【摘要】：骨關節(jié)炎(osteoarthritis,OA)是一種常見的關節(jié)疾病之一,也是導致中老年人關節(jié)功能障礙的主要原因。長期以來,骨關節(jié)炎都被認為是一種典型的非炎癥性關節(jié)病,但近年來的研究證實,炎癥對骨關節(jié)的癥狀及進展起到了非常重要的作用,骨關節(jié)炎是一種炎癥相關性疾病這一概念已被普遍接受。因此,針對骨關節(jié)炎炎癥過程進行干預可能是一種治療及預防骨關節(jié)炎的有效手段。雙特異性磷酸酶1(dual specificity phosphatase1,DUSP1),也被稱為絲裂原活化蛋白激酶磷酸酶1(mitogen-activated protein kinase phosphatase1,MKP1),能夠通過對絲裂原活化蛋白激酶中特異性酪氨酸/蘇氨酸殘基的去磷酸化作用而抑制其活性,是細胞中MAPK信號通路的重要抑制因子。大量研究表明,DUSP1是炎癥反應中重要的負性調控因子,而誘導DUSP1表達可能是一種潛在的新型抗炎方法。本研究首先檢測了DUSP1在人正常及骨關節(jié)成纖維樣滑膜細胞中的表達差異,并進一步探明其作用機制,證實DUSP1通過抑制細胞中p38MAPK及JNK信號通路抑制MMP-13、COX-2等骨關節(jié)炎相關效應分子的表達,從而在骨關節(jié)炎過程中發(fā)揮保護性作用。同時,在研究中我們也證實糖皮質激素地塞米松能夠誘導人骨關節(jié)炎成纖維樣滑膜細胞中DUSP1的表達,并推測地塞米松的抗炎作用可能是部分通過誘導DUSP1表達來實現(xiàn)。因此,DUSP1有望成為治療及預防骨關節(jié)炎新的潛在的作用靶點。目的1.對人正常及骨關節(jié)炎成纖維樣滑膜細胞進行體外培養(yǎng)及鑒定;2.探索DUSP1在人正常及骨關節(jié)炎成纖維樣滑膜細胞中的表達差異;3.證實地塞米松對人骨關節(jié)炎成纖維樣滑膜細胞中DUSP1的誘導表達作用及對p38MAPK、JNK通路和骨關節(jié)炎效應分子MMP-13、COX-2表達的抑制作用;4.構建DUSP1過表達慢病毒載體;5.明確DUSP1通過抑制p38MAPK、JNK信號通路而抑制骨關節(jié)炎相關分子的表達,從而在骨關節(jié)炎中發(fā)揮保護性作用。方法1.采用酶消化法體外培養(yǎng)人正常及骨關節(jié)炎成纖維樣滑膜細胞并傳代,CCK-8法檢測細胞增殖活性,并通過流式細胞術對細胞CD55分子表達進行檢測鑒定并測定細胞純度,以滿足后續(xù)實驗要求;2.采用實時熒光定量PCR、Western blot、細胞免疫熒光染色檢測人正常及骨關節(jié)炎成纖維樣滑膜細胞和地塞米松誘導的人骨關節(jié)炎成纖維樣滑膜細胞中DUSP1的表達;3.采用Western blot、實時熒光定量PCR方法對上述各組細胞中p38MAPK、JNK信號通路活化情況及骨關節(jié)炎相關效應分子MMP-13、COX-2的表達進行檢測;4.采用分子生物學技術構建DUSP1過表達慢病毒載體LV-DUSP1并用PCR、Western blot方法進行鑒定;5.用LV-DUSP1感染人成纖維樣滑膜細胞,探索感染條件并觀察感染效率,用Western blot方法檢測感染后DUSP1表達;6.采用Western blot檢測感染后人骨關節(jié)炎成纖維樣滑膜細胞中p38MAPK、JNK通路的活化情況及相關效應分子表達的變化,并用CCK-8法檢測感染后細胞增殖活性變化。結果1.流式細胞術檢測顯示,培養(yǎng)細胞中CD55陽性率達到98.3%,符合成纖維樣滑膜細胞特征,細胞純度達到后續(xù)實驗要求;2.CCK-8實驗顯示骨關節(jié)炎成纖維樣滑膜細胞的增殖速度明顯高于正常滑膜細胞;3.實時熒光定量PCR、Western blot、細胞免疫熒光染色結果表明,人骨關節(jié)炎成纖維樣滑膜細胞中DUSP1表達明顯受到抑制,地塞米松能夠誘導人骨關節(jié)炎成纖維樣滑膜細胞中DUSP1表達;4.Western blot、實時熒光定量PCR結果顯示地塞米松能夠抑制p38MAPK、JNK信號通路活化及MMP-13、COX-2的表達;5.PCR、Western blot結果表明過表達DUSP1慢病毒載體構建成功;6.熒光顯微鏡觀察顯示過表達DUSP1慢病毒載體成功感染人成纖維樣滑膜細胞;7.CCK-8結果顯示過表達DUSP1能夠抑制人骨關節(jié)炎成纖維樣滑膜細胞增殖活性;8.Western blot結果表明過表達DUSP1能夠抑制p38MAPK、JNK信號通路活性及MMP-13、COX-2的表達。結論1.人正常及骨關節(jié)炎成纖維樣滑膜細胞中DUSP1表達存在差異,在骨關節(jié)炎成纖維樣滑膜細胞中DUSP1表達明顯受到抑制;2.地塞米松能夠誘導人骨關節(jié)炎成纖維樣滑膜細胞中DUSP1表達,并抑制p38MAPK、JNK信號通路的活化及骨關節(jié)炎相關效應分子如MMP-13、COX-2等的表達,初步說明地塞米松至少是部分通路誘導DUSP1的表達發(fā)揮其抗炎及抗分解代謝的作用。3.初步證實了DUSP1能夠通過抑制p38MAPK及JNK信號通路的激活發(fā)揮抗炎及抗分解代謝作用,對骨關節(jié)炎起到保護作用。4.過表達DUSP1能夠明顯抑制人骨關節(jié)炎成纖維樣滑膜細胞的增殖活性,初步說明了DUSP1對人骨關節(jié)炎滑膜增生可能具有一定的抑制作用。
[Abstract]:Osteoarthritis (OA) is one of the common joint diseases and the main cause of joint dysfunction in the elderly.Osteoarthritis has long been considered as a typical non-inflammatory arthropathy.However, recent studies have confirmed that inflammation plays a very important role in the symptoms and progress of osteoarthritis. The concept that osteoarthritis is an inflammation-related disease has been