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An Experimental Study on the Effects of MAPKs Signal Pathway in Espressing MMP-1/-13 of Rabbit Osteoarthritis Chondrocytes

Author: RenZhiWei
Tutor: YuYongLin;JiangJianYuan;QiaoJian
School: Fudan University
Course: Surgery
Keywords: Rabbit Chondrocytes Interleukin -1β (IL-1β) Tumor necrosis factor -α (TNF-α) Matrix metalloproteinase -1 (MMP-1) Matrix metalloproteinase -13 (MMP-13) Nitric oxide (NO) real-time PCR Western blot Mitogen- activated protein kinases (MAPKs) ERK1 / 2 JNK1 / 2 p38 Osteoarthritis Mitogen-activated protein kinases (MAPKs) Signal transduction Real-time PCR
CLC: R684.3
Type: PhD thesis
Year: 2008
Downloads: 549
Quote: 0
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Background Osteoarthritis (osteoarthritiS, OA) is a chronic, degenerative joint disease, the elderly the most common joint disease. OA more common in middle-aged patients, more women than men. Population over the age of 60 the prevalence of up to 50%, 75-year-olds is 80%. With the arrival of aging society, OA has become a cause aging Sick waste of the major diseases, the social burden and health care costs are increasing. Present study suggests that, OA is caused by many factors articular cartilage degeneration caused by joint disease, its etiology is not clear, its occurrence with age, obesity, inflammation, trauma and genetic factors and so on. Many cytokines in the pathogenesis of OA has an important role, in which interleukin -1 (IL-1) and tumor necrosis factor-α (TNF-α) is most important. Them through autocrine or paracrine role in synovial cells and chondrocytes produce metalloproteinases (MMPs), nitric oxide (NO) and prostaglandin E2 (PGE2), etc., inhibition of proteoglycan and collagen type Ⅱ , to promote the extracellular matrix (ECM) degradation and eventually cause arthritis. Although cytokine theory does not fully explain the pathogenesis of OA, but the articular cartilage degeneration without the role of inflammatory cytokines. Related studies show, IL-1β and TNF-α with the corresponding receptor binding, via mitogen-activated protein kinase (mitogen-actirated proteinkinases, MAPKs) pathway and nuclear factor-κB (nuclear factor-κB, NF- κB) pathway of intracellular signal transduction, and ultimately lead MMPs increased free radical production, apoptosis, and a series of processes, and thus participate in the pathogenesis of OA. However, current IL-1β and TNF-α induced MMP-1/-13 specific pathway through MAPKs study the similarities and differences between them are not clear. The experimental application of real-time PCR and western blot techniques to study IL-1β and TNF-α effect on rabbit articular chondrocytes after MMP-1/-13 changes and different MAPKs further study on the impact of each pathway proteins, and applying MAPKs inhibitors and combined effects of IL-1β, namely blocking the MAPKs pathway, observation of the MMP-1/-13 of. Through experiments clarify the MAPKs pathway in osteoarthritis articular chondrocytes MMP-1/-13 increased expression of specific regulatory role for further research in the pathogenesis of OA chondrocytes MMP-1/-13 higher molecular mechanisms provide a theoretical basis. The first part of the IL-1β and TNF-α expression in rabbit articular chondrocytes purpose MMP-1/-13 and NO rabbit articular chondrocytes in vitro to observe the IL-1β and TNF-α on cultured rabbit articular chondrocytes MMP -1 -13 and NO expression, clear differences between them. Materials and Methods New Zealand rabbit articular cartilage, articular chondrocytes were isolated and cultured in vitro and identified. IL-1β were used and the role of TNF-α at different times in the articular chondrocytes (8h, 16h, 24h, 36h). In real-time PCR to detect MMP-1/-13mRNA changes. Also charged cell culture supernatant was detected by Western blot MMP-1/-13 protein changes and NO was measured by Griess reaction changes. Results by inverted microscope cell morphology, toluidine blue staining and type Ⅱ collagen staining proved the cultured cells are chondrocytes. MMP-1/-13 IL-1β can cause increased expression, MMP-1 continued to increase with time, and MMP-13 increased and then maintained at a higher level. TNF-α and the increase does not cause them. IL-1β and TNF-α can cause increased expression of NO. Conclusion rabbit articular chondrocytes can be successfully isolated and cultured. IL-1β can make MMP-1 and MMP-13 expression increased, but both increasing trend differently. TNF-α did not cause the rabbit articular chondrocytes MMP-1/-13 increased. IL-1β and TNF-α can be increased secretion of NO. The second part of the IL-1β in rabbit articular chondrocytes MAPKs signaling pathway protein expression Objective To observe the IL-1β in rabbit articular chondrocytes various MAPKs pathway protein expression. Materials and Methods The cultured chondrocytes were randomly divided into control group and the use of IL-1β (10ng/ml) for different time groups (reaction time was 15min, 30min, 45min, 60min), was detected by Western blot chondrocytes the ERK1 / 2, JNK1 / 2 and p38 pathway total protein and active protein (phosphorylated protein) expression. Results role in IL-1β, compared with control group, 1h MAPKs pathways within the total protein did not change significantly; while the role of the phosphorylated protein expression was significantly increased after 15min, p-ERK1 / 2 increased maintenance to 60min, p-JNK1 / 2 increased to a peak at 15min after, began to decline, to return to normal levels at 60min; p-p38 increased to begin to decline after 30min to 60min, back to normal levels. Each group compared with the control group, the difference was statistically significant. Conclusion IL-1β does not cause changes in the total protein MAPKs pathway, but a significant increase in phosphorylated proteins cause. The third part of the MAPKs pathway in rabbit cartilage cells MMP-1/-13 and the role of NO in Objective MAPKs pathway blockers suppress various osteoarthritis chondrocytes were observed on MMP-1/-13 and NO the impact of various channels to clarify its role in MMP-1/-13 expression. Materials and Methods In vitro cultured rabbit articular chondrocytes, set the blank control group and IL-1β (10ng/ml) action groups, each MAPK pathway blockers ERK, PD98059 (20μM), JNK, SP600125 (25μM), p38, SB230585 ( 10μM) effector cells 30min after adding IL-1β (10ng/ml) together 24h, apply real-time PCR detection MMP-1/-13mRNA changes. Charged cell culture supernatant was measured using Griess reaction NO changes. Results role of IL-1β expression increased after chondrocyte MMP-1/-13mRNA adding blockers, MMP-1/-13 expression was significantly decreased. ERK pathway blocker PD98059 inhibited 29.6% (MMP-1), 39.2% (MMP-13); p38 pathway inhibitors SB203580 inhibited 47.7% (MMP-1), 37.7% (MMP-13); JNK pathway blocker SP600125 inhibition was 55.4% (MMP-1), 52.2% (MMP-13). P38 pathway inhibitor SB203580 can inhibit NO expression. Conclusion rabbit osteoarthritis chondrocytes MMP-1, 13 increased expression in, MAPKs pathway plays an important role. For the expression of MMP-1, JNK and p38 pathways may play a major role, and MMP-13 expression, play a major role in the ERK and JNK pathways. P38 pathway involved in IL-1β-induced NO expression.

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CLC: > Medicine, health > Surgery > Orthopaedic Surgery ( movement system diseases,orthopedic surgery ) > Joint disease and injury > Arthritis
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