【摘要】 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。更多还原

【Abstract】 Objective:To investigate the effect of Heat Shock Protein gp96（HSPgp96）,purified from mouse H22 hepatocarcinoma cell（HCC）,on NO release of mouse peritoneal macrophage（PEMφ）and its relation with intracellular and extracellular free Ca²⁺.Methods:（1）Purified HSPgp96 was obtained by using affinity chromatography and ion exchange chromatography.（2）Labeled with NO fluorescent probe DAFFM-DA,dynamic change in NO signal in single cell was observed during the process when PEMφ was being stimulated by HSPgp96.（3）After using cell membrane and intracellular calcium channel inhibitor and using Calcium ionophore,NO signal change in PEMφ-HSPgp96 treated was observed again.Results:Stimulated with gp96,fluorescence intensity of NO in PEMφ rose immediately and reached its peaking value 87.58%±18.17% at 120s and dropped near to its normal intensity at 830s.Fluorescence peaking intensity of NO dropped obviously as soon as the inflow of extracelluar calcium and the release function of intracellular calcium library were inhibited respectively.The synthesis of NO in PEMφ was induced rapidly by Calcium ionophore A23187 and reached its fluorescence peaking intensity 196.02%±28.68% at 120s followed by a little drop and then a gradual rise.20minutes later when the observation came to the end,the fluorescence peaking intensity was 216.38%±38.39%.The fluorescence peaking intensity of NO almost disappeared when the inflow of extracelluar calcium and the release function of intracellular calcium library were inhibited simultaneously even though PEMφ is still stimulated by Gp96.Conclusion:The release of NO in mouse PEMφ is promoted by gp96 and related to[Ca²⁺]e and[Ca²⁺]i.更多还原