本文選題：Piezo1 + 軟骨細(xì)胞　； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:目的是探究臨床OA患者疾病進(jìn)展中關(guān)節(jié)軟骨軟骨細(xì)胞的機(jī)械信息牽張性離子通道蛋白Piezo1的機(jī)制以及其經(jīng)MAPK/ERK5/ERK1/2的信號(hào)通路介導(dǎo)OA患者中軟骨細(xì)胞過(guò)度凋亡的機(jī)制過(guò)程。方法:取材OA患者的軟骨細(xì)胞、裁剪、培養(yǎng)0A軟骨細(xì)胞,利用Flexcell公司的細(xì)胞周期牽張應(yīng)力加載系統(tǒng),將軟骨細(xì)胞分成空白組,即(0h機(jī)械牽張應(yīng)力組),各加力組(根據(jù)預(yù)實(shí)驗(yàn)結(jié)果分成:2h機(jī)械牽張應(yīng)力組,12h機(jī)械牽張應(yīng)力組,24h機(jī)械牽張應(yīng)力組和48h機(jī)械牽張應(yīng)力組),以及抑制劑組(Piezo1的特異性拮抗劑GsMTx4組,ERK5的特異性抑制劑BIX02188組,ERK1/2的特異抑制劑PD98059組),激光共聚焦顯微鏡觀察定位Piezo1蛋白以及ERK5,ERK1/2在軟骨細(xì)胞的表達(dá)位置;用Western-blot和RT-qPCR檢測(cè)各組細(xì)胞Piezo1和MAPK/ERK信號(hào)通路分子ERK5和ERK1/2的表達(dá)量、凋亡相關(guān)基因Bcl-2、Bcl-2相關(guān)X蛋白(Bcl-associated X protein,Bax)及Bcl-2相關(guān)促凋亡蛋白(Bcl-associated death promoter,BAD)以及Caspase-3的表達(dá)量;用AV-PI凋亡試劑盒檢測(cè)細(xì)胞凋亡情況。結(jié)果:(1)免疫熒光結(jié)果(激光共聚焦顯微鏡觀察)顯示:Piezo1蛋白可以在細(xì)胞核和細(xì)胞質(zhì)表達(dá)。ERK5和ERK1/2可以表達(dá)在細(xì)胞質(zhì)中。(2)Real-time熒光定量PCR檢測(cè)結(jié)果顯示:Pizeo1 mRNA在0A患者軟骨細(xì)胞中有少量表達(dá),2h機(jī)械牽張應(yīng)力組的Piezo1的表達(dá)量與空白組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。12h機(jī)械牽張應(yīng)力組表達(dá)量較2h機(jī)械牽張應(yīng)力組明顯增加(q0.05,P0.05),24h機(jī)械牽張應(yīng)力組的Piezo1蛋白表達(dá)達(dá)峰值,48h機(jī)械牽張應(yīng)力組表達(dá)量較24h機(jī)械牽張應(yīng)力組的Piezo1蛋白的表達(dá)有明顯下降(q0.05,P0.05)。ERK1/2、Bax/Bcl-2、Caspase-3、ERK5、Bcl-2、BAD和Bax mRNA表達(dá)也表現(xiàn)出相同趨勢(shì)。抑制劑組表達(dá)均下降。Western-blot蛋白檢測(cè)顯示Piezo1蛋白在2h機(jī)械牽張應(yīng)力組的表達(dá)量要比空白組略有增加(P0.05),48h機(jī)械牽張應(yīng)力組與24h機(jī)械牽張應(yīng)力組相比較,Piezo1蛋白的表達(dá)量明顯下降(q0.001,P0.05)。(3)流式細(xì)胞儀檢測(cè)細(xì)胞凋亡結(jié)果顯示2h機(jī)械牽張應(yīng)力組細(xì)胞的早期凋亡細(xì)胞凋亡率開始增加,12h機(jī)械牽張應(yīng)力組的軟骨細(xì)胞的晚期調(diào)亡率明顯增加,其中,24h機(jī)械牽張應(yīng)力組晚期細(xì)胞凋亡率最高,48h機(jī)械牽張應(yīng)力組晚期凋亡率比24h機(jī)械牽張應(yīng)力組相比降低(P0.05,q0.05)。(4)熒光定量PCR(RT-PCR)檢測(cè)結(jié)果和Western-blot蛋白檢測(cè)均表明Piezo1、ERK1/2、Bax/Bcl-2、Caspase-3、ERK5、Bcl-2、BAD和Bax mRNA的表達(dá)量與晚期凋亡具有相同的趨勢(shì)。結(jié)論:新型機(jī)械敏感性離子通道Piezo1蛋白參與OA患者的軟骨細(xì)胞晚期凋亡過(guò)程,且通過(guò)MAPK/ERK5信號(hào)通路和MAPK/ERK1/2信號(hào)通路啟動(dòng)軟骨細(xì)胞凋亡。
[Abstract]:Aim: to investigate the mechanism of mechanical information distraction ion-channel protein (Piezo1) in articular chondrocytes during the progression of OA and the mechanism of its signaling pathway through MAPK/ERK5/ERK1/2 to mediate the excessive apoptosis of chondrocytes in OA patients. Methods: the chondrocytes of OA patients were cut and cultured. The chondrocytes were divided into blank group by Flexcell cell cycle strain stress loading system. That is, 0 h mechanical stretch stress group, each stress group is divided into two groups according to the experimental results: 1: 2 h mechanical stretch stress group, 12 h mechanical stretch stress group, 24 h mechanical stretch stress group and 48 h mechanical stretch stress group, and the specificity of Piezo1 in inhibitor group In GsMTx4 group, ERK5 specific inhibitor BIX02188 group, ERK1 / 2 specific inhibitor PD98059 group, the localization of Piezo1 protein and ERK5 ERK1 / 2 expression in chondrocytes were observed by confocal laser microscopy. The expressions of ERK5 and ERK1/2 in Piezo1 and MAPK/ERK signaling