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目的初步探索Notch信号对树突状细胞(dendritic cell,DC)介导的抗寄生虫感染免疫反应的影响。方法体外培养骨髓来源的小鼠DC细胞,利用γ-分泌酶抑制剂N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine tbutyl ester,DAPT于不同时间段处理细胞,阻断DC的Notch信号通路,培养第7d时收集DC,用细粒棘球蚴重组抗原铁蛋白(ferritin protein of Echinococcus granulosus,Eg.ferritin)体外刺激DC,利用扫描电镜观察DC形态的改变,采用流式细胞术检测DC表面分子MHCⅡ、CD40及CD80/CD86的表达情况。结果 DC细胞数量随DAPT浓度的增高而降低;经不同浓度DAPT于不同时间段处理后,DC细胞中Notch1mRNA的表达受到抑制,以50μmol/L DAPT在第0d处理DC,对Notch1mRNA的抑制效果更显著(P<0.05);Eg.ferritin和LPS均能刺激DC细胞表面树突的生成及表面分子MHCⅡ、CD40及CD80/CD86的表达,且Eg.ferritin比LPS的刺激作用更显著(P<0.05),而加入DAPT后,DC细胞表面树突的生成减少,各表面分子的表达水平降低(P<0.05),并削弱了Eg.ferritin和LPS的刺激作用。结论Eg.ferritin能促进DC细胞的分化成熟,而阻断Notch信号通路会影响DC细胞的分化成熟,并降低DC细胞对Eg.ferritin的反应能力。其分子机制涉及Notch信号通路。
Objective To explore the effect of Notch signaling on dendritic cell (DC) -mediated anti-parasite immune response. Methods Bone marrow-derived mouse DC cells were cultured in vitro, and the cells were treated with γ-secretase inhibitor N- [N- (N- difluorophenacetyl) -L-alanyl] -S-phenylglycine tbutyl ester for different time periods. (DCs) were harvested. The DCs were harvested on the 7th day after culturing. DCs were stimulated with E. Ferritin (recombinant E.coli ferritin) in vitro and the morphological changes of DCs were observed by scanning electron microscopy Flow cytometry was used to detect the expression of MHCⅡ, CD40 and CD80 / CD86 on DCs. Results The number of DCs decreased with the increase of DAPT concentration. After treated with different concentrations of DAPT for different time periods, the expression of Notch1 mRNA in DCs was inhibited. When DCs were treated with 50μmol / L DAPT for 0d, the inhibitory effect of Notch1 mRNA was more significant (P <0.05). Eg.ferritin and LPS stimulated the generation of dendrites and the expression of surface molecules MHCⅡ, CD40 and CD80 / CD86 on DCs, and Eg.ferritin stimulated more significantly than LPS (P <0.05) , While the addition of DAPT reduced the generation of dendrites on DCs and decreased the expression of various surface molecules (P <0.05), and attenuated the stimulation of Eg.ferritin and LPS. Conclusion Eg.ferritin can promote the differentiation and maturation of DCs. Blocking Notch signaling pathway can affect the differentiation and maturation of DCs and decrease the ability of DCs to respond to Eg.ferritin. Its molecular mechanism involves the Notch signaling pathway.