三种不同抗原对小鼠γδT细胞体外扩增的比较  

Comparison of mouse spleen γδT cells proliferation with three different antigens in vitro

作　　者：陈小兰[1] 潘磊[1] 张京慧[1] 齐颖[1] 牟丹蕾[2] 

机构地区：[1]首都医科大学附属北京世纪坛医院老年医学科,北京100038 [2]首都医科大学附属北京佑安医院,北京100069

出　　处：《疑难病杂志》2016年第1期52-57,共6页Chinese Journal of Difficult and Complicated Cases

基　　金：国家自然科学基金(81201294)

摘　　要：目的比较异戊烯焦磷酸（IPP）、热休克蛋白70（HSP70）和表位肽（EP6）对小鼠脾脏γδT细胞体外扩增的差异。方法 2011年1月—2015年5月于北京协和医院及北京世纪坛医院选取健康雄性SPF级C57 BL/6小鼠32只,分为16组,每组2只。提取并分离小鼠脾脏γδT细胞,分为对照组、IPP组、HSP70组和EP6组,每组再按浓度及干预方式（连续性刺激及单次刺激）的不同进行分组,IPP浓度分为1.25 ng/ml、2.5 ng/ml、5 ng/ml,HSP70浓度分为6.25μg/ml、12.5μg/ml、25μg/ml,EP6浓度分为0.05μg/μl、0.1μg/μl、0.2μg/μl,连续性刺激即每隔2~3日换液50%并补充所换液体中相应的抗原,单次刺激组即不需补充所换液体中的抗原,共培养10 d。检测对照组与不同浓度抗原、不同干预方式在培养的第0天（D0）、第3天（D3）、第7天（D7）和第10天（D10）的γδT细胞比例。结果不同浓度的抗原作用γδT细胞后第3天出现扩增峰值,达峰时抗原由小到大各浓度的增殖百分比分别为：IPP组（5.46%±0.01%、5.23%±0.01%、5.08%±0.08%）,HSP70组（7.43%±0.11%、4.48%±0.07%、7.78%±0.07%）,EP6组（3.80%±0.10%、6.22%±0.13%、8.42%±0.11%）。达峰时IPP组1.25 ng/ml的扩增效果较2.5ng/ml和5.0 ng/ml明显升高（5.46%±0.01%vs.5.23%±0.01%、5.08%±0.08%,P〈0.05）。3组γδT细胞的扩增效果不随抗原浓度增加而升高,随堵养时间的延长而降低。浓度不变干预方式不同时,连续性刺激组和单次刺激组均在第3天出现扩增峰值,连续性刺激组第3天的增殖百分比分别为：IPP组6.38%±0.58%、HSP70组4.27%±0.26%、EP6组5.41%±0.11%;单次刺激组第3天的增殖百分比分别为：IPP组3.60%±0.27%、HSP70组4.54%±0.16%、EP6组3.20%±0.11%;达峰时IPP组和EP6组均出现连续性刺激组扩增百分比大于一次性单次刺激组（P〈0.05）。结论小鼠模型中抗原体外扩增脾脏γδT细胞时,培养后第3天为扩增的达峰时间,且抗原连续性刺激扩�Objective To compare the difference of in vitro amplification of murine spleen gamma delta T cells in mouse spleen by comparing the expression of IPP and heat shock protein 70（HSP70） and epitope6（EP6）.Methods From January 2011 to May 2015,in Peking Union Hospital Beijing Shijitan Hospital,92 healthy male C57 BL/6 mice,extracted and isolated mouse spleen T cells,then divided into control group,HSP70 group and EP6 group,each group was divided into different groups according to the concentration and intervention（continuous stimulation and single stimulus）,IPP concentration is divided into 1.25 ng/ml,2.5 ng/ml,5 ng/ml.HSP70 concentration is divided into 6.25 μg/ml,12.5 μg/ml,25 μg/ml,EP6 concentration is divided into 0.05 μg/ml,0.1 μg/rnl,0.2 μg/ml,the continuous stimulation group,which is two to three days every 50%days and replaced with the corresponding antigen in the liquid,one time single stimulation group,which does not need to supplement the antigen in the liquid exchange,totally cultured for 10 days.The proportion of gamma delta T cells in the control group and the different concentration of antigen,different intervention methods in the culture of zeroth days（D0）,third days（D3）,seventh days（D7） and tenth days（D10） were detected.Results Different concentrations of the antigen role of gamma delta T cells alter third days emerged of amplification,lime to peak percentage from each concentration group proliferation antigen respectively：IPP group（5.46%±0.01%,5.23%±0.01%,5.08%±0.08%）,HSP70 group（7.43%±0.11%,4.48%±0.07%,7.78%±0.07%）,EP6 group（3.80%±0.10%,6.22%±0.13%,8.42%±0.11%）.IPP group at the peak concentration of 1.25 ng / ml＇s amplify effect was significantly increased than 2.5ng/ml and 5.0 ng/ml[（5.46%±0.01%） vs.（5.23%±0.01%,5.08%±0.08%）]（P 0.05）.The amplification effect of the 3 groups of gamma delta T cells did not increase with the increase of antigen concentration,decreased with the prolongation of culture time.Concentration constant

关 键 词：脾脏 ΓΔT细胞 抗原 增殖 异戊烯焦磷酸 热休克蛋白 表位肽 小鼠 

分 类 号：R392[医药卫生—免疫学]