發(fā)布時間：2018-10-05 07:42

【摘要】：目的： 1.探討外源性EETs在體外對破骨細胞生成及功能的影響和機制。 2.觀察對骨質疏松模型小鼠進行腹腔注射外源性EETs后小鼠體內骨組織及其他的相關變化。 方法： 1.體外實驗：分別用破骨細胞系RAW264.7和小鼠原代骨髓單核細胞加入RANKL被用來誘導破骨細胞生成,此外,小鼠原代骨髓單核細胞還加入mCSF。對以上兩種細胞加入14,15-EET(濃度為0.25μM,0.5μM,1μM,2μM)。通過抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase, TRAP)染色、測細胞培養(yǎng)上清內的TRAP活性、骨吸收實驗、檢測破骨細胞相關基因和蛋白的變化等方法測定EET對破骨細胞的形成及功能的影響。 2.體內實驗：取4月大小C57/BL6雌鼠,進行假手術及去卵巢(ovariectomy. OVX)手術,將14,15-EET(0.17mg/kg)對OVX組小鼠體內進行腹腔注射,于術后六周對實驗組及對照組小鼠進行外周血TRAP酶聯(lián)免疫測試,檢測相關炎性因子的血清水平,骨切片TRAP染色及用μCT掃描評估骨組織形態(tài)學。 結果：通過體外和體內實驗,我們觀察到EETs顯著抑制骨流失及破骨細胞的形成和活性,這一作用與減少NF-κB活化受體配體(RANKL)的活化、骨保護素的比率及血清中TNF-a和IL-1p的水平有關。在破骨細胞形成分子水平,EETs抑制IANKL導致的NF-κB的激活、及激活蛋白-1(AP-1)和絲裂原活化蛋白激酶(MAPKs)信號通路中細胞外調節(jié)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還抑制了RANKL刺激后的活性氧(ROS)的產(chǎn)生。因而,EETs抑制了破骨細胞相關的基因表達：抗酒石酸酸性磷酸酶(TRAP)、組織蛋白酶K(CK)、基質金屬蛋白酶(MMP)-9和NF-κB活化受體(RANK)。 結論： 1.我們的結果證實EETs通過作用于RANKL上下游多種通路抑制了破骨細胞的生成。 2.給予外源性或穩(wěn)定內源性的EETs水平可以成為治療破骨細胞相關性疾病的新策略,比如類風濕性關節(jié)炎和絕經(jīng)后的骨質疏松等疾病。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R580

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