本文關(guān)鍵詞： Prdx5 MMP-13 骨關(guān)節(jié)炎 骨關(guān)節(jié)炎 凋亡 增殖 Prdx5 Prdx5 Wnt/β-catenin通路 骨關(guān)節(jié)炎　出處：《重慶醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分Prdx5在骨關(guān)節(jié)炎軟骨的表達(dá)目的：檢測Prdx5在骨關(guān)節(jié)炎軟骨組織中的表達(dá)情況。方法：1. Western blot, qRT-PCR方法檢測Prdx5在人骨關(guān)節(jié)炎軟骨組織與正常骨關(guān)節(jié)軟骨組織中的表達(dá)情況。2. Western blot方法檢測MMP-13蛋白在人骨關(guān)節(jié)炎軟骨組織與正常骨關(guān)節(jié)軟骨組織中的表達(dá)水平。結(jié)果:1.骨關(guān)節(jié)炎軟骨組織Prdx5 mRNA的表達(dá)量為正常骨關(guān)節(jié)軟骨組織中Prdx5 mRNA表達(dá)量的3.5±0.21倍(P0.05)。骨關(guān)節(jié)炎軟骨組織中Prdx5蛋白表達(dá)水平比正常骨關(guān)節(jié)軟骨組織高2.31±0.18倍(P0.05),提示Prdx5 mRNA和蛋白的表達(dá)水平在骨關(guān)節(jié)炎軟骨組織中與正常的骨關(guān)節(jié)軟骨組織相比明顯升高(P0.05)。2. Western blot檢測結(jié)果顯示在骨關(guān)節(jié)炎軟骨組織中MMP-13蛋白的表達(dá)水平與正常的骨關(guān)節(jié)軟骨組織相比明顯升高(P0.05)。結(jié)論：Prdx5與MMP-13在骨關(guān)節(jié)炎軟骨組織中均高表達(dá),可能與骨關(guān)節(jié)炎的發(fā)生發(fā)展密切相關(guān)。第二部分Prdx5對骨關(guān)節(jié)炎軟骨細(xì)胞增殖和凋亡的影響目的：觀察抑制Prdx5表達(dá)后骨關(guān)節(jié)炎軟骨細(xì)胞增殖和凋亡的變化情況。方法：1.通過流式細(xì)胞術(shù)檢測Prdx5表達(dá)抑制后骨關(guān)節(jié)炎軟骨細(xì)胞凋亡的變化,MTT法檢測Prdx5表達(dá)抑制后骨關(guān)節(jié)炎軟骨細(xì)胞增殖的變化。2.采用ROS檢測試劑盒檢測Prdx5表達(dá)抑制后骨關(guān)節(jié)炎軟骨細(xì)胞中ROS的含量變化。3.通過流式細(xì)胞術(shù)、MTT法檢測抗氧化劑NAC對Prdx5表達(dá)抑制的骨關(guān)節(jié)炎軟骨細(xì)胞凋亡與增殖特性的影響。結(jié)果：1.流式細(xì)胞儀檢測結(jié)果提示骨關(guān)節(jié)炎軟骨細(xì)胞在Prdx5表達(dá)抑制后的凋亡率較對照組明顯增加(P0.05),MTT檢測結(jié)果顯示：與對照組相比,轉(zhuǎn)染Prdx5 shRNA的骨關(guān)節(jié)炎軟骨細(xì)胞的增殖能力明顯降低。2.ROS檢測結(jié)果提示在Prdx5表達(dá)有效抑制后,骨關(guān)節(jié)炎軟骨細(xì)胞內(nèi)ROS的含量與對照組相比顯著增加(P0.05)。3. Prdx5表達(dá)有效抑制后的骨關(guān)節(jié)炎軟骨細(xì)胞加入抗氧化劑NAC作用24 h后,骨關(guān)節(jié)炎軟骨細(xì)胞的增殖能力顯著增強(qiáng),凋亡率明顯下降(P0.05)。結(jié)論：Prdx5表達(dá)有效抑制后,導(dǎo)致骨關(guān)節(jié)炎軟骨細(xì)胞內(nèi)ROS的含量增加,從而致使骨關(guān)節(jié)炎軟骨細(xì)胞增殖抑制和凋亡增加。第三部分Prdx5調(diào)控骨關(guān)節(jié)炎軟骨細(xì)胞增殖和凋亡的機(jī)制目的：觀察Prdx5表達(dá)有效抑制后Wnt/p-catenin通路關(guān)鍵因子的變化情況,探討Prdx5對骨關(guān)節(jié)炎軟骨細(xì)胞增殖和凋亡影響的作用機(jī)制。