Prdx5调控骨关节炎软骨细胞增殖与凋亡的机制研究

发布时间：2018-02-05 03:08

本文关键词： Prdx5 MMP-13 骨关节炎骨关节炎凋亡增殖 Prdx5 Prdx5 Wnt/β-catenin通路骨关节炎　出处：《重庆医科大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分Prdx5在骨关节炎软骨的表达目的：检测Prdx5在骨关节炎软骨组织中的表达情况。方法：1. Western blot, qRT-PCR方法检测Prdx5在人骨关节炎软骨组织与正常骨关节软骨组织中的表达情况。2. Western blot方法检测MMP-13蛋白在人骨关节炎软骨组织与正常骨关节软骨组织中的表达水平。结果:1.骨关节炎软骨组织Prdx5 mRNA的表达量为正常骨关节软骨组织中Prdx5 mRNA表达量的3.5±0.21倍(P0.05)。骨关节炎软骨组织中Prdx5蛋白表达水平比正常骨关节软骨组织高2.31±0.18倍(P0.05),提示Prdx5 mRNA和蛋白的表达水平在骨关节炎软骨组织中与正常的骨关节软骨组织相比明显升高(P0.05)。2. Western blot检测结果显示在骨关节炎软骨组织中MMP-13蛋白的表达水平与正常的骨关节软骨组织相比明显升高(P0.05)。结论：Prdx5与MMP-13在骨关节炎软骨组织中均高表达,可能与骨关节炎的发生发展密切相关。第二部分Prdx5对骨关节炎软骨细胞增殖和凋亡的影响目的：观察抑制Prdx5表达后骨关节炎软骨细胞增殖和凋亡的变化情况。方法：1.通过流式细胞术检测Prdx5表达抑制后骨关节炎软骨细胞凋亡的变化,MTT法检测Prdx5表达抑制后骨关节炎软骨细胞增殖的变化。2.采用ROS检测试剂盒检测Prdx5表达抑制后骨关节炎软骨细胞中ROS的含量变化。3.通过流式细胞术、MTT法检测抗氧化剂NAC对Prdx5表达抑制的骨关节炎软骨细胞凋亡与增殖特性的影响。结果：1.流式细胞仪检测结果提示骨关节炎软骨细胞在Prdx5表达抑制后的凋亡率较对照组明显增加(P0.05),MTT检测结果显示：与对照组相比,转染Prdx5 shRNA的骨关节炎软骨细胞的增殖能力明显降低。2.ROS检测结果提示在Prdx5表达有效抑制后,骨关节炎软骨细胞内ROS的含量与对照组相比显著增加(P0.05)。3. Prdx5表达有效抑制后的骨关节炎软骨细胞加入抗氧化剂NAC作用24 h后,骨关节炎软骨细胞的增殖能力显著增强,凋亡率明显下降(P0.05)。结论：Prdx5表达有效抑制后,导致骨关节炎软骨细胞内ROS的含量增加,从而致使骨关节炎软骨细胞增殖抑制和凋亡增加。第三部分Prdx5调控骨关节炎软骨细胞增殖和凋亡的机制目的：观察Prdx5表达有效抑制后Wnt/p-catenin通路关键因子的变化情况,探讨Prdx5对骨关节炎软骨细胞增殖和凋亡影响的作用机制。方法：1. Prdx5 shRNA转染骨关节炎软骨细胞后应用Western blot方法检测Wnt/β-catenin通路核心蛋白β-catenin在细胞核和细胞质中表达水平的变化以及其它关键因子Wnt-4, Frizzled-2, GSK-3β, p-GSK-3βser9, p-β-cateninser33/37与下游目的基因CyclinD1,MMP-13的蛋白表达情况；qRT-PCR检测CyclinD1, MMP-13 mRNA表达变化情况。2.通过细胞免疫荧光实验检测Prdx5沉默后Wnt/p-catenin通路核心蛋白β-catenin在骨关节炎软骨细胞内的定位变化。3.应用TOPflash荧光素酶报告基因系统检测Prdx5沉默后Wnt/β-catenin通路核心蛋白β-catenin转录活性的变化情况。4. Western blot方法检测Prdx5 shRNA转染正常骨关节软骨细胞后Wnt/β-catenin通路核心蛋白P-catenin在细胞核和细胞质中表达水平的变化以及该通路其它关键因子GSK-3β, p-β-cateninser33/37的蛋白表达情况。5.采用Wnt/β-catenin通路抑制剂XAV-939作用于转染Prdx5 shRNA的骨关节炎软骨细胞后,通过ROS检测试剂盒观察细胞内源性ROS含量的变化情况。结果：1. Western blot结果显示：Prdx5表达有效抑制后,骨关节炎软骨细胞中β-catenin蛋白表达水平在细胞核中明显升高,细胞质中明显降低(P0.05)；而骨关节炎软骨细胞中β-catenin的总蛋白水平在Prdx5抑制前后无明显变化；p-β-cateninser33/37和GSK-3β的蛋白表达水平在Prdx5抑制后明显降低,Wnt-4, Frizzled-2, p-GSK-3βser9, CyclinD1与MMP-13的蛋白表达水平则显著升高(P0.05)；qRT-PCR结果提示CyclinD1和MMP-13 mRNA表达在Prdx5抑制后显著升高(P0.05)。2.细胞免疫荧光结果显示：Prdx5的表达有效抑制后,骨关节炎软骨细胞核内的P-catenin的荧光强度较对照组明显增强,说明骨关节炎软骨细胞中的P-catenin由细胞质移位到细胞核。3.荧光素酶报告基因系统检测结果显示Prdx5基因沉默后,骨关节炎软骨细胞中β-catenin的转录活性显著升高(p0.05)。4. Western blot结果显示：Prdx5 shRNA转染正常骨关节软骨细胞后,Wnt/β-catenin通路核心蛋白β-catenin在细胞质和细胞核中的表达水平以及该通路其它关键因子p-β-cateninser33/37,GSK-3β的蛋白表达水平没有明显变化。5.ROS检测结果提示：单独应用XAV-939能明显降低骨关节炎软骨细胞的ROS含量(P0.05),然而XAV-939与Prdx5 shRNA共同作用于骨关节炎软骨细胞后,ROS的含量没有进一步降低。结论：Prdx5表达抑制后能上调Wnt/β-catenin信号通路,Prdx5可能通过调控Wnt/β-catenin通路从而影响骨关节炎软骨细胞的增殖和凋亡。
[Abstract]:Part 1 expression of Prdx5 in osteoarthritis cartilage Objective: to detect the expression of Prdx5 in osteoarthritis. Methods: 1. Western blot, the expression level of MMP-13 protein detection method for the detection of qRT-PCR Prdx5 in human osteoarthritic cartilage tissue and normal articular cartilage in the expression of.2. Western blot in human bone articular cartilage tissue and normal articular cartilage tissue. Results: the expression of 1. osteoarthritis