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目的:探讨从小鼠H22肝癌细胞中提纯的热休克蛋白gp96(HSPgp96)对小鼠腹腔巨噬细胞(PEMφ)NO释放的影响及其与细胞内外游离钙的关系。方法:(1)用亲和层析和离子交换层析等方法从小鼠H22肝癌细胞中获得纯化的HSPgp96。(2)用细胞内NO荧光探针DAF-FM-DA监测HSPgp96作用于小鼠PEMφ过程中,单个细胞NO信号的动态变化。(3)使用细胞膜和细胞内钙通道抑制剂及钙离子载体后,再观察HSPgp96作用后PEMφ内NO信号的变化。结果:小鼠PEMφ受Gp96刺激后显示NO的荧光强度立即上升,120 s时达峰值87.58%±18.17%,在830 s时降低接近正常水平。当分别抑制细胞外钙的内流和阻断细胞内钙库释放功能时,NO荧光值高峰明显降低。钙离子载体A23187可迅速诱导PEMφ内NO的合成,120 s时达峰值196.02%±28.68%,随后轻度下降后又逐渐升高,20分钟观察结束时增幅达峰值216.38%±38.39%。如同时阻断胞外钙的内流及胞内钙库释钙功能时,PEMφ受Gp96刺激后NO荧光值高峰几乎消失。结论:Gp96可促使小鼠PEMφ的NO生成快速增加,并与细胞内钙的浓度升高有关。
Objective: To investigate the effect of heat shock protein gp96 (HSPgp96) purified from mouse H22 hepatoma cells on NO release from mouse peritoneal macrophages (PEMφ) and its relationship with intracellular free calcium. Methods: (1) Purified HSPgp96 was obtained from mouse H22 hepatoma cells by affinity chromatography and ion exchange chromatography. (2) The intracellular NO fluorescent probe DAF-FM-DA was used to monitor the dynamic changes of NO signal in single cells during the process of HSPgp96 in mouse PEMφ. (3) After using cell membrane and intracellular calcium channel inhibitor and calcium ionophore, observe the change of NO signal in PEMφ after HSPgp96. Results: After stimulated by Gp96, the fluorescence intensity of NO in mouse PEMφ immediately increased, reached the peak of 87.58% ± 18.17% at 120 s and decreased to the normal level at 830 s. When inhibiting the influx of extracellular calcium and blocking the release of intracellular calcium stores, the peak of NO fluorescence decreased significantly. Calcium ionophore A23187 rapidly induced the NO synthesis in PEMφ, reaching a peak value of 196.02% ± 28.68% at 120 s, then slightly decreased and then increased gradually, and reached the peak value of 216.38% ± 38.39% at the end of 20 min. If at the same time blocking the influx of extracellular calcium and intracellular calcium release of calcium function, PEMφ by Gp96 peak NO fluorescence almost disappeared. Conclusion: Gp96 can promote NO production in PEMφ mice rapidly, which is related to the increase of intracellular calcium concentration.