唑来膦酸/白介素-2诱导的中国恒河猴外周血γδT细胞体外扩增

In vitro Expansion of Peripheral Blood γδT Cells from Chinese Rhesus Macaque Stimulated by Zoledronate and Interleukin-2

建立了一种培养及大量扩增中国恒河猴外周血γδT细胞的方法。采用Ficoll密度梯度离心法分离中国恒河猴(rhesus macaque)外周血单核细胞(peripheral blood mononuclear cells,PBMCs),用含5%猴自体血清、1 000 U·mL^-1白介素-2(IL-2)和不同浓度唑来膦酸(1,5,10μmol·L^-1)的OpTmizer细胞培养基进行培养,并使用流式细胞仪进行纯度及表型分析。结果显示:使用含不同浓度唑来膦酸培养基诱导第8 d时,1μmol·L^-1和5μmol·L^-1处理组的猴γδT细胞分别扩增至总细胞的91. 4%和93. 1%;5μmol·L^-1处理组表达CD56、CD69及HLA-DR蛋白的猴γδT细胞占总细胞的比例分别为75. 6%、75. 5%以及78. 0%;5μmol·L^-1处理组表达干扰素IFN-γ的猴γδT细胞比例为84. 8%。本研究通过优化培养基的组成成分,仅添加猴自体血清和IL-2即能有效诱导猴γδT细胞在体外的大量扩增,且培养得到的猴γδT细胞能够显著表达CD56、CD69、HLADR及IFN-γ,并具有较强的免疫激活和细胞杀伤作用。

In this study, we developed a technique for in vitro culture and large-scale expansion of γδT cells from rhesus macaques of China origin. Peripheral blood mononuclear cells(PBMCs) were isolated from rhesus macaques blood by Ficoll gradient centrifugation, cultured with OpTmizer culture medium containing 5% autologous serum, 1 000 U·mL^-1 interleukin-2(IL-2)and zoledronate acid(ZA) with indicated concentration(1, 5, 10 μmol·L^-1). The purity and typical surface phenotype of monkeyγδT cells were analyzed by flowcytometry. The results show that the purity of monkey γδT cells under 1 or 5 μmol·L^-1 ZA culture condition is 91.4% or 93.1%, respectively on the 8 th day. The expression of CD56, CD69 and HLA-DR on monkey γδT cells are 75.6%, 75.5% or 78.0% with 5 μmol·L^-1 ZA stimulation, respectively on the 8 th day. In addition, 84.8% of monkey γδT cells could release interferon-γ(IFN-γ) with 5 μmol·L^-1 ZA stimulation. To be concluded, this method does not need extra cytokines for growth factor and highly effective for rhesus macaques PBMC-derived γδT cells large-scale expansion in the OpTmizer culture with autologous monkey serum and IL-2. The cultured Rhesus macaques γδT cells are activated with the high expression of CD56, CD69, HLA-DR on the surface and show the effect of cytotoxicity with inducible IFN-γ releasing.