不同荧光标记检测抗体组合及不同抗凝剂对流式测定食蟹猴T淋巴细胞亚群的影响  

Impact of different fluorescence labeled antibody combinations and different anticoagulants on the detection of T lymphocyte subsets in cynomolgus monkeys by flowcytometry

作　　者：姜华[1] 刘丽[1] 李路路[1] 苗玉发[1] 文海若 孙立[1] 王欣[1] 张颖丽[1] 李伟[1] 霍艳[1] 耿兴超[1] JIANG Hua;LIU Li;LI Lu-lu;MIAO Yu-fa;WEN Hai-ruo;SUN Li;WANG Xin;ZHANG Ying-li;LI Wei;HUO Yan;GENG Xing-chao(Key Laboratory of Beijing for Nonclinical Safety Evaluation Research of Drugs,National Center for Safety Evaluation of Drugs,National Institutes for Food and Drug Control,Beijing 100176,China)

机构地区：[1]中国食品药品检定研究院/国家药物安全评价监测中心药物非临床安全评价研究北京市重点实验室,北京100176

出　　处：《中国医药生物技术》2021年第5期391-399,共9页Chinese Medicinal Biotechnology

基　　金：国家重点研发计划(2016YFAO101503);中国科学院战略性先导科技专项(XDA16040502)。

摘　　要：目的比较不同荧光标记检测抗体组合与不同抗凝剂对检测食蟹猴外周血T淋巴细胞亚群的影响,为相关检测提供方法参考。方法取肝素钠抗凝猴血50μl分别与两组荧光检测抗体PerCP-CD3/FITC-CD8/PE-CD4(检测抗体组合1)和PerCP-CD3/FITC-CD4/PE-CD8(检测抗体组合2)孵育,流式细胞仪测定CD3^(+)CD4^(+)和CD3^(+)CD8^(+)淋巴细胞百分比和平均荧光强度(MFI),并计算CD3^(+)CD4^(+)和CD3^(+)CD8^(+)淋巴细胞比值,比较不同荧光标记抗体组合检测食蟹猴外周血T淋巴细胞亚群的结果。取肝素钠和乙二胺四乙酸二钾(EDTA-K2)抗凝猴血各50μl分别与上述两组荧光检测抗体组合进行孵育,测定CD3^(+)CD4^(+)和CD3^(+)CD8^(+)淋巴细胞百分比和MFI,计算这两种淋巴细胞亚群比值,比较不同抗凝剂对食蟹猴外周血T淋巴细胞亚群检测结果的影响。结果⑴肝素钠抗凝猴血荧光检测抗体测定T淋巴细胞亚群的结果显示,FITC标记抗CD4抗体检测到的CD4^(+)T淋巴细胞百分比和MFI均显著低于PE标记抗CD4抗体检测到的CD4^(+)T淋巴细胞百分比和MFI,同样,FITC标记抗体检测到的CD8^(+)T淋巴细胞百分比和MFI均显著低于PE标记抗体检测到的相应结果,应用检测抗体组合2的CD3^(+)CD4^(+)/CD3^(+)CD8^(+)淋巴细胞比值显著低于应用检测抗体组合1的,雌、雄动物均出现前述结果,雌性动物的变化更为显著。⑵应用抗体组合1检测时,与肝素钠抗凝组相比,EDTA-K2抗凝剂组CD3^(+)CD4^(+)淋巴细胞百分比显著增加、CD3^(+)CD8^(+)淋巴细胞百分比和CD8^(+)MFI显著降低,CD3^(+)CD4^(+)/CD3^(+)CD8^(+)淋巴细胞比值显著增加;应用抗体组合2检测时,仅发现EDTA-K2抗凝组比肝素钠组CD8^(+)MFI显著降低,未见其他有意义的变化。结论FITC标记抗CD4抗体和抗CD8抗体检测相应细胞百分比和荧光强度均小于PE标记的抗体,在应用这两种荧光标记抗体进行T淋巴细胞亚群检测时应注意对试验结果的影响和合理�Objective The objective of the study is to compare the effects of different fluorescence labeled antibody combinations and different anticoagulants on the detection of peripheral blood T lymphocyte subsets in cynomolgus monkeys and to supply a method reference for related determinations.Methods 50μl of heparin sodium anticoagulant monkey blood was incubated with two groups of fluorescence labeled detection antibodies PerCP-CD3/FITC-CD8/PE-CD4(detection antibody combination 1)and PerCP-CD3/FITC-CD4/PE-CD8(detection antibody combination 2),respectively.The percentage and mean fluorescence intensity(MFI)of CD3^(+)CD4^(+)and CD3^(+)CD8^(+)lymphocytes were measured by flow cytometry,and the ratio of CD3^(+)CD4^(+)and CD3^(+)CD8^(+)lymphocytes was calculated.The results of peripheral blood T lymphocyte subsets in cynomolgus monkeys were compared with those of different fluorescent detection antibody combinations.50μl heparin sodium and EDTA-K_(2)anticoagulant monkey blood were incubated with the above two groups of fluorescence detection antibody combinations,respectively.The percentage and MFI of CD3^(+)CD4^(+)and CD3^(+)CD8^(+)lymphocytes were measured,and the ratio of these two lymphocyte subsets was calculated.The effects of different anticoagulants on the detection results of peripheral blood T lymphocyte subsets in cynomolgus monkeys were compared.Results⑴The results from using the above two groups of fluorescent detection antibodies to detect T lymphocyte subsets in heparin sodium anticoagulant monkey blood showed that the percentage of CD4^(+)T lymphocytes and MFI detected by FITC-labeled anti-CD4 antibody were significantly lower than those detected by PE-labeled anti-CD4 antibody.Similarly,the percentage of CD8^(+)T lymphocytes and MFI detected by FITC-labeled antibody were significantly lower than those detected by PE-labeled antibody.The CD3^(+)CD4^(+)/CD3^(+)CD8^(+)lymphocyte ratio of antibody combination 2 was significantly lower than that of antibody combination 1.The above results were observed in b

关 键 词：外周血T淋巴细胞亚群 流式细胞测定法 食蟹猴 荧光标记检测抗体 抗凝剂 

分 类 号：R392[医药卫生—免疫学]