大鼠骨髓来源树突状细胞的分离与免疫学特征

Isolation,culture,and immunological character of dendritic cells derived from rat bone marrow

目的:探讨大鼠骨髓来源树突状细胞(DC)的体外分离培养方法,为DC的免疫学研究及临床应用提供技术参考。方法:取大鼠四肢骨髓细胞悬液,经密度梯度离心得到大鼠骨髓单个核细胞(BMMNC);应用大鼠重组粒细胞-巨噬细胞集落刺激因子(rrGM-CSF)、大鼠重组白细胞介素-4(rrIL-4)及大鼠重组肿瘤坏死因子-α(rrTNF-α)进行体外培养;肿瘤抗原(TAA)/DC组于诱导分化的第3天加入大鼠卵巢癌细胞株NuTu-19的冻融抗原,获得负载TAA的成熟DC;DC组为对照组,未加入NuTu-19。对两组进行形态学观察、流式细胞学表型鉴定及体外淋巴细胞增殖反应测定。结果:大鼠骨髓来源的BMMNC在相关细胞因子的作用下可以培养出成熟的DC,TAA/DC组高表达表面抗原,其OX62、CD86和MHC-Ⅱ表达高于对照组(P<0.01);在效靶比(SC∶RC)为1∶3、1∶10、1∶30和1∶100时,淋巴细胞刺激指数(SI)TAA/DC组均高于对照组(P<0.01)。结论:成功地建立了大鼠骨髓DC的培养方法,大鼠骨髓来源的BMMNC可以培养出成熟的DC。

Objective To explore the methods for isolating and culturing dendritic cells derived from rat bone marrow in vitro, and provide reference for immunological research and clinical application. Methods The bone marrow mononuclear cells （BMMNC） were obtained from bone marrow of Fischer344 rat＇ s limbs through density gradient centrifugation; and the mature DC was gained by t3MMNC cultured in medium with recombinant rat granulocyte-macrophage colony stimulating factor （rrGM-CSF）, recombinant rat interleukin-4 （rrIL-4） and recombinant rat tumor necrosis factor -α（rrTNF-α）. In group 1, the rat＇ s ovarian cancer cell line NuTu-19 freeze- melt antigens were added on the third day and the tumor cells lysates pulsed DC was acquired; in group 2, as control, nothing was added. Then the shape of DC was observed, the cell-surface phenotype and the proliferation rate were detected. Results The mature DC could be induced by BMMNC co-culturing with relative cytokines, and the expressions of cell-surface phenotypes such as OX62, CD86 and MHC- Ⅱ in group 1 were higther than those in control group （P〈0.01）. When the ratios of stimulating cells （SC） to reaction cells （RC） were 1 ： 3, 1 ： 10, 1 ： 30 and 1 ： 100, the stimulating indexes （SI） in group 1 were higher than those in control group （P〈0.01）. Conclusion The culture method of DC from bone marrow of Fischer344 rats is established, which means the mature DC can be acquired from BMMNC.