微柱阵列型拓扑结构对细胞通道功能响应性的影响要点分析.doc

摘 要 制备了微柱名义直径为4 μm或10 μm，名义间距为 4 μm或 7 μm，名义高度为4 μm 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。 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。相同微柱间距(4 μm、7 μm)，较大微柱直径(10μm)的基底促进细胞铺展，同时伴随着较高的TRPV1或TRPV4基因表达水平；相同微柱直径(4 μm或10 μm)，较小微柱间距(4μm)的基底一定程度促进细胞铺展，但却伴随着较低的TRPV1或TRPV4基因表达水平。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。 关键词：HepG2细胞，PDMS微柱阵列拓扑结构，激光共聚焦显微技术，TRPV1通道，TRPV4通道  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. Our experiments showed that the morphological spreading and degree of polarization of HepG2 cells changed significantly on the topographic substrates, as compared to cells on flat PDMS 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 substrates. In the same dimensions of 4 or 10μm, the topographic substrates with smaller spacing (4μm) were related to higher spreading areas and lower mRNA levels of TRPV1 and TRPV4, when compared with the cells grown on the topographic substrates with larger spacing (7μm). In the same spacing of 4 or 7μm, the topographic substrates with larger dimensions (10μm) were related to higher spreading areas and