热休克蛋白gp96对巨噬细胞功能的影响

背景与目的:热休克蛋白(heatshockprotein,HSP)gp96在抗原递呈和肿瘤特异性免疫治疗中具有重要作用,但其对巨噬细胞的作用机制尚待深入研究。本研究旨在探讨HSPgp96对小鼠腹腔巨噬细胞(peritonealelicitedmacrophages,PEMφ)功能的影响。方法:应用激光扫描共聚焦显微术,结合特异性荧光探针Fluo3-AM、DAF-FM-DA和H2DCF-DA,检测在HSPgp96作用下,腹腔巨噬细胞内钙离子(Ca2 )、一氧化氮(NO)和活性氧(ROS)的动态变化,并用荧光标记技术检测小鼠腹腔巨噬细胞MHCⅡ及CD86的表达强度和分布的变化。结果:小鼠腹腔巨噬细胞受HSPgp96刺激后,细胞内Ca2 、NO和ROS的生成量都快速升高,分别在刺激后30s、80s和580s时达峰值,增幅分别为(161.06±70.99)%、(77.58±31.17)%和(647.28±185.70)%。小鼠腹腔巨噬细胞受HSPgp96刺激后MHCⅡ阳性表达率:对照组为(40.9±3.5)%,HSPgp96(60mg/L)组为(61.8±5.1)%;CD86阳性表达率:对照组为(23.1±3.1)%,HSPgp96(60mg/L)组为(54.9±2.0)%。结论:HSPgp96在体外可使小鼠腹腔巨噬细胞内Ca2 、NO及ROS生成量明显增加;使抗原递呈分子MHCⅡ、CD86的表达明显上调。

Effects of heat shock protein gp96 on function of macrophages from mouse

BACKGROUND & OBJECTIVE: Heat shock protein gp96 (HSPgp96) plays an important role in antigen presenting and specific antitumor immunotherapy, but its effects on macrophages need further investigation. This study was to investigate the effects of HSPgp96 derived from H22 tumor cells on mouse peritoneal elicited macrophages (PEMphi) by detecting some factors that are related to the function of the macrophages. METHODS: After macrophages were stimulated by HSPgp96, dynamic changes of intracellular free calcium (Ca2+), nitric oxide (NO) and reactive oxygen species (ROS) in the macrophages were measured by the laser scanning confocal microscopy (LSCM) with sensitive fluorescent dye Fluo-3/AM, DAF-FM-DA, and H2DCF-DA. The color immunofluorescence assay was used to observe the expression intensity and distribution of MHC II and CD86 in the macrophages. RESULTS: The production of Ca2+, NO and ROS increased rapidly in the macrophages after stimulation of HSPgp96. Their concentration peaks appeared at 30 s, 80 s, and 580 s after stimulation with the increase scopes of (161.06+/-70.99)%, (77.58+/-31.17)%, and (647.28+/-185.70)%, respectively. The positive rates of MHC II and CD86 were significantly higher in 60 mg/L HSPgp96-stimulated macrophages than in control macrophages [(61.8+/-5.1)% vs. (40.9+/-3.5)%, P<0.01; (54.9+/-2.0)% vs.(23.1+/-3.1)%, P<0.01]. CONCLUSION: The in vitro stimulation of HSPgp96 may enhance the production of Ca2+, NO and ROS in mouse PEMphi, and up-regulate the expression of MHC II and CD86.