环氧类二十烷酸对破骨细胞生成的抑制及防止卵巢切除所致的骨流失

发布时间：2018-10-05 07:42

【摘要】：目的： 1.探讨外源性EETs在体外对破骨细胞生成及功能的影响和机制。 2.观察对骨质疏松模型小鼠进行腹腔注射外源性EETs后小鼠体内骨组织及其他的相关变化。方法： 1.体外实验：分别用破骨细胞系RAW264.7和小鼠原代骨髓单核细胞加入RANKL被用来诱导破骨细胞生成,此外,小鼠原代骨髓单核细胞还加入mCSF。对以上两种细胞加入14,15-EET(浓度为0.25μM,0.5μM,1μM,2μM)。通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色、测细胞培养上清内的TRAP活性、骨吸收实验、检测破骨细胞相关基因和蛋白的变化等方法测定EET对破骨细胞的形成及功能的影响。 2.体内实验：取4月大小C57/BL6雌鼠,进行假手术及去卵巢(ovariectomy. OVX)手术,将14,15-EET(0.17mg/kg)对OVX组小鼠体内进行腹腔注射,于术后六周对实验组及对照组小鼠进行外周血TRAP酶联免疫测试,检测相关炎性因子的血清水平,骨切片TRAP染色及用μCT扫描评估骨组织形态学。结果：通过体外和体内实验,我们观察到EETs显著抑制骨流失及破骨细胞的形成和活性,这一作用与减少NF-κB活化受体配体(RANKL)的活化、骨保护素的比率及血清中TNF-a和IL-1p的水平有关。在破骨细胞形成分子水平,EETs抑制IANKL导致的NF-κB的激活、及激活蛋白-1(AP-1)和丝裂原活化蛋白激酶(MAPKs)信号通路中细胞外调节蛋白激酶(Extracellular Regulated Protein Kinase, ERK)和c-Jun氨基末端激酶(c-Jun N-terminal Kinase, JNK)的活化。此外,EETs还抑制了RANKL刺激后的活性氧(ROS)的产生。因而,EETs抑制了破骨细胞相关的基因表达：抗酒石酸酸性磷酸酶(TRAP)、组织蛋白酶K(CK)、基质金属蛋白酶(MMP)-9和NF-κB活化受体(RANK)。结论： 1.我们的结果证实EETs通过作用于RANKL上下游多种通路抑制了破骨细胞的生成。 2.给予外源性或稳定内源性的EETs水平可以成为治疗破骨细胞相关性疾病的新策略,比如类风湿性关节炎和绝经后的骨质疏松等疾病。
[Abstract]:Objective: 1. To investigate the effect and mechanism of exogenous EETs on osteoclast formation and function in vitro. 2. To observe the changes of bone tissue and other related changes of osteoporosis model mice after intraperitoneal injection of exogenous EETs. Methods: 1. In vitro experiments: the osteoclast cell line RAW264.7 and mouse primary bone marrow monocytes were added to RANKL to induce osteoclast formation. In addition, mouse primary bone marrow monocytes were added to mCSF.. The above two kinds of cells were added with 145- EET (0. 25 渭 MU 0. 5 渭 MU 1 渭 MU 2 渭 M). The effects of EET on the formation and function of osteoclasts were determined by tartrate-resistant acid phosphatase, TRAP) staining, TRAP activity in the supernatant of cell culture, bone resorption assay and changes of osteoclast related genes and proteins. 2. In vivo experiment: four months old C57/BL6 female rats were sham-operated and ovariectomized (ovariectomy.). OVX). The mice in OVX group were injected intraperitoneally with 14 OVX 15-EET (0.17mg/kg). The peripheral blood TRAP enzyme-linked immunosorbent assay (Elisa) was performed in the experimental group and the control group six weeks after the operation, and the serum levels of inflammatory factors were detected. Bone sections were stained with TRAP and bone histomorphology was evaluated by 渭 CT scanning. Results: in vitro and in vivo experiments, we observed that EETs significantly inhibited bone loss and the formation and activity of osteoclasts, which was related to the reduction of the activation of NF- 魏 B receptor ligand (RANKL), the ratio of osteoprotegerin and the levels of TNF-a and IL-1p in serum. At the molecular level of osteoclast formation, EETs inhibit the activation of NF- 魏 B induced by IANKL, and the activation of extracellular regulated protein kinase (Extracellular Regulated Protein Kinase, ERK) and c-Jun amino-terminal kinase (c-Jun N-terminal Kinase, JNK) in the activator protein 1 (AP-1) and mitogen-activated protein kinase (MAPKs) signaling pathway. In addition, EETs inhibited the production of reactive oxygen (ROS) stimulated by RANKL. Therefore, EETs inhibited the expression of osteoclast related genes: tartrate-resistant acid phosphatase (TRAP), cathepsin K (CK), matrix metalloproteinase (MMP) -9 and NF- 魏 B activated receptor (RANK). Conclusion: 1. Our results demonstrate that EETs inhibits osteoclast formation by acting on multiple upstream and downstream pathways of RANKL. 2. Exogenous or stable endogenous EETs levels may become a new strategy for the treatment of osteoclast-related diseases such as rheumatoid arthritis and postmenopausal osteoporosis.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R580

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