阻断p38丝裂原激活蛋白激酶通路抑制脾脏树突状细胞介导的OT-Ⅱ细胞增殖

Blocking p38MAPK pathway inhibits the proliferation of OT-Ⅱ cells mediated by splenic dendritic cells

目的探讨阻断p38丝裂原活化蛋白激酶（p38MAPK）信号通路对脾脏树突状细胞（DC）介导的CD4＋-鸡卵清蛋白转基因小鼠T（OT-Ⅱ）细胞增殖的影响。方法应用CD11c＋免疫磁珠从C57BL/6小鼠脾脏中分选DC。应用CD4＋分离试剂盒从OT-Ⅱ转基因小鼠脾脏中分选出OT-Ⅱ细胞。p38MAPK抑制剂SB203580处理DC后,脂多糖（LPS）刺激。流式细胞术检测DC表面共刺激分子（CD80、CD86）、MHCⅡ分子的表达,及DC提呈组织相容性复合体Ⅱ类分子Eα链第52~68位抗原肽（Eα52-68）的能力。ELISA检测细胞培养上清中肿瘤坏死因子α（TNF-α）、白细胞介素1α（IL-1α）、IL-6、转化生长因子β（TGF-β）的水平。DC经OVA323-339多肽处理后,与OT-Ⅱ细胞共培养,采用流式细胞术检测OT-Ⅱ细胞增殖。结果分选后获得较高纯度的DC和OT-Ⅱ细胞（〉90%）。SB203580降低DC表面CD80、CD86、MHCⅡ的表达,抑制其抗原提呈功能,同时TNF-α、IL-1α、IL-6表达下调,TGF-β表达上调,并且抑制DC介导的OT-Ⅱ细胞增殖。结论 SB203580阻断p38MAPK通路,可能通过调节DC表面CD80、CD86、MHCⅡ的表达及其抗原提呈功能和细胞因子的合成,从而抑制DC介导的OT-Ⅱ细胞增殖。

Objective To investigate the effect of blocking p38 mitogen-activated protein kinase（ p38MAPK） pathway on the proliferation of OT-Ⅱ cells mediated by splenic dendritic cells（ DCs）. Methods Splenic DCs of C57 BL /6 mice were purified with anti-CD11 c immunomagnetic beads,and OT-Ⅱ cells were isolated from the splenic of CD4＋-ovalbumin transgenic mice（ OT-Ⅱ transgenic mice） by mouse CD4 T cell isolation kits. After being pretreated with SB203580,an inhibitor of p38 MAPK,DCs were stimulated with lipopolysaccharides（ LPS）. Then the expression levels of co-stimulatory molecules（ CD80,CD86） and MHC Ⅱ in DCs,and antigen-presenting ability of DCs treated with the 52-68 fragment of the E alpha-chain of I-E class Ⅱ molecules（ Eα52-68 peptide） were detected by flow cytometry. The protein levels of tumor necrosis factor α（ TNF-α）,interleukin 1α（ IL-1α）,interleukin 6（ IL-6） and transforming growth factor β（ TGF-β） in the culture supernatants were measured by ELISA. The proliferation of OT-Ⅱcel s which were co-cultured with OVA323-339-treated DCs was analyzed by flow cytometry. Results The purity of both DCs and OT-Ⅱ cells reached over 90% after isolation. SB203580 downregulated the expressions of CD80,CD86 and MHC Ⅱ,and suppressed the antigen-presenting ability of DCs. The expressions of TNF-α,IL-1α and IL-6 were downregulated,while the expression of TGF-β was raised. Finally,SB203580 inhibited DCs-mediated proliferation of OT-Ⅱ cells. Conclusion Blocking p38 MAPK pathway with SB203580 could inhibit DCs-mediated proliferation of OT-Ⅱ cells,which might be involved in modulating the expressions of CD80,CD86 and MHCⅡ,the antigen-presenting ability,as well as the expressions of pro-inflammatory and anti-inflammatory cytokines.