Piezo1蛋白通过MAPK信号通路介导软骨细胞凋亡的机制研究

发布时间：2018-05-20 21:57

  本文选题：Piezo1 + 软骨细胞　； 参考：《青岛大学》2017年硕士论文

【摘要】：目的:目的是探究临床OA患者疾病进展中关节软骨软骨细胞的机械信息牵张性离子通道蛋白Piezo1的机制以及其经MAPK/ERK5/ERK1/2的信号通路介导OA患者中软骨细胞过度凋亡的机制过程。方法:取材OA患者的软骨细胞、裁剪、培养0A软骨细胞,利用Flexcell公司的细胞周期牵张应力加载系统,将软骨细胞分成空白组,即(0h机械牵张应力组),各加力组(根据预实验结果分成:2h机械牵张应力组,12h机械牵张应力组,24h机械牵张应力组和48h机械牵张应力组),以及抑制剂组(Piezo1的特异性拮抗剂GsMTx4组,ERK5的特异性抑制剂BIX02188组,ERK1/2的特异抑制剂PD98059组),激光共聚焦显微镜观察定位Piezo1蛋白以及ERK5,ERK1/2在软骨细胞的表达位置;用Western-blot和RT-qPCR检测各组细胞Piezo1和MAPK/ERK信号通路分子ERK5和ERK1/2的表达量、凋亡相关基因Bcl-2、Bcl-2相关X蛋白(Bcl-associated X protein,Bax)及Bcl-2相关促凋亡蛋白(Bcl-associated death promoter,BAD)以及Caspase-3的表达量;用AV-PI凋亡试剂盒检测细胞凋亡情况。结果:(1)免疫荧光结果(激光共聚焦显微镜观察)显示:Piezo1蛋白可以在细胞核和细胞质表达。ERK5和ERK1/2可以表达在细胞质中。(2)Real-time荧光定量PCR检测结果显示:Pizeo1 mRNA在0A患者软骨细胞中有少量表达,2h机械牵张应力组的Piezo1的表达量与空白组比较差异无统计学意义(P0.05)。12h机械牵张应力组表达量较2h机械牵张应力组明显增加(q0.05,P0.05),24h机械牵张应力组的Piezo1蛋白表达达峰值,48h机械牵张应力组表达量较24h机械牵张应力组的Piezo1蛋白的表达有明显下降(q0.05,P0.05)。ERK1/2、Bax/Bcl-2、Caspase-3、ERK5、Bcl-2、BAD和Bax mRNA表达也表现出相同趋势。抑制剂组表达均下降。Western-blot蛋白检测显示Piezo1蛋白在2h机械牵张应力组的表达量要比空白组略有增加(P0.05),48h机械牵张应力组与24h机械牵张应力组相比较,Piezo1蛋白的表达量明显下降(q0.001,P0.05)。(3)流式细胞仪检测细胞凋亡结果显示2h机械牵张应力组细胞的早期凋亡细胞凋亡率开始增加,12h机械牵张应力组的软骨细胞的晚期调亡率明显增加,其中,24h机械牵张应力组晚期细胞凋亡率最高,48h机械牵张应力组晚期凋亡率比24h机械牵张应力组相比降低(P0.05,q0.05)。(4)荧光定量PCR(RT-PCR)检测结果和Western-blot蛋白检测均表明Piezo1、ERK1/2、Bax/Bcl-2、Caspase-3、ERK5、Bcl-2、BAD和Bax mRNA的表达量与晚期凋亡具有相同的趋势。结论:新型机械敏感性离子通道Piezo1蛋白参与OA患者的软骨细胞晚期凋亡过程,且通过MAPK/ERK5信号通路和MAPK/ERK1/2信号通路启动软骨细胞凋亡。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R684.3

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