钙结合蛋白S100A4调节小鼠骨髓源肥大细胞活化的作用

The Effect of Calcium Binding Protein S100A4 in Regulating Bone Marrow-Derived Mast Cell Activation of Mice

目的:探讨钙结合蛋白S100A4调节小鼠骨髓源肥大细胞(BMMC)活化的作用。方法:8~10周龄野生型(WT)和S 100 A 4基因敲除(S 100 A 4-/-)C57BL/6健康雄性小鼠各2只,取胫骨与股骨提取骨髓进行骨髓源肥大细胞(BMMC)培养并鉴定;以成熟WT BMMC为模型细胞,实验分为S100A4蛋白处理组(1500μg/L S100A4蛋白)和磷酸盐缓冲液(PBS)对照组(S100A4蛋白等量体积PBS),分别于培养第1、2及3周采用甲苯胺蓝染色鉴定小鼠成熟BMMC,采用化学发光法检测各组小鼠成熟BMMC中β-氨基己糖苷酶(β-hex)的吸光度(OD),采用流式细胞术检测各组小鼠成熟BMMC的S100A4蛋白表达和白细胞介素-5(IL-5)、白细胞介素-6(IL-6)、白细胞介素-13(IL-13)及肿瘤坏死因子-α(TNF-α)的荧光强度并计算相对荧光指数(rFI);以成熟WT和S 100 A 4-/-BMMC为模型细胞,实验分为WT离子霉素(ION)处理组(1500μg/L ION)、WT PBS对照组(ION等量体积PBS)、S 100 A 4-/-ION处理组(1500μg/L ION)及S 100 A 4-/-PBS对照组(ION等量体积PBS),采用化学发光法检测各组小鼠成熟BMMC中β-hex的吸光度(OD)。结果:BMMC培养至3周时基本成熟,并表达S100A4蛋白;S100A4蛋白处理组成熟BMMC中β-hex、IL-5、IL-6、IL-13及TNF-α水平高于PBS对照组(P<0.05或P<0.01);WT ION处理组成熟BMMCβ-hex、IL-5、IL-6、IL-13及TNF-α水平高于WT PBS对照组(P<0.01),S 100 A 4-/-ION处理组成熟BMMC中β-hex、IL-5及IL-6明显高于S 100 A 4-/-PBS对照组(P<0.05),S 100 A 4-/-ION处理组成熟BMMC中β-hex、IL-5、IL-6、IL-13及TNF-α水平均低于WT ION处理组(P<0.05)。结论:S100A4蛋白能够直接诱导成熟BMMC活化,S 100 A 4基因的缺失可抑制成熟BMMC活化及炎性因子的产生。

Objective:To investigate the effect of calcium-binding protein S100A4 in regulating bone marrow-derived mast cells(BMMCs)activation of mice.Methods:BMMCs were cultured and identified from mice of wild type(WT)C57BL/6 or S100A4 knockout(S100A4-/-).With mature WT BMMCs as the model cells,the experiment was divided into S100A4 protein-treated group(1500μg/L S100A4 protein)and PBS control group(S100A4 protein equal volume of PBS).The mature BMMCs were identified by toluidine blue staining the 1 st,2 nd,and 3 rd weeks after culturing respectively.Chemiluminescence was used to measure the optical density(OD)ofβ-aminohexosidase(β-hex)in mature BMMCs.The expression of S100A4 protein and the relative fluorescence index(rFI)of interleukin-5(IL-5)and interleukin-6(IL-6),interleukin-13(IL-13)and tumor necrosis factor-α(TNF-α)were detected by flow cytometry in each group.In addition,mature WT and S100A4-/-BMMCs were used as model cells.The experiment was divided into WT ionomycin(ION)-treated group(1500μg/L ION),WT PBS control group(ION equal volume of PBS),S100A4-/-ION-treated group(1500μg/L ION)and S100A4-/-PBS control group(ION equal volume of PBS).Theβ-hex and rFI of IL-5,IL-6,IL-13,and TNF-αwere detected by the above methods.Results:BMMCs were basically mature through 3 week's culture so as to express S100A4 protein.S100A4 protein-treated mature WT BMMCs results showed thatβ-hex,IL-5,IL-6,IL-13,and TNF-αlevels in the S100A4 protein-treated group were higher than those in PBS control group(P<0.05).The results of ION-treated of mature WT and S100A4-/-BMMCs showed thatβ-hex,IL-5,IL-6,IL-13,and TNF-αlevels in WT ION-treated group were significantly higher than those in WT PBS control group(P<0.01),andβ-hex,IL-5 and IL-6 in the S100A4-/-ION-treated group were higher than those in the S100A4-/-PBS control group(P<0.05),while theβ-hex,IL-5,IL-6,IL-13,and TNF-αlevels in the S100A4-/-ION-treated group were lower than WT ION-treated group(P<0.05).Conclusion:S100A4 protein can directly induce mature BMMCs activation,and its gene deletion inhibits mature BMMCs activation and the production of inflammatory factors.