DUSP1在人正常及骨关节炎滑膜细胞中的表达及对骨关节炎的保护作用
发布时间：2018-08-26 08:08
【摘要】：骨关节炎(osteoarthritis,OA)是一种常见的关节疾病之一,也是导致中老年人关节功能障碍的主要原因。长期以来,骨关节炎都被认为是一种典型的非炎症性关节病,但近年来的研究证实,炎症对骨关节的症状及进展起到了非常重要的作用,骨关节炎是一种炎症相关性疾病这一概念已被普遍接受。因此,针对骨关节炎炎症过程进行干预可能是一种治疗及预防骨关节炎的有效手段。双特异性磷酸酶1(dual specificity phosphatase1,DUSP1),也被称为丝裂原活化蛋白激酶磷酸酶1(mitogen-activated protein kinase phosphatase1,MKP1),能够通过对丝裂原活化蛋白激酶中特异性酪氨酸/苏氨酸残基的去磷酸化作用而抑制其活性,是细胞中MAPK信号通路的重要抑制因子。大量研究表明,DUSP1是炎症反应中重要的负性调控因子,而诱导DUSP1表达可能是一种潜在的新型抗炎方法。本研究首先检测了DUSP1在人正常及骨关节成纤维样滑膜细胞中的表达差异,并进一步探明其作用机制,证实DUSP1通过抑制细胞中p38MAPK及JNK信号通路抑制MMP-13、COX-2等骨关节炎相关效应分子的表达,从而在骨关节炎过程中发挥保护性作用。同时,在研究中我们也证实糖皮质激素地塞米松能够诱导人骨关节炎成纤维样滑膜细胞中DUSP1的表达,并推测地塞米松的抗炎作用可能是部分通过诱导DUSP1表达来实现。因此,DUSP1有望成为治疗及预防骨关节炎新的潜在的作用靶点。目的1.对人正常及骨关节炎成纤维样滑膜细胞进行体外培养及鉴定;2.探索DUSP1在人正常及骨关节炎成纤维样滑膜细胞中的表达差异;3.证实地塞米松对人骨关节炎成纤维样滑膜细胞中DUSP1的诱导表达作用及对p38MAPK、JNK通路和骨关节炎效应分子MMP-13、COX-2表达的抑制作用;4.构建DUSP1过表达慢病毒载体;5.明确DUSP1通过抑制p38MAPK、JNK信号通路而抑制骨关节炎相关分子的表达,从而在骨关节炎中发挥保护性作用。方法1.采用酶消化法体外培养人正常及骨关节炎成纤维样滑膜细胞并传代,CCK-8法检测细胞增殖活性,并通过流式细胞术对细胞CD55分子表达进行检测鉴定并测定细胞纯度,以满足后续实验要求;2.采用实时荧光定量PCR、Western blot、细胞免疫荧光染色检测人正常及骨关节炎成纤维样滑膜细胞和地塞米松诱导的人骨关节炎成纤维样滑膜细胞中DUSP1的表达;3.采用Western blot、实时荧光定量PCR方法对上述各组细胞中p38MAPK、JNK信号通路活化情况及骨关节炎相关效应分子MMP-13、COX-2的表达进行检测;4.采用分子生物学技术构建DUSP1过表达慢病毒载体LV-DUSP1并用PCR、Western blot方法进行鉴定;5.用LV-DUSP1感染人成纤维样滑膜细胞,探索感染条件并观察感染效率,用Western blot方法检测感染后DUSP1表达;6.采用Western blot检测感染后人骨关节炎成纤维样滑膜细胞中p38MAPK、JNK通路的活化情况及相关效应分子表达的变化,并用CCK-8法检测感染后细胞增殖活性变化。结果1.流式细胞术检测显示,培养细胞中CD55阳性率达到98.3%,符合成纤维样滑膜细胞特征,细胞纯度达到后续实验要求;2.CCK-8实验显示骨关节炎成纤维样滑膜细胞的增殖速度明显高于正常滑膜细胞;3.实时荧光定量PCR、Western blot、细胞免疫荧光染色结果表明,人骨关节炎成纤维样滑膜细胞中DUSP1表达明显受到抑制,地塞米松能够诱导人骨关节炎成纤维样滑膜细胞中DUSP1表达;4.Western blot、实时荧光定量PCR结果显示地塞米松能够抑制p38MAPK、JNK信号通路活化及MMP-13、COX-2的表达;5.PCR、Western blot结果表明过表达DUSP1慢病毒载体构建成功;6.荧光显微镜观察显示过表达DUSP1慢病毒载体成功感染人成纤维样滑膜细胞;7.CCK-8结果显示过表达DUSP1能够抑制人骨关节炎成纤维样滑膜细胞增殖活性;8.Western blot结果表明过表达DUSP1能够抑制p38MAPK、JNK信号通路活性及MMP-13、COX-2的表达。结论1.人正常及骨关节炎成纤维样滑膜细胞中DUSP1表达存在差异,在骨关节炎成纤维样滑膜细胞中DUSP1表达明显受到抑制;2.地塞米松能够诱导人骨关节炎成纤维样滑膜细胞中DUSP1表达,并抑制p38MAPK、JNK信号通路的活化及骨关节炎相关效应分子如MMP-13、COX-2等的表达,初步说明地塞米松至少是部分通路诱导DUSP1的表达发挥其抗炎及抗分解代谢的作用。3.初步证实了DUSP1能够通过抑制p38MAPK及JNK信号通路的激活发挥抗炎及抗分解代谢作用,对骨关节炎起到保护作用。4.过表达DUSP1能够明显抑制人骨关节炎成纤维样滑膜细胞的增殖活性,初步说明了DUSP1对人骨关节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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R684.3
，
本文编号：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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R684.3
，
本文编号：2204254
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