Piezo1蛋白在人退变软骨细胞应力模型中的表达特点

目的:探索新型机械敏感性离子通道Piezo1蛋白在人退变软骨细胞应力模型中的表达特点。方法:构建人退变软骨细胞体外培养-应力刺激模型,采用多通道细胞牵张应力加载系统FX-4000T处理软骨细胞,根据预试验结果加载0.5 Hz的加载频率和20%的细胞拉伸率。按细胞的处理时间分成0 h,2 h,12 h,24 h和48 h机械应力组。采用RT-PCR技术,Western-blot检测Piezo1蛋白在周期性牵张应力作用下的表达水平。利用激光共聚焦显微镜检测Piezo1蛋白荧光的强弱。结果:(1)RT-PCR结果显示,2 h牵张应力组Piezo1的基因相对表达量较0 h牵张应力组增加(F=13.917,q=0.037 1,P0.05),24 h牵张应力组Piezo1基因相对表达量达峰值,而48 h牵张应力组Piezo1基因相对表达量较24 h牵张应力组降低,差异有统计学意义(F=13.917,q=0.049 5,P0.05)。(2)Western-blot结果显示,2 h牵张应力组Piezo1的蛋白表达量较空白对照组(0 h牵张应力组)略有增加(F=19.341,q=0.037 1,P0.05),24 h牵张应力组的Piezo1蛋白的表达量最多,而48 h牵张应力组的Piezo1蛋白表达量较24 h牵张应力组降低(F=19.341,q=0.017 7,P0.05)。(3)Piezo1蛋白表达于髓核细胞的细胞质与细胞核,并且随着加力时间的增加,蛋白的荧光强度也有相应的增加。结论:在人类退变软骨细胞中,新型机械敏感性离子通道Piezo1蛋白有微量表达,加载周期性机械拉伸力后,Piezo1蛋白的表达量增多,并且呈现时间依赖性。

Expression characteristics of Piezo1 protein in stress models of human degenerative chondrocytes

Objective: To explore the expression characteristics of new mechanosensitive ion channel Piezo1 protein in stress models of human degenerative chondrocytes.Methods: The stress stimulation model of human degenerative chondrocytes in vitro was constructed. Multi-channel cell stretch stress loading system FX-4000T was used to treat chondrocytes. According to the results of pre-test,the loading frequency of 0.5 Hz and the cell elongation of 20% were loaded. According to cell processing time,it was divided into 0 h,2 h,12 h,24 h and 48 h mechanical stress group. The RT-PCR and Western-blot were used to test the expression of the Piezo1,also the Laser scanning confocal microscope (LSCM) was used to test the intensity of the fluorescence of the Piezo1.Results: (1)The result of the RT-PCR showed that the expression of the Piezo1 in the 2 h group was higher than the 0 h group(F=13.917,q=0.037 1,P<0.05). The expression of the piezo1 in the 24 h group was the highest. While the expression of the piezo1 in the 48 h group was lower than the expression of the piezo1 in the 24 h group(F=13.917,q=0.049 5,P<0.05). (2)The result of the Western-blot showed that the 2 h group was higher than the 0 h group(F=19.341,q=0.037 1,P<0.05). The expression of the 24 h had the highest expression which was higher than the 48 h group(F=19.341,q=0.017 7,P<0.05). (3) The Piezo1 protein was extensively expressed in the cytoplasm and nucleus of the nucleus pulposus cells. And with the increase of stress processing time,the fluorescence intensity of the protein also increased.Conclusion: In human degeneration cartilage cells,the new mechanio sensitive ion channel Piezo1 protein has a trace expression. After loading periodic mechanical tensile force,the expression of Piezo1 protein increases with time dependence.