RNA干扰抑制IL-23表达对感染后内脏高敏感小鼠肠黏膜固有层DC活化Th17细胞功能的影响

Effect of IL-23 RNA Interference on Activation of Th17 Cells Induced by Dendritic Cells from Intestinal Lamina Propria in Postinfectious Visceral Hypersensitivity Mice

背景:前期研究发现感染后内脏高敏感小鼠肠黏膜固有层树突细胞(DC)诱导活化Th17细胞与肠道感染消退后肠黏膜免疫系统的持续激活有关。推测DC可能系通过分泌白细胞介素-23(IL-23)活化Th17细胞。目的:应用RNA干扰技术抑制DC分泌IL-23,探讨感染后内脏高敏感小鼠肠黏膜固有层DC活化Th17细胞的机制。方法:建立旋毛虫感染后内脏高敏感小鼠模型,以免疫磁珠分选肠黏膜固有层DC和脾脏CD4+T细胞。构建、鉴定小鼠IL-23小发夹RNA(shRNA)干扰质粒,以脂质体法转染DC(A组)以抑制IL-23表达,同时设置转染空脂质体的DC(B组)和转染无关序列shRNA干扰质粒的DC(C组)作为对照。各组DC与CD4+T细胞共培养120 h,以单独培养的CD4+T细胞(D组)作为对照。以ELISA方法检测DC转染前后培养上清液中的IL-23水平,以及DC与CD4+T细胞共培养上清液和CD4+T细胞单独培养上清液中的IL-17水平。结果:A组DC培养上清液中的IL-23水平较转染前显著降低(P<0.05),B、C两组转染前后IL-23水平无明显变化。A、B、C组DC与CD4+T细胞共培养上清液中的IL-17水平均较D组显著增高(P<0.05),其中A组显著低于B、C两组(P<0.05),B、C组间差异无统计学意义。结论:感染后内脏高敏感小鼠肠黏膜固有层DC可能通过分泌IL-23活化Th17细胞,参与维持肠道感染消退后肠黏膜免疫系统的持续激活。

Background: Previous study indicated that in postinfectious visceral hypersensitivity mice the dendritic cells(DCs) from intestinal lamina propria could induce the activation of Th17 cells and associated with the sustained activation of intestinal mucosal immune system after recovery from intestinal infection.It was presumed that the effect of DCs on activation of Th17 cells might be mediated by interleukin-23(IL-23).Aims: To investigate the mechanism of activation of Th17 cells by DCs from intestinal lamina propria in postinfectious visceral hypersensitivity mice through inhibiting the IL-23 secretion in DCs by RNA interference technique.Methods: Postinfectious visceral hypersensitivity was induced by Trichinella spiralis infection in mice.DCs from intestinal lamina propria and splenic CD4+ T cells were isolated and purified by MACS MicroBeads.Mouse IL-23 small hairpin RNA(shRNA) plasmid was constructed,identified and transfected into DCs(group A) by Lipofectamine TM 2000 to suppress the IL-23 expression.DCs transfected with liposome only(group B) and with shRNA plasmid irrespective to mouse IL-23 gene(group C) were served as controls.Then DCs in group A,B and C were cocultured with CD4+ T cells for 120 hours in vitro,and CD4+ T cells cultured alone(group D) were served as controls.ELISA was used to determine the IL-23 level in supernatants of DCs before and after transfection,as well as the IL-17 level in supernatants of cocultured DCs and CD4+ T cells and CD4+ T cells cultured alone.Results: IL-23 level in supernatant of DCs decreased significantly after transfection(P<0.05) in group A,while that in group B and group C did not change obviously.IL-17 levels in supernatants of cocultured DCs and CD4+ T cells in group A,B and C were all significantly higher than that in group D(P<0.05),among them group A was lower than group B and group C(P< 0.05),and no significant difference was found between group B and group C.Conclusions: In postinfectious visceral hypersensitivity mice,DCs from intestinal lamina propria might secrete IL-23 to activate Th17 cells and thus maintaining the activation of intestinal mucosal immune system after recovery from intestinal infection.