探讨其在结核病(TB)患者中的应用,以了解产IL-22的淋巴细胞亚群在抗结核感染免疫中的作用。 方法 采集22例TB患者和18名健康对照者外周血,与相同体积RPMI-1640培养液混匀,加入佛波醇酯(PMA)和钙离子霉素,混匀后于37 ℃ 5% CO2静置培养2 h,再加入莫能霉素后继续培养4 h,收集细胞,用荧光素标记进行细胞表面抗原和胞内细胞因子染色,用FCM检测表达IL-22、IL-17的不同淋巴细胞亚群比例。 结果 PMA浓度为100 ng/mL,钙离子霉素浓度为1 μg/mL,刺激培养2 h可最有效刺激IL-22的产生;TB患者外周血中产IL-22的γδT细胞比例(7.37%)明显高于正常人(1.81%,P<0.05),而在CD4T、CD8T、自然杀伤(NK)细胞和B细胞等其他细胞亚群中,产IL-22的阳性细胞比例在TB患者和健康对照者之间差异无统计学意义(P>0.05);Th22和Th17细胞比例在TB患者和健康对照者之间差异无统计学意义(P>0.05)。 结论 建立了一套简便、完整、可靠的FCM检测产IL-22的淋巴细胞亚群的方法,产IL-22的γδT细胞在抗结核感染免疫中可能发挥免疫效应。

关键词: 白细胞介素22, 白细胞介素17, 淋巴细胞, 结核病, 流式细胞术

Abstract: Objective To establish a method of detecting different interleukin 22(IL-22)producing lymphocyte subpopulations in human peripheral blood by flow cytometry(FCM),and investigate its application in patients with tuberculosis(TB)and realize the role of IL-22 producing lymphocyte subpopulations in anti-infection immunity against TB.   Methods Peripheral blood samples collected from 22 TB patients and 18 healthy controls were mixed with RPMI-1640 culture medium at the scale of the same volume. After adding phorbol ester(PMA)and calcium ionomycin mixed for 2 h culturing on the conditions of 37℃ and 5% CO2,monensin was added and continued being cultured for 4 h. The cells were collected and stained surface molecular or intracellular with fluorochrome-conjugated monoclonal antibodies. The respective proportions of different IL-22 and IL-17 producing lymphocyte subpopulations were detected by FCM. Results The condition that the concentration of PMA was 100ng/mL and that of ionomycin was 1μg/mL with 2 h being stimulated and cultured could produce IL-22 effectively. The proportions of IL-22 producing gamma delta T cells in TB patients(7.37%)were obviously higher than those in healthy controls(1.81%,P<0.05). No statistical significance was found between the proportions of IL-22 producing positive cells in CD4T,CD8T,natural killer(NK)cells and B cells in TB patients and those in healthy controls (P>0.05). No statistical significance was found between the proportions of Th22 and Th17 in TB patients and those in healthy controls (P>0.05). Conclusions The established easy,complete and reliable method for the detection of IL-22 producing lymphocyte subpopulations and IL-22 producing gamma delta T cells could act immunological effects in anti-infection immunity against TB.

Key words: Interleukin 22, Interleukin 17, Lymphocyte, Tuberculosis, Flow cytometry