人乳腺癌OX40L转基因细胞株的构建及其对T细胞的共刺激作用

Construction of Human Breast Carcinoma Cell Line Transfected with OX40L and Its Costimulatory Effect on T Cells

背景与目的:共刺激分子OX40(CD134)和OX40配体(OX40L、CD134L)分属TNFR、TNF家族,OX40-OX40L信号能促进细胞因子的产生及提高抗原特异性记忆T细胞的数量,发挥重要的免疫调节作用。本实验旨在构建稳定表达OX40配体(OX40L)的人乳腺癌转基因细胞株,进而研究OX40-OX40L信号对T细胞体外活化和功能的调节作用。方法:运用逆转录聚合酶链反应(RT-PCR)技术从人成熟树突细胞(dendriticcell,DC)获得全长人OX40L基因,构建真核表达载体pcDNA3.1-OX40L转染人乳腺癌细胞株MDA-MB-435;经过G418抗性筛选和亚克隆,获得稳定表达OX40L的转基因细胞,并经RT-PCR和免疫荧光标记确证靶分子的表达;采用MTT、酶联免疫吸附试验(ELISA)和免疫荧光标记等方法分析转基因细胞对T细胞的增殖、IL-2、IL-4、IFN-γ的分泌和T细胞上Fas分子表达的影响。结果:成功地获得稳定表达人OX40L分子的人乳腺癌MDA-MB-435转基因细胞株,该细胞株可有效地促进T细胞增殖,促进T细胞分泌IL-2和IFN-γ,在第7天,T细胞、T细胞+M435、T细胞+M435-mock、T细胞+M435-OX40L各组IL-2的浓度依次为:315ng/ml、322ng/ml、586ng/ml、973.4ng/ml;IFN-γ浓度依次为:(2518±117.6)pg/ml、(2490±124.2)pg/ml、(2695±134.5)pg/ml、(3755±187.75)pg/ml,该基因转染细胞同时还能下调T细胞上Fas分子的表达[(68.3±5.6)%,P<0.05]。结论:稳定表达人OX40L的乳腺癌转基因细胞在体外能有效地活化T细胞,介导其增殖、分泌细胞因子及抑制T细胞活化诱导的细胞死亡。

BACKGROUND ＆ OBJECTIVE. Costimulator OX40 （CD134） belongs to TNFR family, and its ligand OX40L （CD134L） belongs to TNF family. OX40-OX40L signal plays an important role in immune regulation, which can increase the production of cytokines and enhance the survival of antigen-specific T cells. This research was to transfect OX40L into human breast cancer cell line MDA-MB-435 to construct OX40L-MDA-MB-435 cells, and to investigate the costimulatory effect of OX40-OX40L signal on biological activity of T cells in vitro. METHODS. cDNA fragment encoding OX40L was obtained from human mature dendritic cells by reverse transcriptionpolymerase chain reaction （RT-PCR）, and inserted into eukaryotic expression vector pcDNA3.1. The recombinant plasmid pcDNA3.1-OX40L was transfected into MDA-MB-435 cells, and verified by indirect immunophenotyping and RTPCR. The effect of OX40L-MDA-MB-435 cells to the biological activity of T cells was investigated by MTT, enzyme-linked immunosorbent assay （ELISA）, and direct immunophenotyping. RESULTS, OX40L-MDA-MB-435 cells were successfully constructed. These cells efficiently promoted the proliferation of T cells, enhanced the secretion of interleukin-2 （IL-2） （973.4 ng/ml at the 7th day） and interferon-γ（IFN-γ） （3 689.167 pg/ml at the 3rd day）, and down-regulated the expression of Fas on T cells [ （68.3±5.6）%, P〈0.05]. CONCLUSION. OX40L-MDA-MB-435 cells could activate T cells in vitro, promote T cell proliferation and cytokine secretion, and suppress T cell activation-induced cell death.