cartilage Prdx5 mRNA for expression of Prdx5 in normal articular cartilage in mRNA 3.5 + 0.21 times (P0.05). The protein level of Prdx5 than the normal articular cartilage was 2.31 + 0.18 times high expression of osteoarthritis cartilage tissue (P0.05), the expression level of Prdx5 mRNA and protein in osteoarthritic cartilage and bone joint cartilage tissue were significantly higher than normal (P0.05).2. Western blot respectively. The results show that in osteoarthroitis expression level of MMP-13 protein and bone joint cartilage tissue were significantly higher than normal (P0.05). Conclusion: Prdx5 and MMP-13 were highly expressed in osteoarthritic cartilage tissues may be closely related to the development of osteoarthritis. The second part of the effect of Prdx5 on proliferation and apoptosis Osteoarthritis Chondrocytes Objective: changes of proliferation and apoptosis of articular cartilage cells were observed after inhibition of Prdx5 expression. Methods: 1. by flow cytometry to detect the expression of Prdx5 in chondrocyte apoptosis of osteoarthritis after inhibition of Prdx5 MTT expression detection method after inhibition of.3. content in articular cartilage of osteoarthritis ROS by flow cytometry in.2. proliferation of Osteoarthritis Chondrocytes by ROS Kit to detect the inhibition of Prdx5, inhibition of bone and joint detection of antioxidant NAC on the expression of Prdx5 by MTT Effect of cartilage cell apoptosis and proliferation characteristics. Results: 1. flow cytometry showed cartilage cells after inhibition of expression of apoptosis rate significantly increased compared with the control group in Prdx5 (P0.05), MTT test results showed that: compared with the control group, transfection of Prdx5 shRNA cartilage cell proliferation results showed that the expression of.2.ROS decreased significantly after Prdx5 effectively inhibited, Osteoarthritis Chondrocytes in ROS were significantly increased compared with the control group (P0.05.3.) Prdx5 expression of anti oxidant NAC 24 h after adding effective inhibition of articular cartilage cells, significantly enhanced cartilage cell proliferation and the apoptosis rate was significantly decreased (P0.05). Conclusion: the expression of Prdx5 was effectively inhibited, resulting in the increase of the content of articular cartilage cells of ROS, so as a result of osteoarthritis cartilage cell proliferation and apoptosis The third part of the Prdx5 control mechanism. The purpose of osteoarthritis cartilage cell proliferation and apoptosis: To observe the expression of Prdx5 effectively inhibited Wnt/p-catenin pathway key factor, effect of Prdx5 on proliferation and apoptosis of articular cartilage cells. Methods: the expression of Wnt-4 and other key factors, detection of Wnt/ beta -catenin pathway beta -catenin core protein in the nucleus and cytoplasm of the application of Western blot method 1. Prdx5 shRNA transfected cartilage cells after Frizzled-2, GSK-3 beta, p-GSK-3 beta ser9, p- beta -cateninser33/37 and downstream target gene CyclinD1, expression of