小鼠骨髓干细胞体外诱导成熟的树突状细胞对T细胞增殖的影响

目的：研究小鼠骨髓干细胞体外诱导分化的树突状细胞对T细胞增殖的影响，为进一步研究树突状细胞的功能及临床肿瘤的治疗提供有效的实验方法。 方法：应用IL-4和GM-CSF培养小鼠骨髓细胞5~7 d，光镜下观察细胞形态学变化；培养至第5~6天，加入黑色素瘤（B16）冻融抗原继续培养1~2 d，流式细胞仪检测细胞表面分子CD83、CD86，并与同种异体T细胞混合培养72 h，终止培养前16 h加入³H-TdR（18.5 kBq/孔），γ-液体闪烁仪测定cpm值。 结果：用IL-4和GM-CSF培养3 d可见细胞形态发生改变，细胞形状不规则，培养5~6 d时，有刺状突起、拉长，为典型树突状细胞形态学特征；抗原装载后流式细胞仪检测CD83、CD86表达，IL-4+GM-CSF+TNF-α组（61.68%、71.25%）及IL-4+GM-CSF+冻融抗原组（63.11%、76.88%）明显高于对照组（2.41%、3.88%）及单纯IL-4+GM-CSF培养组（21.86%、28.69%）（P<0.001），IL-4+GM-CSF+TNF-α组与IL-4+GM-CSF+冻融抗原组比较，CD83和CD86表达差异无显著性；液闪仪检测结果显示，IL-4+GM-CSF+冻融抗原组刺激T细胞增殖的能力明显强于其他组，且差异具有显著性（P<0.001, P<0.05）。 结论：小鼠骨髓细胞体外培养成功地诱导扩增出足量树突状细胞，经肿瘤抗原刺激后绝大部分成熟，并能够刺激同种异体T细胞大量增殖，参与免疫应答。

Effects of mature dendritic cells derived from mouse bone marrow on T cells proliferation in vitro

Objective To study the effects of mature dendritic cells (DCs) derived from mouse bone marrow on T cell proliferation in vitro , and provide techinological methods for tumor therapy.Methods With IL 4 and GM CSF, the cells from mouse bone marrow were cultured for 5 7 days. The morphological changes were observed under light microscope on 3rd and 5th day after culture; immature DCs on 5th or 6th day after culture were loaded with tumor antigen for 1 2 days; CD83 and CD86 were tested by flowcytometric analysis; T cell stimulatory activity in DCs derived from mouse bone marrow and loaded with tumor antigen was analysed by γ scintillomete. Results Morphological changes were found and dendritic protrusion appeared on 3rd day after culture; dendritic protrusion prolonged on 5th day after culture; flow cytometric analysis showed that expressions of CD83 and CD86 of IL 4+GM CSF+TNFα group (61 68% and 71 25%) and IL 4+GM CSF+tumor antigen group (63 1% and 76 88%) had significant differences compared with control group (2 41% and 3 88%) and IL 4+GM CSF group (21 86% and 28 69%) ( P <0 001), and IL 4+GM CSF+tumor antigen group had also significant difference compared with IL 4+GM CSF+TNFα group( P <0 01); proliferation of T cells in IL 4+GM CSF+tumor antigen group strongly enhanced compared with other groups ( P <0 001, P <0 05). Conclusion Sufficient DCs generated from bone marrow cells are induced in vitro and strongly stimulate proliferation of T cells after tumor antigen are loaded.