吴通前,金筱茜,马岚,周萍萍,李静,袁锐,余芳
WU Tongqian,JIN Xiaoqian,MA Lan,ZHOU Pingping,LI Jing,YUAN Rui,YU Fang

• 1:贵州医科大学附属医院临床研究中心
• 2:贵州医科大学医学检验学院临床微生物及免疫学教研室
• 3:贵州医科大学附属医院临床检验中心
• 4:湖北医科大学附属东风医院临床检验中心

摘要(Abstract)：

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S100A4基因敲除(S100A4-/-) 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(1 500μg/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(r FI);以成熟WT和S100A4-/-BMMC为模型细胞,实验分为WT离子霉素(ION)处理组(1 500μg/L ION)、WT PBS对照组(ION等量体积PBS)、S100A4-/-ION处理组(1 500μg/L ION)及S100A4-/-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),S100A4-/-ION处理组成熟BMMC中β-hex、IL-5及IL-6明显高于S100A4-/-PBS对照组(P <0. 05),S100A4-/-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,S100A4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 S100 A4 in regulating bone marrow-derived mast cells( BMMCs) activation of mice. Methods: BMMCs were cultured and identified from mice of wild type( WT) C57 BL/6 or S100 A4 knockout( S100 A4-/-) . With mature WT BMMCs as the model cells,the experiment was divided into S100 A4 protein-treated group( 1 500 μg/L S100 A4 protein) and PBS control group( S100 A4 protein equal volume of PBS). The mature BMMCs were identified by toluidine blue staining the 1 st,2 nd,and 3 rdweeks after culturing respectively.Chemiluminescence was used to measure the optical density( OD) of β-aminohexosidase( β-hex) in mature BMMCs. The expression of S100 A4 protein and the relative fluorescence index( r FI) 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 S100 A4-/-BMMCs were used as model cells. The experiment was divided into WT ionomycin( ION)-treated group( 1 500 μg/L ION),WT PBS control group( ION equal volume of PBS),S100 A4-/-IONtreated group( 1 500 μg/L ION) and S100 A4-/-PBS control group( ION equal volume of PBS). Theβ-hex and r FI of IL-5,IL-6,IL-13,and TNF-α were detected by the above methods. Results:BMMCs were basically mature through 3 week 's culture so as to express S100 A4 protein. S100 A4 protein-treated mature WT BMMCs results showed that β-hex,IL-5,IL-6,IL-13,and TNF-α levels in the S100 A4 protein-treated group were higher than those in PBS control group( P < 0. 05). The results of ION-treated of mature WT and S100 A4-/-BMMCs showed that β-hex,IL-5,IL-6,IL-13,and TNF-α levels in WT ION-treated group were significantly higher than those in WT PBS control group( P < 0. 01),and β-hex,IL-5 and IL-6 in the S100 A4-/-ION-treated group were higher than those in the S100 A4-/-PBS control group( P < 0. 05),while the β-hex,IL-5,IL-6,IL-13,and TNF-α levels in the S100 A4-/-ION-treated group were lower than WT ION-treated group( P < 0. 05). Conclusion:S100 A4 protein can directly induce mature BMMCs activation,and its gene deletion inhibits mature BMMCs activation and the production of inflammatory factors.

关键词(KeyWords)： 小鼠,基因敲除;肥大细胞;钙结合蛋白;β-氨基己糖苷酶;炎性因子;肥大细胞活化
mice,knockout;bone marrow-derived mast cells(BMMCs);calcium-binding protein;β-hexosaminidase(β-hex);inflammatory cytokine;activation

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81760294)

作者(Author): 吴通前,金筱茜,马岚,周萍萍,李静,袁锐,余芳
WU Tongqian,JIN Xiaoqian,MA Lan,ZHOU Pingping,LI Jing,YUAN Rui,YU Fang

DOI: 10.19367/j.cnki.2096-8388.2020.12.001

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