犬淋巴细胞的体外活化及增殖研究

Study on Activation and Proliferation of Canine Lymphocytes in Vitro

为探讨犬淋巴细胞的分离、激活和增殖以进一步研究犬肿瘤的过继免疫疗法,用Ficoll密度梯度离心液对犬的全血进行分离,得到淋巴细胞。克隆犬CD86基因并构建了人工抗原递呈细胞K562.CD32.GFP-cCD86稳定细胞系。分别通过刀豆蛋白(ConA),抗CD28抗体,或者结合CD3抗体的K562.CD32.GFP-cCD86细胞激活犬淋巴细胞,通过Coulter Counter对T细胞进行计数和体积分析,并在显微镜下观察细胞激活后的形态变化。结果显示,在体外成功分离到犬外周血淋巴细胞。在不同扩增条件下,K562.CD32.GFP-cCD86结合抗CD28和CD3抗体对淋巴细胞有最好的激活效果和增殖效率,在激活第8天细胞可以达到44倍的增殖。研究结果为犬淋巴细胞的体外分离和增殖提供了有效方法,并为犬的肿瘤免疫治疗研究奠定了基础。

To study adoptive T cell transfer in the treatment of canine malignance,the successful isolation and expansion of canine lymphocytes are required.Canine peripheral blood mononuclear cells(PBMC)were isolated from whole blood by Ficoll-Pague density gradient centrifuge.cCD86 was cloned from PBMC and inserted into a lentiviral vector.The artificial antigen presentation cell line K562.CD32.GFP-cCD86 was constructed.Canine peripheral blood lymphocytes were stimulated by concanavalin A(ConA),or antihCD28 antibody,or K562.CD32.GFP-cCD86 coupled with anti-cCD3 antibody.The cell number was measured by Coulter Counter based on cell volume,and then further confirmed by morphological change through microscope.Results showed that canine peripheral blood lymphocytes were successfully separated in vitro.Among the three stimulating methods,K562.CD32.GFP-cCD86 loaded with anti-canine CD3 antibody could stimulate lymphocytes to the highest stimulation and expansion level.By this method,an average of44 folds increasing was achieved after 8 days proliferation,accompanied with the increase of cell volume.The study provided tools and effective way for the isolation and proliferation of canine peripheral blood lymphocytes in vitro to further facilitate the development of canine cancer immunotherapy.