破骨细胞分化过程中丙酮酸的作用  

Role of pyruvic acid in osteoclast differentiation

作　　者：刘官娟 夏千禧 宋娜 霍花 洪伟[2] 廖健 Liu Guanjuan;Xia Qianxi;Song Na;Huo Hua;Hong Wei;Liao Jian(School of Stomatology/Stomatological Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Key Laboratory of Molecular Biology,Guizhou Medical University,Guiyang 550004,Guizhou Province,China)

机构地区：[1]贵州医科大学口腔医学院/附属口腔医院,贵州省贵阳市550004 [2]贵州医科大学分子生物学重点实验室,贵州省贵阳市550004

出　　处：《中国组织工程研究》2023年第31期5015-5021,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(82060207),项目负责人:廖健;贵州省卫生健康委科学技术基金(gzwkj2022-165),项目负责人:廖健;大学生创新创业训练项目(202110660022),项目负责人:夏千禧,指导老师:廖健。

摘　　要：背景:糖酵解在破骨细胞分化过程中起关键作用,成熟破骨细胞骨吸收主要依赖于糖酵解。丙酮酸作为糖酵解的终末产物,在三大营养物质的代谢中起重要的枢纽作用。目的:探讨丙酮酸对破骨细胞分化的影响。方法:采用CCK-8法检测不同浓度(0.1,1,10,20,30,50 mmol/L)丙酮酸对RAW264.7细胞活性的影响,筛选出安全浓度丙酮酸。将RAW264.7细胞分组干预:空白对照组加入完全培养基(含体积分数10%胎牛血清、1%双抗的α-MEM培养基),对照组加入含核因子κB受体活化因子配体的完全培养基,实验组加入核因子κB受体活化因子配体与不同浓度丙酮酸的完全培养基,分别进行抗酒石酸酸性磷酸酶染色、细胞骨架纤维性肌动蛋白染色、RT-qPCR检测、Western blot检测与骨吸收陷窝实验。结果与结论:①CCK-8检测显示,丙酮酸对RAW264.7细胞的最大半数抑制浓度为8.923 mmol/L,后续实验选择0.1,1,5 mmol/L为安全浓度;②抗酒石酸酸性磷酸酶染色显示,1 mmol/L丙酮酸促进RAW264.7细胞向破骨细胞分化;③细胞骨架纤维性肌动蛋白染色显示,1 mmol/L丙酮酸促进破骨细胞肌动蛋白环的形成,5 mmol/L丙酮酸不影响破骨细胞分化也不干扰破骨细胞肌动蛋白环的形成;④RT-qPCR检测显示,安全浓度范围内的丙酮酸对破骨分化相关基因核转录因子活化T细胞核因子C1、抗酒石酸酸性磷酸酶的mRNA相对表达量无明显影响;⑤Western blot检测显示,0.1,1 mmol/L丙酮酸对破骨分化相关蛋白核转录因子活化T细胞核因子C1的表达量无明显影响,5 mmol/L丙酮酸抑制核转录因子活化T细胞核因子C1蛋白的表达,0.1,1,5 mmol/L丙酮酸促进破骨分化相关蛋白c-fos的表达;⑥1 mmol/L丙酮酸促进破骨细胞骨吸收陷窝的形成;⑦结果表明,1 mmol/L丙酮酸在体外对核因子κB受体活化因子配体诱导的RWA264.7细胞向破骨细胞分化具有促进作用。BACKGROUND:Glycolysis is essential for osteoclast differentiation and bone resorption of mature osteoclasts is mainly dependent on glycolysis.Pyruvic acid,as the end product of glycolysis,plays an important role in the metabolism of the three nutrients.OBJECTIVE:To observe the effect of pyruvic acid on osteoclast differentiation.METHODS:The toxicity of different concentrations of pyruvic acid(0.1,1,10,20,30,50 mmol/L)to RAW264.7 cells was detected by cell counting kit-8 assay,and the safe concentration of pyruvic acid was selected.RAW264.7 cells were grouped into interventions:blank control group was cultured with a complete medium(α-MEM medium containing volume fraction 10%fetal bovine serum and 1%double antibodies);control group was cultured with a complete medium containing nuclear factorκB receptor activating factor ligand,and experimental group was cultured with a complete medium with nuclear factorκB receptor activating factor ligand and different concentrations of pyruvic acid,followed by tartrate-resistant acid phosphatase staining,cytoskeletal F-actin staining,RTqPCR assay,western blot assay and bone resorption lacuna assay.RESULTS AND CONCLUSION:(1)The maximum half inhibitory concentration of pyruvic acid on RAW264.7 cells was 8.923 mmol/L,and 0.1,1,5 mmol/L were selected as the safe concentrations for subsequent experiments.(2)Tartrate-resistant acid phosphatase staining revealed that 1 mmol/L pyruvic acid promoted the differentiation of RAW264.7 cells into osteoclasts.(3)F-actin staining showed that 1 mmol/L pyruvic acid promoted the formation of osteoclast F-actin ring,while 5 mmol/L pyruvic acid did not interfere with the formation of osteoclast F-actin ring.(4)RT-qPCR assay indicated that pyruvic acid within the safe concentration range had no significant effect on the relative mRNA expression of osteoclast differentiation related genes nuclear factor of activator T-cells and tartrate-resistant acid phosphatase.(5)Western blot assay showed that 0.1 and 1 mmol/L pyruvic acid within the safe conce

关 键 词：破骨细胞 糖酵解 丙酮酸 骨代谢 骨稳态 

分 类 号：R459.9[医药卫生—治疗学] R329.2[医药卫生—临床医学] R589.5