小鼠骨髓成熟与未成熟树突状细胞的生物免疫学功能比较研究

The studies of comparison in bio-inununology function of bone marrow immature and mature dendritic cells in mice

目的比较成熟和未成熟状态小鼠骨髓树突状细胞（Dc）的生物免疫学功能的差异。方法粒细胞一巨噬细胞刺激因子（GM—CSF）和白细胞介素（IL）-4联合诱导培养小鼠骨髓未成熟Dc，经LPS刺激制备成熟Dc。分别收集培养第6天的两组Dc并用小鼠CD11c免疫磁珠纯化。电镜下观察成熟和未成熟DC的细胞形态。经流式细胞术检测两组细胞表面分子（MHC—Ⅱ、CD86和CD40）的表达水平。另将收集的DC分别与异基因小鼠脾脏T细胞共培养72h，四唑蓝（MTF）比色法检测T细胞增殖程度。取细胞上清液，液相芯片法检测Th1／Th2细胞因子表达水平。结果电镜下可见两组Dc在形态上存在很大差异。同成熟DC相比，未成熟DC细胞表面较光滑，细胞内存在大量的吞噬小泡。流式细胞术分析显示未成熟DC细胞表面3类分子（MHC—Ⅱ、CD86和CD40）的表达水平均明显低于成熟DC组。混合淋巴细胞反应（MLR）显示．未成熟DC刺激T细胞增殖显著低于相应反应比例的成熟DC，差异有统计学意义（P〈0．05）。细胞因子表达谱表明未成熟DC组分泌Th1细胞因子（IL-2和IFN-γ）较成熟DC组显著下降（P〈0．01）．Th2细胞因子相对占主导。结论未成熟DC倾向于诱导T细胞免疫耐受，有效抑制DC的成熟并维持DC的未成熟状态将有望用于治疗类风湿关节炎等由Th1细胞介导的自身免疫性疾病。

Objective To compare the immunological function differences between marine bone marrow mature and immature dendritic ceils （DCs）. Methods Murine bone marrow ceils were cultured for 6 days in RPMI-1640 medium with GM-CSF and IL-4. Immature DCs were obtained. Immature DCs stimulated by LPS, the mature DCs were obtained. In day 6, the immature and mature DCs were harvested and purified by mouse CDllc microbeads, respectively. Cells＇ morphous characteristics were observed through the electronic microscope. The expression of surface molecules in DC, including MHC-Ⅱ, CD86 and CD40, was examined by flow cytometry. The two groups DCs were cocuhured with mouse spleen T lymphocytes for 72h, respectively. T cells proliferation capacities were detected by MTT approach. The culture supernatant was collected and Th cytokine detected by liquid chip approach. Results Compared with mature DCs, a relatively smooth surface and a large number of phagocytic vesicles were observed under electronic microscope in immature DCs. Flow cytometric analysis indicated three class molecules in immature DCs, namely, MHC-Ⅱ, CD86 and CD40, all had a lower expression level than those in mature DCs. MLR indicated T cells proliferation capacities in immature DCs were significantly lower than those in mature DCs （P〈0.05）. In contrast to mature DCs, cytokine expression profile analysis indicated the expression levels of Thl cytokine （IL-2 and IFN-γ） in immature DCs was significantly decreased （P〈0.01）, Th2 cells were dominant relatively. Conclusions Immature DCs tend to induce T cells immunological tolerance. To suppress the mature state of DCs effectively and keep DCs in immature state can be used to treat autoimmune diseases mediated by Thl effectively such as rheuma- toid arthritis.