曹东林1，陈 伟2，王 玲2
Expansion and function analysis of CD4+CD25+ T cells
Cao Dong-lin1, Chen Wei2, Wang Ling2
1Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, Guangdong Province, China; 2Department of Hematology, Second People’s Hospital, Guangzhou 510317, Guangdong Province, China
transplantation. OBJECTIVE: To investigate the proliferation of CD4+CD25+T cells from C57BL/6 mice and function changes in vitro.
DESIGN, TIME AND SETTING: The cytology in vitro study was performed at the Department of Hematology, Zhujiang Hospital,
;CD25+ T 细胞增殖能力低，且在人外周血中仅占单个核细胞的 4%左右。若能在体外高效扩增 CD4+CD25+ T 细 胞，并保持其免疫调节特性，将会对临床移植产生积极的影响。 目的：观察 C57BL/6 小鼠来源的 CD4+CD25+ T 细胞体外增殖情况及其扩增后的功能变化。 设计、时间及地点：细胞学体外观察，于 2007-10/2008-05 在南方医科大学珠江医院血液科完成。 材料：SPF 级 C57BL/6 及 BALB/C 雄性小鼠购自南方医科大学动物所。小鼠白血病细胞 EL9611 由珠江医院血液科惠赠。 方法：利用免疫磁珠法分选小鼠 CD4+CD25+ T 细胞；以抗鼠 CD3ε单抗、抗鼠 CD28 单抗、鼠重组白细胞介素 2 及辐射 过的 BALB/C 小鼠脾细胞为共刺激因子，通过实时定量 RT-PCR 检测扩增后 CD4+CD25+ T 细胞 FoxP3 基因 mRNA 表达变 化，以确定增殖效率；3H-TdR 掺入法检测扩增后的 CD4+CD25+ T 细胞对 CD4+CD25-T 细胞增殖的影响；LDH 释放法检测 扩增后的 CD4+CD25+ T 细胞对 CD4+CD25-T 细胞杀伤小鼠白血病细胞 EL9611 的影响，以 CD4+CD25-T 细胞为效应细胞， 以 EL9611 细胞为靶细胞。 结果：经免疫磁珠分选可获得高纯度及较强活性的 CD4+CD25+ T 细胞。扩增后 CD4+CD25+T 细胞 FoxP3 基因 mRNA 的表 达平均为扩增前的 5.46 倍，最高可达 14.39 倍。扩增后 CD4+CD25+T 细胞可明显抑制 CD4+CD25-T 细胞的增殖，且随着 CD4+CD25+T 细胞数的增加，这种抑制增殖的能力也逐渐增强，当两者比例为 1:1 时抑制率最大，达 62.05%。与单纯 CD4+CD25- T 细胞对 EL9611 细胞杀伤率比较，效靶比为 10:1 时扩增后的 CD4+CD25+ T 细胞联合 CD4+CD25- T 细胞的杀 伤率无明显变化（t=2.199，P > 0.05）；效靶比为 5:1 时扩增后的 CD4+CD25+ T 细胞联合 CD4+CD25- T 细胞的杀伤率则 明显降低（t=5.839，P < 0.05）。 结论：单抗加异源性抗原能有效扩增 CD4+CD25+T 细胞；扩增后的 CD4+CD25+T 细胞比新鲜分离的 CD4+CD25+T 细胞能 更有效地抑制 CD4+CD25-T 细胞的增殖，其对 CD4+CD25-T 细胞杀伤白血病细胞的作用则取决于其与 CD4+CD25-T 细胞的 相对比例。
Supported by: the Scientific and Technological Plan Program of Guangzhou City, No. 005Z3-E0371*
Received: 2008-05-21 Accepted: 2008-08-10
Abstract BACKGROUND: CD4+CD25+ T cells have low proliferation ability, and are about 4% of mononuclear cells in the peripheral blood. To amplify CD4+CD25+T cells in a high level and with its immunological regulation in vitro can have active effects on clinical
Southern Medical University from October 2007 to May 2008.
MATERIALS: The SPF, C57BL/6 and BALB/C male mice were purchased from Animal Room of Southern Medical University.
Cao Dong-lin☆, Studying for doctorate, Lecturer, Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, Guangdong Province, China caodl＠
Cao DL, Chen W, Wang L.Expansion and function analysis of CD4+CD25+ T cells.Zhongguo Zuzhi Gongcheng Yanjiu yu
Linchuang Kangfu 2009;13(1):96-100(China)
基础医学
中国组织工程研究与临床康复 第 13 卷 第 1 期 2009–01–01 出版 Journal of Clinical Rehabilitative Tissue Engineering Research January 1, 2009 Vol.13, No.1
CD4+CD25+ T细胞的扩增及其功能分析*☆
关键词：T 细胞；体外增殖；功能
曹东林，陈伟，王玲.CD4+CD25+ T 细胞的扩增及其功能分析[J].中国组织工程研究与临床康复，2009，13(1):96-100 [ ]
Correspondence to: Wang Ling, Doctor, Chief physician, Master’s supervisor, Department of Hematology, Second People’s Hospital, Guangzhou 510317, Guangdong Province, China gzlwang@
monoclonal antibody, anti-mouse CD28 monoclonal antibody, mouse recombinant interleukin-2 and irradiated spleen cells from
BALB/C mouse were employed as co-stimulating factors. Following real time quantitative reverse transcription-polymerase chain reaction, CD4+CD25+T cells FoxP3 mRNA expression changed to define proliferation efficiency. The inhibitory effect of CD4+CD25+ T on CD4+CD25-T cells was detected by 3H-thymidine incorporation assay and on CD4+CD25-T cells killing leukemic cells EL9611 was assayed by LDH cytotoxicity assay. CD4+CD25-T cells were used as effector cells, and EL9611 cells as target
Leukemic cell EL9611 was presented by Department of Hematology, Zhujiang Hospital. METHODS: Immunomagnetic beads sorting method was used to separate CD4+CD25+T cells from mice. Anti-mouse CD3ε
cells. RESULTS: After sorting, CD4+CD25+T cells with high purity and cell vitality rate were collected. The relative expression of FoxP3 mRNA in expanded CD4+CD25+ T cells was averagely 5.46 times than that of freshly CD4+CD25+ T cells, and the highest 14.39 times. After amplification, CD4+CD25+T cells obviously inhibited the CD4+CD25-T cell proliferation in vitro. The inhibitory rate increased with the more CD4+CD25+T cells and achieved to maximum in coculture of CD4+CD25+ T cells together with CD4+CD25-T cells (ratio of 1∶1), and the inhibitory rate was about 62.05%. There was no significantly difference between CD4+CD25+T cell plus CD4+CD25-T cell group and CD4+CD25-T cell group when effect-target ratio was10:1（t=2.199，P > 0.05）. But there was significantly difference between this two groups when effect-target ratio was 5:1 （t=5.839，P < 0.05）. CONCLUSION: CD4+CD25+T cells can be expanded by monoclonal antibody and heterogenetic antigen. Expanded CD4+CD25+T cells can obviously inhibit the proliferation of CD4+CD25-T cells compared to freshly CD4+CD25+ T cells. Its effect on CD4+CD25-T cells to EL9611 leukemic cells depend on the ratio to CD4+CD25-T cells.