pathway, Bcl-associated X protein (Bcl-associated X protein), Bcl-2 associated apoptosis promoting protein (Bcl-associated death promoter Badad) and Caspase-3 were detected by Western-blot and RT-qPCR. AV-PI apoptosis kit was used to detect apoptosis. Results the results of immunofluorescence (laser confocal microscopy) showed that: Piezo1 protein could be expressed in nucleus and cytoplasm. ERK5 and ERK1/2 could be expressed in cytoplasm. Real-time quantitative PCR analysis showed that: Pizeo1 mRNA was expressed in cartilage of 0A patients. There was no significant difference in the expression of Piezo1 between the 2 h mechanical stress group and the blank group. Compared with the 2 h mechanical stretch stress group, the expression of Piezo1 in the 2 h mechanical stretch stress group was significantly higher than that in the 2 h mechanical stretch stress group. The expression of Piezo1 in the 24 h mechanical stretch stress group was significantly higher than that in the 2 h mechanical stretch stress group. The expression of Piezo1 protein in the group of mechanical stretch stress at 48h was significantly decreased compared with that in the group of 24 h mechanical stretch stress. The expression of Piezo1 protein was similar to that in the group of 24 hours of mechanical stretch stress. The expression of Piezo1 protein was similar to that in the group of 24 h mechanical stretch stress, and the expression of Piezo1 protein was also decreased in the same trend as that in the group of 24 hours of mechanical stretch stress. The expression of Piezo1 protein in the 2 h mechanical stretch stress group was slightly higher than that in the control group. The expression of Piezo1 protein in the 48 h mechanical stretch stress group was significantly higher than that in the 24 h mechanical stretch stress group. The results of flow cytometry showed that the apoptosis rate of the cells in 2 h mechanical stretch stress group began to increase, and the late apoptosis rate of chondrocytes in 12 h mechanical stretch stress group increased significantly. The apoptotic rate of late stage cells in mechanical stress group was the highest in 24 h mechanical stretch stress group. Compared with 24 h mechanical stretch stress group, the late apoptosis rate of mechanical strain stress group was lower than that of 24 h mechanical stretch stress group. The results of fluorescence quantitative PCRRT PCR and Western-blot protein detection showed that Piezo1 / ERK1 / 2BaxBcl-2Caspase-3ERK5 Bcl-2BAD and Bax mRNA were also detected by Piezo1 / ERK1 / 2Bcl-2Caspase-3ERK5BAD and Bax mRNA. There is the same trend between the quantity of reaching and the late stage of apoptosis. Conclusion: the novel mechanically sensitive ion channel Piezo1 protein is involved in the late apoptosis of chondrocytes in OA patients and chondrocytes apoptosis is initiated through MAPK/ERK5 signaling pathway and MAPK/ERK1/2 signaling pathway.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R684.3

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相關(guān)期刊論文 前1條

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本文編號(hào)：1916381