DUSP1在人正常及骨關(guān)節(jié)炎滑膜細(xì)胞中的表達(dá)及對(duì)骨關(guān)節(jié)炎的保護(hù)作用
發(fā)布時(shí)間：2018-08-26 08:08
【摘要】：骨關(guān)節(jié)炎(osteoarthritis,OA)是一種常見的關(guān)節(jié)疾病之一,也是導(dǎo)致中老年人關(guān)節(jié)功能障礙的主要原因。長(zhǎng)期以來,骨關(guān)節(jié)炎都被認(rèn)為是一種典型的非炎癥性關(guān)節(jié)病,但近年來的研究證實(shí),炎癥對(duì)骨關(guān)節(jié)的癥狀及進(jìn)展起到了非常重要的作用,骨關(guān)節(jié)炎是一種炎癥相關(guān)性疾病這一概念已被普遍接受。因此,針對(duì)骨關(guān)節(jié)炎炎癥過程進(jìn)行干預(yù)可能是一種治療及預(yù)防骨關(guān)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(duì)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(xì)胞中MAPK信號(hào)通路的重要抑制因子。大量研究表明,DUSP1是炎癥反應(yīng)中重要的負(fù)性調(diào)控因子,而誘導(dǎo)DUSP1表達(dá)可能是一種潛在的新型抗炎方法。本研究首先檢測(cè)了DUSP1在人正常及骨關(guān)節(jié)成纖維樣滑膜細(xì)胞中的表達(dá)差異,并進(jìn)一步探明其作用機(jī)制,證實(shí)DUSP1通過抑制細(xì)胞中p38MAPK及JNK信號(hào)通路抑制MMP-13、COX-2等骨關(guān)節(jié)炎相關(guān)效應(yīng)分子的表達(dá),從而在骨關(guān)節(jié)炎過程中發(fā)揮保護(hù)性作用。同時(shí),在研究中我們也證實(shí)糖皮質(zhì)激素地塞米松能夠誘導(dǎo)人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1的表達(dá),并推測(cè)地塞米松的抗炎作用可能是部分通過誘導(dǎo)DUSP1表達(dá)來實(shí)現(xiàn)。因此,DUSP1有望成為治療及預(yù)防骨關(guān)節(jié)炎新的潛在的作用靶點(diǎn)。目的1.對(duì)人正常及骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞進(jìn)行體外培養(yǎng)及鑒定;2.探索DUSP1在人正常及骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中的表達(dá)差異;3.證實(shí)地塞米松對(duì)人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1的誘導(dǎo)表達(dá)作用及對(duì)p38MAPK、JNK通路和骨關(guān)節(jié)炎效應(yīng)分子MMP-13、COX-2表達(dá)的抑制作用;4.構(gòu)建DUSP1過表達(dá)慢病毒載體;5.明確DUSP1通過抑制p38MAPK、JNK信號(hào)通路而抑制骨關(guān)節(jié)炎相關(guān)分子的表達(dá),從而在骨關(guān)節(jié)炎中發(fā)揮保護(hù)性作用。方法1.采用酶消化法體外培養(yǎng)人正常及骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞并傳代,CCK-8法檢測(cè)細(xì)胞增殖活性,并通過流式細(xì)胞術(shù)對(duì)細(xì)胞CD55分子表達(dá)進(jìn)行檢測(cè)鑒定并測(cè)定細(xì)胞純度,以滿足后續(xù)實(shí)驗(yàn)要求;2.采用實(shí)時(shí)熒光定量PCR、Western blot、細(xì)胞免疫熒光染色檢測(cè)人正常及骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞和地塞米松誘導(dǎo)的人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1的表達(dá);3.采用Western blot、實(shí)時(shí)熒光定量PCR方法對(duì)上述各組細(xì)胞中p38MAPK、JNK信號(hào)通路活化情況及骨關(guān)節(jié)炎相關(guān)效應(yīng)分子MMP-13、COX-2的表達(dá)進(jìn)行檢測(cè);4.采用分子生物學(xué)技術(shù)構(gòu)建DUSP1過表達(dá)慢病毒載體LV-DUSP1并用PCR、Western blot方法進(jìn)行鑒定;5.用LV-DUSP1感染人成纖維樣滑膜細(xì)胞,探索感染條件并觀察感染效率,用Western blot方法檢測(cè)感染后DUSP1表達(dá);6.采用Western blot檢測(cè)感染后人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中p38MAPK、JNK通路的活化情況及相關(guān)效應(yīng)分子表達(dá)的變化,并用CCK-8法檢測(cè)感染后細(xì)胞增殖活性變化。結(jié)果1.流式細(xì)胞術(shù)檢測(cè)顯示,培養(yǎng)細(xì)胞中CD55陽性率達(dá)到98.3%,符合成纖維樣滑膜細(xì)胞特征,細(xì)胞純度達(dá)到后續(xù)實(shí)驗(yàn)要求;2.CCK-8實(shí)驗(yàn)顯示骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞的增殖速度明顯高于正常滑膜細(xì)胞;3.實(shí)時(shí)熒光定量PCR、Western blot、細(xì)胞免疫熒光染色結(jié)果表明,人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1表達(dá)明顯受到抑制,地塞米松能夠誘導(dǎo)人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1表達(dá);4.Western blot、實(shí)時(shí)熒光定量PCR結(jié)果顯示地塞米松能夠抑制p38MAPK、JNK信號(hào)通路活化及MMP-13、COX-2的表達(dá);5.PCR、Western blot結(jié)果表明過表達(dá)DUSP1慢病毒載體構(gòu)建成功;6.熒光顯微鏡觀察顯示過表達(dá)DUSP1慢病毒載體成功感染人成纖維樣滑膜細(xì)胞;7.CCK-8結(jié)果顯示過表達(dá)DUSP1能夠抑制人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞增殖活性;8.Western blot結(jié)果表明過表達(dá)DUSP1能夠抑制p38MAPK、JNK信號(hào)通路活性及MMP-13、COX-2的表達(dá)。結(jié)論1.人正常及骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1表達(dá)存在差異,在骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1表達(dá)明顯受到抑制;2.地塞米松能夠誘導(dǎo)人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞中DUSP1表達(dá),并抑制p38MAPK、JNK信號(hào)通路的活化及骨關(guān)節(jié)炎相關(guān)效應(yīng)分子如MMP-13、COX-2等的表達(dá),初步說明地塞米松至少是部分通路誘導(dǎo)DUSP1的表達(dá)發(fā)揮其抗炎及抗分解代謝的作用。3.初步證實(shí)了DUSP1能夠通過抑制p38MAPK及JNK信號(hào)通路的激活發(fā)揮抗炎及抗分解代謝作用,對(duì)骨關(guān)節(jié)炎起到保護(hù)作用。4.過表達(dá)DUSP1能夠明顯抑制人骨關(guān)節(jié)炎成纖維樣滑膜細(xì)胞的增殖活性,初步說明了DUSP1對(duì)人骨關(guān)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(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R684.3
，
本文編號(hào)：2204254
[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(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R684.3
，
本文編號(hào)：2204254
本文鏈接：http://sikaile.net/yixuelunwen/waikelunwen/2204254.html
最近更新
教材專著