方法：1. Prdx5 shRNA轉(zhuǎn)染骨關(guān)節(jié)炎軟骨細(xì)胞后應(yīng)用Western blot方法檢測Wnt/β-catenin通路核心蛋白β-catenin在細(xì)胞核和細(xì)胞質(zhì)中表達(dá)水平的變化以及其它關(guān)鍵因子Wnt-4, Frizzled-2, GSK-3β, p-GSK-3βser9, p-β-cateninser33/37與下游目的基因CyclinD1,MMP-13的蛋白表達(dá)情況；qRT-PCR檢測CyclinD1, MMP-13 mRNA表達(dá)變化情況。2.通過細(xì)胞免疫熒光實驗檢測Prdx5沉默后Wnt/p-catenin通路核心蛋白β-catenin在骨關(guān)節(jié)炎軟骨細(xì)胞內(nèi)的定位變化。3.應(yīng)用TOPflash熒光素酶報告基因系統(tǒng)檢測Prdx5沉默后Wnt/β-catenin通路核心蛋白β-catenin轉(zhuǎn)錄活性的變化情況。4. Western blot方法檢測Prdx5 shRNA轉(zhuǎn)染正常骨關(guān)節(jié)軟骨細(xì)胞后Wnt/β-catenin通路核心蛋白P-catenin在細(xì)胞核和細(xì)胞質(zhì)中表達(dá)水平的變化以及該通路其它關(guān)鍵因子GSK-3β, p-β-cateninser33/37的蛋白表達(dá)情況。5.采用Wnt/β-catenin通路抑制劑XAV-939作用于轉(zhuǎn)染Prdx5 shRNA的骨關(guān)節(jié)炎軟骨細(xì)胞后,通過ROS檢測試劑盒觀察細(xì)胞內(nèi)源性ROS含量的變化情況。結(jié)果：1. Western blot結(jié)果顯示：Prdx5表達(dá)有效抑制后,骨關(guān)節(jié)炎軟骨細(xì)胞中β-catenin蛋白表達(dá)水平在細(xì)胞核中明顯升高,細(xì)胞質(zhì)中明顯降低(P0.05)；而骨關(guān)節(jié)炎軟骨細(xì)胞中β-catenin的總蛋白水平在Prdx5抑制前后無明顯變化；p-β-cateninser33/37和GSK-3β的蛋白表達(dá)水平在Prdx5抑制后明顯降低,Wnt-4, Frizzled-2, p-GSK-3βser9, CyclinD1與MMP-13的蛋白表達(dá)水平則顯著升高(P0.05)；qRT-PCR結(jié)果提示CyclinD1和MMP-13 mRNA表達(dá)在Prdx5抑制后顯著升高(P0.05)。2.細(xì)胞免疫熒光結(jié)果顯示：Prdx5的表達(dá)有效抑制后,骨關(guān)節(jié)炎軟骨細(xì)胞核內(nèi)的P-catenin的熒光強(qiáng)度較對照組明顯增強(qiáng),說明骨關(guān)節(jié)炎軟骨細(xì)胞中的P-catenin由細(xì)胞質(zhì)移位到細(xì)胞核。3.熒光素酶報告基因系統(tǒng)檢測結(jié)果顯示Prdx5基因沉默后,骨關(guān)節(jié)炎軟骨細(xì)胞中β-catenin的轉(zhuǎn)錄活性顯著升高(p0.05)。4. Western blot結(jié)果顯示：Prdx5 shRNA轉(zhuǎn)染正常骨關(guān)節(jié)軟骨細(xì)胞后,Wnt/β-catenin通路核心蛋白β-catenin在細(xì)胞質(zhì)和細(xì)胞核中的表達(dá)水平以及該通路其它關(guān)鍵因子p-β-cateninser33/37,GSK-3β的蛋白表達(dá)水平?jīng)]有明顯變化。5.ROS檢測結(jié)果提示：單獨應(yīng)用XAV-939能明顯降低骨關(guān)節(jié)炎軟骨細(xì)胞的ROS含量(P0.05),然而XAV-939與Prdx5 shRNA共同作用于骨關(guān)節(jié)炎軟骨細(xì)胞后,ROS的含量沒有進(jìn)一步降低。結(jié)論：Prdx5表達(dá)抑制后能上調(diào)Wnt/β-catenin信號通路,Prdx5可能通過調(diào)控Wnt/β-catenin通路從而影響骨關(guān)節(jié)炎軟骨細(xì)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R684.3

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本文編號：1491945