不同级别小鼠来源的骨髓细胞对制备优势DC2的影响

Study of the Effect of Bone Marrow Cells from Different Grade of Mice on the Preparation of Predominant DC2

目的探索不同级别小鼠来源的骨髓细胞对体外制备优势DC2细胞的影响。方法取SPF级和健康普通级C57BL/6小鼠股、胫骨骨髓细胞及脾细胞,设细胞因子诱导的实验组、低浓度GM-CSF对照组、无外源细胞因子对照组及脾细胞组。骨髓细胞采用GM-CSF,IL-4,Flt3L和SCF的不同细胞因子浓度和组合体外培养诱导。脾细胞培养2d去除大部分淋巴细胞,第3天采用流式细胞术4色荧光标记法分析细胞表型,骨髓细胞诱导的各组同样于第3天采用流式细胞术分析细胞表型,比较不同级别小鼠骨髓细胞所诱导的DC2细胞的表型和比例。结果SPF来源的小鼠骨髓细胞诱导的各组第3d在CD11c+的DC细胞群中CD8α+DC2所占比例及不成熟DC2的比例均高于普通级小鼠,显示普通鼠来源的骨髓细胞更倾向于产生成熟的DC,不易诱导产生高比例的不成熟的DC2。对两种动物脾细胞DC2的分析结果表明,普通鼠脾脏中CD8α-MHCⅡ+的成熟DC1比例为75.58%,远远超过SPF鼠的30.60%。结论小鼠的微生物级别可影响耐受性DC的诱导效果,采用SPF级小鼠来源的骨髓细胞体外制备优势DC2细胞效果优于普通级鼠,原因可能与普通鼠接触微生物较多,免疫系统更易于产生免疫应答而非免疫耐受有关。

Objective To explore the preparation of predominant DC2 from different grade of mice. Method Bone marrow （BM） cells of femur and tibia and spleen cells from SPF and conventional healthy C57BL/6 mice were isolated. Cell samples were divided into cytokine induced experimental group, low GM-CSF control and no exogenous cytokine control and spleen cell group. Different cytokine combination and concentration of GM-CSF, IL-4, Fh3L and SCF were used to induce predominant DC2 cells in vitro. The spleen cells were cultured for 2 days to remove majority of lymphoeytes, and their surface markers were analyzed by 4-color flow cytometry （FCM） on day 3. Cell surface markers of the groups of BM cells were also analyzed by FCM on day 3, to compare the surface markers and proportion of DC2 induced by BM cells of different grade of mice. Result On day 3, the proportions of CD8α^＋DC2 and immature DC2 among CDllc^＋ DC were all higher than that of conventional mice, showing the BM cells from conventional mice tended to generate more mature DC than high proportion of immature DC2. The spleen cell analysis showed that the proportion of mature CD8α^-MHC Ⅱ· DCI in the spleen of conventional mice was 75.58%, much higher than 30.60% of SPF mice. Conclusion The microorganism grade of mice could affect the induction Results of tolerogenic DC. BM cells from SPF mice were better than those from conventional mice when prepare predominant DC2 in vitro, and the reason may lie in the more microorganisms in conventional mice, which made their immune system easier to generate immune response rather than tolerance.