中国肿瘤生物治疗杂志2018，Vol.25Issue(4)：407-413,7.DOI:10.3872/j.issn.1007-385x.2018.04.015
人源性CD3抗体诱导食蟹猴T淋巴细胞培养方法的建立
Establishment of a method for culturing cynomolgus T lymphocytes induced by human CD3Ab
摘要
Abstract
Objective:To establish a method for in vitro isolation and culture of T lymphocytes from peripheral blood of cynomolgus monkeys that induced by human CD3 antibody based on the foundation of protein homology of CD3 from human,cynomolgus monkey and porcine.Methods:The amino acid sequences of human,cynomolgus monkeys and porcine CD3 proteins were obtained from NCBI,and the sequence,homology and phylogenetic tree were analyzed by DNAMAN software.Western blotting was used to detect the expression of CD3 protein on T cell membranes from the three species.PBMCs of healthy cynomolgus were isolated and divided into three groups:group A was stimulated with anti-human CD3Ab alone,group B was stimulated with IL-2 alone,and group C was costimulated with human CD3Ab and IL-2.Cell morphology and growth status were observed under inverted microscope and the cell growth curve was plotted.Cell viability was detected by trypan blue staining and the expressions of CD3,CD4 and CD8 on T cell surface were detected by flow cytometry.Results:The homology of the amino acid sequence of human CD3 protein to cynomolgus monkey and porcine were 86.9％ and 65.6％ respectively.The expression levels of CD3 protein on cynomolgus and porcine T cell membrane were 79％ and 17％ contrast to human,respectively.Cells of group A did not proliferate.Proliferation,viability and CD3 expression [(93.8±3.6)％ vs (70.3±4.7)％,P＜0.01] in T cells of group C were significantly higher than those in group B.Growth curve of T cells in group C showed an S-shape,which is consistent with Logistic growth curve.T cells in group C exhibited high purity and expressed high level CD3;moreover,the CD8+T cell took a high proportion.Conclusion:The membrane ofT lymphocytes from peripheral blood of cynomolgus can express CD3 protein that highly homological to human.Co-stimulation of human CD3Ab,IL-2 and 1％ PHA can induce the proliferation and differentiation of T lymphocytes of cynomolgus,and obtain T lymphocytes with good growth status,high proliferation ability and high purity.关键词
同源性/抗人CD3抗体/T淋巴细胞/食蟹猴/诱导培养Key words
homology/anti-human CD3Ab/T lymphocyte/cynomolgus monkey/induced culture分类
医药卫生引用本文复制引用
缪怡,董坚,角德灵,宋倩,高嫦娥,赵恒,孟小琴..人源性CD3抗体诱导食蟹猴T淋巴细胞培养方法的建立[J].中国肿瘤生物治疗杂志,2018,25(4):407-413,7.基金项目
云南省科技计划项目重大项目资助(No.2016FC007) （No.2016FC007）
云南省细胞治疗技术转化医学重点实验室资助(No.2015DG034). Project supported by the Key Foundation of Science and Technology Program in Yunnan Province (No.2016FC007),and the Yunnan Key Laboratory of Cell Therapy Technology Translational Medicine (No.2015DG034) （No.2015DG034）