widely accepted. Therefore, intervention in the inflammatory process of osteoarthritis may be an effective means of treatment and prevention of osteoarthritis. Dual specific phosphatase 1 (DUSP1), also known as mitogen-activated protein kinase phosphatase 1 (mitogen-a) Activated protein kinase phosphatase 1 (MKP1), which can inhibit the activity of mitogen-activated protein kinase through dephosphorylation of specific tyrosine/threonine residues, is an important inhibitor of MAPK signaling pathway in cells. This study first examined the differential expression of DUSP1 in human normal and osteoarthritic fibroblast-like synovial cells, and further explored its mechanism of action. It was confirmed that DUSP1 inhibited the expression of MMP-13, COX-2 and other osteoarthritis-related effectors by inhibiting p38 MAPK and JNK signaling pathways in cells. At the same time, we also confirmed that dexamethasone can induce the expression of DUSP1 in fibroblast-like synovial cells of osteoarthritis, and speculated that the anti-inflammatory effect of dexamethasone may be partly achieved by inducing the expression of DUSP1. Therefore, DUSP1 is expected to be a therapeutic and prognostic agent. Objective 1. To culture and identify fibroblast-like synoviocytes from human normal and osteoarthritis in vitro; 2. To investigate the expression of DUSP1 in fibroblast-like synoviocytes from human normal and osteoarthritis; 3. To confirm the induction of DUSP1 expression by dexamethasone in fibroblast-like synoviocytes from human osteoarthritis. Effect and inhibition of p38MAPK, JNK pathway and osteoarthritis effector MMP-13, COX-2 expression; 4. Construction of DUSP1 overexpression lentiviral vector; 5. Clear DUSP1 through inhibiting p38MAPK, JNK signaling pathway to inhibit the expression of osteoarthritis-related molecules, thereby playing a protective role in osteoarthritis. Methods 1. Enzyme digestion method in vitro Human osteoarthritis fibroblast-like synovial cells were cultured and subcultured, and the proliferation activity was detected by CCK-8 method, and the expression of CD55 molecule was detected and identified by flow cytometry to meet the following experimental requirements. 2. Real-time fluorescence quantitative PCR, Western blot and immunofluorescence staining were used to detect the normal and human osteoarthritis fibroblast-like synovial cells. The expression of DUSP1 in osteoarthritis fibroblast-like synoviocytes and dexamethasone-induced fibroblast-like synoviocytes was detected by Western blot and real-time quantitative PCR. Lentiviral vector LV-DUSP1 was constructed by molecular biology and identified by PCR and Western blot. 5. Human fibroblast-like synoviocytes were infected with LV-DUSP1 