树突状细胞MyD88介导热休克蛋白60的信号转导

Myeloid differentiation factor 88 of dendritic cells mediate the signal transduction of HSP60

目的观察热休克蛋白60(HSP60)刺激对树突状细胞(DC)活性的影响,探讨髓性分化因子88(MyD88)在介导HSP60信号转导中的作用。方法培养小鼠骨髓源性DC,分为对照组、HSP60组及RNA干扰组,对照组在正常条件下继续培养2d,HSP60组加入终质量浓度为10 mg/L的HSP60,RNA干扰组于加入MyD88 siRNA4h后给予10mg/L HSP60刺激,均继续培养2d。流式细胞术检测DC细胞表面CD11c、CD80、CD86及MHC-Ⅱ分子的变化,ELISA法检测DC分泌肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)和白细胞介素-12(IL-12)的浓度,免疫细胞化学检测DC MyD88、核因子-κB(NF-κB)的表达,Western blot检测DC MyD88的变化,混合淋巴细胞培养检测T细胞增殖能力。结果3组CD11c阳性率差异无统计学意义(P>0.05),分别为78.65%、82.40%及85.72%,HSP60组CD80、CD86及MHC-Ⅱ分子的阳性率分别为70.28%、76.49%及38.24%,较对照组(阳性率分别为28.42%、34.56%及16.28%)及RNA干扰组(阳性率分别为32.16%、40.47%及20.76%)显著增高(P<0.05);HSP60组TNF-α、IFN-γ及IL-12浓度显著高于对照组及RNA干扰组(P<0.05);HSP60组可见MyD88高表达及NF-κB核移位;HSP60组SI显著高于对照组及RNA干扰组(P<0.01)。结论HSP60通过MyD88依赖的途径活化DC,MyD88是HSP60信号转导中的重要调节分子。

Objective To explore the role of myeloid differentiation factor 88 （MyD88） in activating dendritic ceils in heat shock proteins60 （HSP60） signal transduction. Methods Mouse DCs were generated from bone marrow ceils and were divided into control group, HSWoO group and RNA interference group. Control group was cultured at normal conditions, and HSP60 group was added HSP60 of the final concentrations of 10 mg/L. MyD88 siRNA was added in RNA interference group, and 4 hours later, HS of the final concentrations of 10 mg/L was added. All groups were continued to culture for 2 days. Flow cytometry and mixed lymphocyte reaction （MLR） was used to detect the phenotype and functional properties of DCs. ELISA was used to detect the concentration of TNF-α, IFN-γ and IL-12 in the supernatant. Immtmochemistry was used to detect the concentration of MyD88 and NF-κB. Western blot was used to detect the concentration of MyD88. Results The expression of CD80, CD86 and MHC- Ⅱ in HSP60 group （ 70. 28%, 76.49%, 38. 24% ） were higher than that in control group （ 28. 42%, 34. 56%, 16.28% ） and RNA interference group （ 32. 16%, 40.47%, 20.76% ）. HSP60 stimulation increased TNF-α, IFN-γ and IL-12 concentration in the supernatant. HSP60 stimulation also increased the MyD88 in the cytoplasm and promoted the shift of NF-κB to karyon. MyD88 siRNA can inhibit these effects. Conclusion HSP60 activated DCs through MyD88- dependent pathway. MyD88 was critical modulatory molecule in HSWoO signal transduction.