摘要
目的:绝经后骨质疏松PMOP(postmenopause osteoporosis)是一种慢性炎症性疾病,破骨细胞的异常活化是发病的重要原因之一.病理环境下骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)外泌体对破骨细胞的作用及影响尚不明确.本研究分析了PMOP环境下,BMSCs外泌体(exosome,Exo)对破骨细胞分化的调控作用,对于明确骨质疏松的发病机制具有至关重要的意义.方法:分别从正常和绝经后骨质疏松患者骨髓中提取BMSCs,通过超速离心联合Exoquick试剂盒分别提取两种细胞的Exo,即正常BMSCs外泌体(healthy-Exo,H-Exo)和绝经后骨质疏松骨髓间充质干细胞外泌体(postmenopause osteoporosis-Exo,P-Exo),并通过透射电子显微镜(TEM),纳米颗粒示踪分析技术(NTA)、Western blot对分离的外泌体进行鉴定;两种不同的外泌体被引入RAW264.7细胞中,以便通过实时荧光定量聚合酶链反应(RT-qPCR)、Western blot分析和抗酒石酸酸性磷酸酶(TRAP)染色来评估RAW264.7细胞中破骨分化相关指标的表达.同时,使用Western blot和ELISA方法来测定这两种外泌体中OPG/RANKL蛋白的表达水平.实验数据通过SPSS 24.0软件进行统计分析.结果:对提取后的两种外泌体进行形态学比较发现,与H-Exo相比,P-Exo直径更大,P-Exo能够促进RAW264.7细胞破骨分化标志性基因NFATC1和Trap mRNA及蛋白的表达,Trap染色可见分化的破骨细胞,Western blot及ELISA检测两种外泌体O PG/RANKL的表达发现,与H-Exo相比,P-Exo内OPG的表达量下降,而RANKL表达量升高.结论:绝经后骨质疏松中骨吸收增加,可能是炎症微环境下,BMSCs外泌体通过传递RANKL,促进破骨细胞分化引起的.此项研究为深入研究骨质疏松病因的开辟了新的途径,并为骨质疏松的治疗提供了新的治疗靶点.
Abstract
Objective:Postmenopausal osteoporosis(PMOP)is a chronic inflammatory disease.Abnormal activation of osteoclasts is one of the important reasons for PMOP.In pathological environment,the effect of exosomes(Exo)derived from bone marrow mesenchymal stem cell(BMSCs)on osteoclasts is still unclear.In this study,we analyzed the regulatory effect of BMSCs exo on osteoclast differentiation in PMOP environment,which is of vital significance for clarifying the pathogenesis of osteoporosis.Methods:BMSCs were extracted from the bone marrow of normal people and postmenopausal osteoporosis patients,and Exo(H-Exo and P-Exo)of the two cells were extracted by ultracentrifugation combined with Exoquick kit,and the isolated exosomes were identified by transmission electron microscopy(TEM)and Western blot.Two exosomes were introduced into RAW264.7 culture medium,and osteoclast differentiation markers were identified using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),Western blot,and tartrate-resistant acid phosphatase(TRAP)staining after a week.The presence of OPG/RANKL in exosomes was identified using Western blotting and ELISA techniques.The experimental data was statistically analyzed using the SPSS 24.0 software.Results:A morphological analysis of the two kinds of isolated exosomes revealed that P-Exo possessed a greater diameter compared to H-Exo,and P-Exo was capable of markedly enhancing the gene and protein expression levels of osteoclasts differentiation marker in RAW264.7 cells.The application of TRAP staining revealed the presence of distinct osteoclasts.The presence of OPG/RANKL in a pair of exosomes was identified using WB and ELISA techniques.In contrast to H-Exo,there was a reduction in OPG expression in P-Exo and an elevation in RANKL expression.Conclusion:Increased bone resorption in postmenopausal osteoporosis may be caused by the transfer of RANKL by exosomes secreted by BMMSC to promote osteoclast differentiation in the inflammatory microenvironment.This study provides a new idea for further studying the pathogenesis of osteoporosis and providing new therapeutic targets.
基金项目
国家自然科学基金面上项目(82071084)
北京市海淀区卫生健康发展科研培育计划项目(HP2021-12-80702)
维普
万方数据