【摘要】 制备了微柱名义直径为4μm或10μm,名义间距为4μm或7μm,名义高度为4μm的聚二甲基硅氧烷微柱阵列型拓扑结构基底,研究了HepG2细胞与拓扑结构基底复合后细胞瞬时受体电位通道TRPV1、TRPV4在基因和蛋白水平的表达及其功能响应性。细胞TRPV1和TRPV4在基因水平表达的评价采用定量PCR技术进行;TRPV1和TRPV4在蛋白水平的表达以免疫印迹和免疫荧光染色确认;TRPV1和TRPV4功能响应性的研究系以TRPV1和TRPV4激动剂辣椒素和4α-佛波醇-12,13-二葵酸酯刺激细胞,采用钙离子染料钙绿-1结合激光共聚焦显微技术记录钙内流动态过程,以钙内流荧光响应幅度及阳性响应比率进行评价。实验结果表明,在四种拓扑结构基底上细胞TRPV1和TRPV4的mRNA表达量均显著高于平面基底上相应值。免疫印迹实验证实了TRPV1和TRPV4在蛋白水平的表达,且拓扑结构基底上TRPV1和TRPV4免疫荧光染色强度较之平面基底相应值明显增高或趋于增高。在激动剂作用下,TRPV1介导的钙内流表现为快速去敏感化(25秒内)的瞬态内流,且拓扑结构基底上阳性响应细胞比例或相对荧光响应幅度较之平面基底相应值增高;而拓扑结构基底上细胞TRPV4阳性响应细胞比例和相对荧光响应幅度较之平面基底均全面明显升高。上述结果表明,TRPV介导的离子信号可能是基底拓扑结构优化HepG2细胞功能表型的重要信号机制。更多还原

【Abstract】 Polydimethylsiloxane( PDMS) micropillar arrayed topographic substrates were fabricated,with dimensions of 4 and 10μm in nominal pillar diameter,4 and 7μm in spacing and 4μm in height,to investigate the expression and responsiveness of transient receptor potential( TRP) V1 and TRPV4 channels of HepG2 cells cultured on the substrates. Quantitative real-time PCR( qRT-PCR) analysis revealed that the mRNA levels of TRPV1 and TRPV4 were significantly up-regulated in the cells grown on all four topographic substrates,when compared with the cells grown on the flat PDMS substrates. The presence of TRPV1 and TRPV4 proteins in HepG2 cells was confirmed with Western blotting,and similar up-regulation of these two channel proteins by the substrate topography also revealed by stronger immunofluorescence staining. Subsequently, the channel responsiveness of TRPV1 and TRPV4 was quantified by using the calcium inflow-responding magnitudes and percentages of cells having a defined responding magnitude upon stimulation by the channel agonists capsaicin and 4-α-phorbol- 12,13-didecanoate( 4αPDD), respectively. Herein, Calcium Green-1 as a fluorescent indicator was employed in its dynamic assessment by confocal laser scanning microscopy. The results displayed that upon stimulation by the channel agonist,the calcium influx through TRPV1 exhibits a dynamic characteristic of rapid desensitization with a transient within 25 seconds,and either the relative fluorescence responding amplitudes or the percentages of responsive cells on the topographic substrates are greater than those observed from the cells on the flat substrates. By contrast,stimulation by the TRPV4 agonist caused a calcium response with much slower recovery within 5 minutes,and both the relative fluorescence responding amplitudes and the percentages of responsive cells on the topographic substrates are higher than those of the cells on the flat substrates. Collectively,these data indicate that the TRPV-mediated ion signaling elicits an important role in the cellular phenotypic and functional regulation by the substrate topography.更多还原