MMP-13 protein; qRT-PCR MMP-13 mRNA to detect CyclinD1 expression of.2. by changing the location of Wnt/p-catenin core protein pathway beta -catenin in articular cartilage cells in Prdx5 silenced cells was detected using immunofluorescence experiments The application of.3. TOPflash luciferase reporter gene system for detecting Prdx5 gene silencing of Wnt/ beta -catenin pathway core protein beta -catenin transcription activity changes of.4. Western blot Prdx5 method for the detection of shRNA transfected normal articular cartilage cells after Wnt/ beta -catenin pathway core protein P-catenin expression and the other key factor GSK-3 beta pathway in the nucleus and cytoplasm, expression p- beta -cateninser33/37 protein.5. by Wnt/ beta -catenin signaling pathway inhibitor XAV-939 in transfected Prdx5 shRNA cartilage cells, observe the change of endogenous ROS content by ROS kit. Results: 1. Western blot showed that the expression of Prdx5 effectively inhibited, significantly increased -catenin expression in the nucleus protein in osteoarthritic cartilage cells, the cytoplasm was significantly reduced (P0.05) and bone; Beta -catenin arthritis cartilage cells in the total protein level in Prdx5 inhibition had no significant change before and after; the expression level of p- beta -cateninser33/37 and GSK-3 beta protein decreased significantly in Prdx5, Frizzled-2, p-GSK-3 after inhibition of Wnt-4 beta ser9, the expression level of CyclinD1 and MMP-13 protein were significantly increased (P0.05); the results suggest that CyclinD1 and qRT-PCR MMP-13 mRNA expression was significantly increased in Prdx5 after inhibition of.2. (P0.05) immunofluorescence showed that the expression of Prdx5 effectively inhibited, the fluorescence intensity of osteoarthritic cartilage in the nucleus of P-catenin were markedly increased compared to control group, indicating the Osteoarthritis Chondrocytes in the P-catenin cytoplasmic translocation to the nucleus by.3. luciferase reporter gene system test results Prdx5 gene silencing, the transcriptional activity of beta -catenin in articular cartilage of osteoarthritis was significantly higher (P0.05).4. Western blot showed that Prdx 5 shRNA normal articular cartilage cells after transfection, the expression level of Wnt/ beta -catenin pathway beta -catenin core protein in the cytoplasm and in the nucleus and the other key factor p- beta -cateninser33/37 pathway, the expression of GSK-3 beta protein levels did not change significantly in.5.ROS test results: XAV-939 alone can significantly reduce the content of ROS in Osteoarthritis Chondrocytes the (P0.05) XAV-939 and Prdx5, but shRNA together in osteoarthritic cartilage cells, ROS content was not further reduced. Conclusion: the expression of Prdx5 after inhibition can upregulate Wnt/ beta -catenin signaling pathway, Prdx5 may affect the proliferation and apoptosis of articular cartilage cells by regulating Wnt/ beta -catenin pathway.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R684.3

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