γδT细胞体外抑制未成熟树突状细胞向破骨细胞转分化

γδ T cells inhibit transdifferentiation of immature dendritic cells into osteoclasts in vitro

目的:探讨γδT细胞对人外周血来源未成熟树突状细胞(Immature dendritic cells,im DC)转分化为破骨细胞(Osteoclasts,OC)过程的影响。方法:(1)采用唑来膦酸(Zoledronate,Zol)及重组人白细胞介素2(Interleukin-2,IL-2)体外激活扩增外周血γδT细胞,并采用粒细胞-巨噬细胞集落刺激因子(Granulocyte macrophage colony-stimulating factor,GM-CSF)及重组人白细胞介素4(Interleukin-4,IL-4)诱导PBMNC(Peripheral blood mononuclear cell,PBMNC)分化为im DC,后者在细胞核因子кB受体活化因子配体(Receptor activator of nuclear factorкB ligand,RANKL)和巨噬细胞集落刺激因子(Macrophage colonystimulating factor,M-CSF)作用下继续培养转分化为OC,采用流式细胞术分别检测γδT细胞扩增前后纯度及PBMNC经由imDC转分化为OC过程中的细胞表型变化;(2)免疫磁珠分选法收集培养至第7天的γδT细胞,建立γδT细胞及im DC共培养(γδT细胞数量∶im DC数量=10∶1)体系,采用耐酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRAP)染色和甲苯胺蓝染色分析γδT细胞对im DC转分化为OC过程的影响;并收集各培养组培养上清,采用酶联免疫吸附实验检测肿瘤坏死因子(Tumor necrosis factor-alpha,TNF-α)浓度。结果:(1)Zol及重组人IL-2可在体外培养条件下扩增MM患者外周血γδT细胞,扩增产率与健康志愿者无统计学差异(68.87%±20.94%vs 69.33%±16.84%,P>0.05);(2)外周血诱导分化的im DC在RANKL和M-CSF作用下可转分化为OC;(3)γδT细胞与im DC间接接触共培养条件下,TRAP+多核细胞数和牙本质骨吸收面积均显著低于对照组(分别为5.67±0.58个vs 28.33±2.08个;4.97%±4.3%vs 28.47%±12.8%);(4)在γδT细胞与im DC间接共培养条件下,TNF-α分泌水平显著增高。结论:激活的γδT细胞在体外培养条件下可能抑制im DC转分化为OC过程,γδT细胞免疫治疗有望成为骨髓瘤骨病治疗的新手段。

Objective： To explore the role of γδ T cells in the transdifferentiation of immature dendritic cells（ im DC） into osteoclasts（ OC）. Methods：（ 1） Peripheral blood mononuclear cells（ PBMNC） were cultured with zoledronate（ Zol） and recombinant human interleukin-2（ IL-2）,and PBMNC from healthy volunteers were cultured with granulocyte macrophage colony-stimulating factor（ GM-CSF） and recombinant human interleukin-4（ IL-4） to differentiate into im DC,which were then cultured with receptor activator nuclear factor к B ligand（ RANKL） and macrophage colony-stimulating factor（ M-CSF） to differentiate into OC. The purity of γδ T cells,and phenotype changing of OC transdifferentiated from im DC were investigated by flow cytometry.（ 2） Co-culture system was established using millicell inserts. γδ T cells isolated with immune magnetic bead were placed in the upper compartment and im DC in the lower compartment in the ratio of 10∶ 1. To explore the role of γδ T cells during differentiation of im DC into OC,tartrate resistant acid phosphatase（ TRAP） staining and bone resorption observation staining were used. Tumor necrosis factor-alpha（ TNF-α） of supernatant liquid from different cultures was measured using ELISA（ Enzyme linked immunosorbent assay） kit. Results：（ 1） γδ T cells can be expanded from PBMNC of MM patients,and the production capacity was similar to that of healthy volunteers（ 68. 87% ± 20. 94% vs69. 33% ± 16. 84%,P〉0. 05）.（ 2） OC could be transdifferentiated from im DC when cultured with RANKL and M-CSF.（ 3） The number of TRAP ＋ multinuclear cell and the absorption area of dentine were significantly lower in the group of im DC indirectly co-cultured with γδ T cells than in the group of control im DC（ 5. 67 ± 0. 58 vs 28. 33 ± 2. 08,4. 97% ± 4. 3% vs 28. 47% ± 12. 8%,respectively）.（ 4） Under the circumstance of γδ T cell-im DC indirect coculture,TNF-α got significantly higher. Conclusion： γδ T cells might inhibit the transdifferentiation of im DC into OC. γδ T cells-based immunotherapy is expected to be a new treatment for myeloma bone disease.