目的： 在初步分析人、食蟹猴和猪CD3蛋白同源性的基础上，建立利用人源性CD3抗体诱导食蟹猴外周血T淋巴细 胞的体外分离培养技术。 方法： 从NCBI 查询获取人类、食蟹猴和猪CD3蛋白各自的氨基酸序列，并用DNAMAN 软件进行序列 比对、同源性分析和构建系统进化树。Western blotting检测三种T细胞膜上CD3蛋白的表达水平。分离健康食蟹猴PBMC，分为 3组，A组加入抗人CD3抗体单刺激、B组加入IL-2单刺激、C组加入抗人CD3抗体和IL-2共刺激。倒置显微镜下观察细胞生 长状态并计数，绘制细胞生长曲线，锥虫蓝染色检测细胞活性，流式细胞术检测T细胞表面标志物CD3、CD4、CD8的表达。 结 果： 食蟹猴、猪的CD3蛋白氨基酸序列与人类的同源性分别为86.9% 、65.6%，T细胞膜上CD3蛋白的表达量分别为人的79%、 17%。A组细胞不增殖；C组细胞增殖能力、细胞活性及CD3表达率［(93.8±3.6)% vs (70.3±4.7)%，P<0.01］均显著高于B组，细胞 生长曲线呈S形，符合Logistic生长曲线；C组T细胞高表达CD3，T细胞纯度较高，且CD8 + T 细胞占比较多。 结论： 食蟹猴外周血 T淋巴细胞膜表面能表达和人同源性很高的CD3蛋白，在人源性CD3抗体、IL-2和1%PHA共刺激下能诱导食蟹猴外周血T淋巴 细胞的增殖分化，获得生长状态良好、增殖能力强、纯度较高的T淋巴细胞。

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 mon- key 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 in- to three groups: group A was stimulated with anti-human CD3Ab alone, group B was stimulated with IL-2 alone, and group C was co- stimulated 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 sur- face were detected by flow cytometry. Results: The homology of the amino acid sequence of human CD3 protein to cynomolgus mon- key and porcine were 86.9% and 65.6% respectively. The expression levels of CD3 protein on cynomolgus and porcine T cell mem- brane were 79% and 17% contrast to human, respectively. Cells of group A did not proliferate. Proliferation, viability and CD3 expres- sion [(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 of T lymphocytes from periph- eral 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 sta- tus, high proliferation ability and high purity.

云南省科技计划项目重大项目资助(No.2016FC007);云南省细胞治疗技术转化医学重点实验室资助(No.2015DG034)