to explore the infection conditions and observe the infection efficiency. The expression of DUSP1 was detected by Western blot. 6. Fibrillation of osteoarthritis was detected by Western blot. The activation of p38 MAPK, JNK pathway and the expression of related effector molecules were detected by CCK-8 method. Results 1. The positive rate of CD55 in cultured cells was 98.3%, which accorded with the characteristics of fibroblast-like synoviocytes, and the purity of the cells reached the following experimental requirements. The proliferation rate of osteoarthritis fibroblast-like synoviocytes was significantly higher than that of normal synoviocytes. 3. Real-time fluorescence quantitative PCR, Western blot and immunofluorescence staining showed that the expression of DUSP1 in fibroblast-like synoviocytes of osteoarthritis was significantly inhibited, and dexamethasone could induce fibroblast-like synoviocytes of osteoarthritis. The results of Western blot and real-time fluorescence quantitative PCR showed that dexamethasone could inhibit the expression of p38 MAPK, JNK signaling pathway activation and MMP-13, COX-2; 5. PCR and Western blot showed that over-expression of DUSP1 lentiviral vector was successfully constructed; 6. Fluorescence microscopy showed that over-expression of DUSP1 lentiviral vector successfully infected human fibroblasts. Vitamin-like synoviocytes; 7. CCK-8 results showed that over-expression of DUSP1 could inhibit the proliferation of fibroblast-like synoviocytes in osteoarthritis; 8. Western blot results showed that over-expression of DUSP1 could inhibit p38 MAPK, JNK signaling pathway activity and the expression of MMP-13, COX-2. Conclusion 1. DUSP1 expression exists in human normal and osteoarthritis fibroblast-like synoviocytes. Dexamethasone can induce the expression of DUSP1 in fibroblast-like synoviocytes of osteoarthritis, and inhibit the activation of p38 MAPK, JNK signaling pathway and the expression of osteoarthritis-related effector molecules such as MMP-13, COX-2, etc. It is preliminarily suggested that dexamethasone is at least partial. DUSP1 expression was induced by different pathways to play its anti-inflammatory and anti-catabolic roles. 3. Preliminary results showed that DUSP1 can play an anti-inflammatory and anti-catabolic role in osteoarthritis by inhibiting the activation of p38 MAPK and JNK signaling pathways. 4. Overexpression of DUSP1 can significantly inhibit the proliferation of fibroblast-like synovial cells in osteoarthritis. The activity indicates that DUSP1 may inhibit the synovial hyperplasia of human osteoarthritis.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R684.3
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本文編號：2204254