SRB法检测鸡外周血T淋巴细胞增殖试验最佳条件的筛选  

Screening the optimum SRB assay for chicken peripheral T-lymphocytes proliferation

作　　者：刘宸铄[1] 刘芳[1] 杨鸣琦[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2013年第7期15-18,24,共5页Journal of Northwest A&F University(Natural Science Edition)

基　　金：农业部"948"引进项目(2009-Z45(2))

摘　　要：【目的】探讨磺酰罗丹明B法(Sulforhodamine B,SRB)检测鸡外周血T淋巴细胞增殖反应的最佳条件。【方法】体外培养鸡外周血T淋巴细胞,选用SRB法对淋巴细胞含量(1×105,1×106,2.5×106,5×106,7.5×106和1×107 mL-1)、ConA质量浓度(5.0,7.5,10.0,12.5和15.0μg/mL)、培养时间(36,48,60h)3个因素进行组合设计培养T淋巴细胞,每个组合设3个重复,SRB显色后用酶标仪在492nm波长处测量OD492值,计算刺激指数(Stimulation index,SI),筛选SRB法检测鸡外周血T淋巴细胞增殖的最佳条件。【结果】SRB法检测鸡外周血T淋巴细胞增殖试验的最佳条件为:淋巴细胞含量1×106 mL-1,ConA质量浓度5.0μg/mL,培养时间48h。【结论】确定了体外培养鸡外周血T淋巴细胞增殖的最佳ConA质量浓度、培养时间及淋巴细胞含量,证实用SRB法检测鸡外周血T淋巴细胞增殖结果可靠,是切实可行的淋巴细胞数量与活性检测方法。【Objective】 The optimal experiment conditions for SRB assay in chicken peripheral T-lymphocyte proliferation were investigated.【Method】 The technology of cell in vitro culture to the peripheral T-lymphocyte of chicken was used.Three major factors which affect the proliferation,including the concentration of cell（1×105,1×106,2.5×106,5×106,7.5×106,and 1×107 mL-1）,the ConA density（5.0,7.5,10.0,12.5 and 15.0 μg/mL）,and the cell culture time（36,48 and 60 h）,were studied.The SRB assay and micro-plate reader with a wavelength of 492 nm were also performed.Meanwhile,the stimulation index was calculated.【Result】 The optimal selection of SRB was：culture for 48 h,5.0 μg/mL ConA and 1×106 mL-1 cell concentration.【Conclusion】 The determinate concentration of cell,ConA density and cell culture time were obtained.It shows that SRB assay is a reliable practical method to analyze the number of lymphocyte and detect their activity.

关 键 词：鸡 外周血T淋巴细胞 细胞增殖 磺酰罗丹明B法 

分 类 号：R446.63[医药卫生—诊断学] S858.31[医药卫生—临床医学]