尘螨抗原对P815肥大细胞维生素D受体和炎症介质分泌的影响及1,25(OH)_2D_3的免疫调节作用

Effects of 1,25(OH)_2D_3 on expression of vitamin D receptor and release of inflammatory mediators in P815 mast cells induced by Dermatophagoides pteronyssinus antigen

目的探讨1,25(OH)_2D_3对尘螨抗原(Der p)直接作用的P815肥大细胞维生素D受体(VDR)表达及IL-4、IL-10分泌的影响及其免疫调节作用。方法用不同浓度Der p、1,25(OH)_2D_3单独或联合作用P815细胞,收集细胞和上清液。采用Western blot、RT-PCR法分别检测P815肥大细胞VDR蛋白及m RNA表达,ELISA法检测细胞悬液上清液中IL-4、IL-10浓度。结果 P815肥大细胞表达VDR。不同浓度Der p对P815肥大细胞VDR表达有调节作用,作用12、24h组VDR m RNA表达呈明显浓度依赖性下调(P<0.05)。不同浓度Der p作用P815肥大细胞36h后,VDR蛋白表达呈浓度依赖性下调。高浓度1,25(OH)_2D_3单独作用对VDR表达有上调作用(P<0.05)。不同浓度1,25(OH)_2D_3单独作用P815肥大细胞可促进IL-10的释放(P<0.05),但对P815肥大细胞IL-4分泌无明显影响(P>0.05)。1,25(OH)_2D_3可抑制Der p引起的IL-4分泌(P<0.05),且1,25(OH)_2D_3浓度越高,抑制作用越强。Der p、1,25(OH)_2D_3联合处理P815肥大细胞36h后,上清液中IL-10浓度无明显变化(P>0.05)。结论 1,25(OH)_2D_3可通过上调肥大细胞上VDR表达,促进IL-10分泌,从而发挥抗炎作用。1,25(OH)_2D_3对Der p引起的肥大细胞免疫反应的干预作用可能是通过抑制IL-4的合成和分泌,从而抑制Th2型免疫应答的。

Objective To investigate the effect of 1,25(OH)2D3 on the expression of vitamin D receptor(VDR) and the release of interleukin-4(IL-4), interleukin-10(IL-10) in P815 mast cells induced by Dermatophagoides pteronyssinus(Der p) antigen. Methods After P815 mast cells were challenged with different concentrations of Der p, 1,25(OH)2D3 alone or combined. The supernatant of challenged cells were collected, the contents of IL-4, IL-10 in supernatant were measured by enzyme-linked immunosorbent assay (ELISA) and the expression of VDR mRNA and protein was detected by real-time RT-PCR and Western blot. Results The expression of VDR mRNA in P815 mast cells was significantly down-regulated after challenged with Der p at different concentrations for 12h and 24h in a concentration-dependent manner(P<0.05). After 36h of challenge with different concentrations of Der p, VDR protein expression in P815 mast cells was down-regulated in a concentration-dependent manner. High concentration of 1,25(OH)2D3 up-regulated VDR expression in P815 cells (P<0.05). 1,25(OH)2D3 at different concentra- tions stimulated the release of IL-10 in P815 mast cells (P<0.05), but no effect on IL-4 release(P >0.05). 1,25(OH)2D3 inhibited the secretion of IL-4 induced by Der p(P<0.05) in a dose-dependent manner. After treatment with Der p and 1,25(OH)2D3 for 36h, there was no significant change in the concentration of IL-10 in the supernatant of P815 mast cells(P >0.05). Conclusion 1,25(OH)2D3 can up-regulate the expression of VDR and increase the secretion of IL-10 in mast cells to exert its anti-inflammatory effect and to suppress the Th2-type responses induced by Der p.