骨碎补通过骨髓间充质干细胞调节RANKL/OPG抑制破骨细胞的实验研究
Rhizoma drynaria inhibits osteoclasts by regulating RANKL/OPG of bone marrow mesenchymal stem cells
  
DOI:10.3969/j.issn.1006-7108.2019.05.009
中文关键词:  中医中药  动物实验  破骨细胞  骨碎补  骨髓间充质干细胞  骨质疏松  大鼠
英文关键词:traditional Chinese medicine  animal experiment  osteoclasts  rhizoma drynariae  bone marrow mesenchymal stem cells  osteoporosis  rat
基金项目:国家自然科学基金项目(81473709);山东省中医药科技发展计划项目(2017-431)
作者单位
张峻玮1,2 李琰2 薛海鹏 3 李朝辉2 聂伟志2 徐展望3* 1.山东中医药大学山东 济南 250014 2.山东省文登整骨医院山东 文登 260014 3.山东中医药大学附属医院山东 济南 250014 
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中文摘要:
      目的 研究骨碎补对绝经后骨质疏松大鼠骨髓间充质干细胞(bone marrow stromal cells,BMSCs)RANKL/OPG及破骨细胞分化成熟的影响并探查其可能的作用机制。方法 实验大鼠去双侧卵巢造模,分为实验组(OVXDF,造模+骨碎补水煎液灌胃)、模型组(OVX,造模+0.9%生理盐水灌胃)、假手术组(SHAM,假手术+0.9%生理盐水灌胃),造模成功后提取BMSCs,将BMSCs和骨髓单核细胞共培养于Transwell小室的上室和下室,分为实验组+破骨细胞(OVXDF+OC)、模型组+破骨细胞(OVX+OC)、假手术组+破骨细胞(SHAM+OC)。下室加入破骨细胞诱导剂,倒置相差显微镜观察破骨细胞的分化成熟情况并计数,酶联免疫吸附剂测定(ELISA)检测下室培养液中骨保护素(osteoprotegerin,OPG)、RANKL的含量,并计算RANKL/OPG,实时荧光定量PCR和蛋白质印迹法(Western blot)检测BMSCs中Wnt10b、β-catenin、RANKL、OPG mRAN及蛋白表达并计算RANKL/OPG。结果 在共培养系统中,与去卵巢灌胃大鼠BMSCs共培养的破骨细胞(OVXDF+OC)数量较单纯模型组+破骨细胞(OVX+OC)明显减少(P<0.05)。下室培养液OPG含量及共培养BMSCs中Wnt10b、β-catenin、OPG mRAN及蛋白表达模型组+破骨细胞(OVX+OC)最低,RANKL及RANKL/OPG最高,经骨碎补灌胃后(OVXDF+OC)培养液中OPG含量及BMSCs细胞中Wnt10b、β-catenin、OPG mRAN及蛋白表达明显升高,培养液及BMSCs细胞中RANKL及RANKL/OPG明显降低(P<0.05)。结论 骨碎补可调节BMSCs细胞OPG、RANKL的表达,激活OPG/RANKL/RANK信号通路抑制破骨细胞的分化和成熟,此作用可能与BMSCs的Wnt/β-catenin信号通路的激活有关。
英文摘要:
      Objective To study the effect of rhizoma drynariae on RANKL/OPG and osteoclast differentiation by bone marrow stromal cells (BMSCs) in postmenopausal osteoporotic rats, and to explore its possible mechanism. Methods Experimental rats were divided into experimental group (OVXDF, ovariectomized and rhizoma drynariae gavage), model group (OVX, ovariectomized and 0.9% NaCl gavage), and sham operation group (SHAM, sham surgery and 0.9% NaCl gavage). After successful modeling, BMSCs were extracted. BMSC and bone marrow mononuclear cells were co-cultured in the upper and lower chambers of the Transwell chamber, respectively. The co-cultured cells were divided into experimental group + osteoclasts (OVXDF+OC), model group + osteoclasts (OVX+OC), and sham operation group + osteoclasts (SHAM+OC). The differentiation and maturation of osteoclasts were observed and counted with a phase-contract microscope. OPG and RANKL in the culture medium of the lower chamber were detected using ELISA method. mRNA and protein expressions of Wnt10b, β-Catenin, RANKL, and OPG in BMSCs were detected using PCR and Western blotting. Results In the co-culture systems, the number of osteoclasts in OVXDF+OC group was significantly less than in OVX+OC group (P<0.05). OPG content in the culture medium of the lower chamber and the expression of wnt10b, β-Catenin, and OPG in the co-culture systems were the lowest, but RANKL and RANKL/OPG were the highest, in OVX+OC group. After rhizoma drynariae gavage, OPG content in OVXDF+OC medium and mRNA and protein expressions of Wnt10b, β-Catenin, and OPG in BMSCs increased significantly (P<0.05), but RANKL content and RANKL/OPG decreased significantly (P<0.05). Conclusion Rhizoma drynariae regulates the expression of OPG and RANKL, and activates OPG/RANKL/RANK signaling pathway to inhibit the differentiation and maturation of osteoclasts in BMSCs. This effect may be related to the activation of Wnt/β-Catenin signaling pathway in BMSCs.
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