聚乳酸三维微结构条件下人神经母细胞瘤细胞电压依从式钙通道的功能响应性

摘要

UV lithography, silicon etching and soft lithography were adopted to fabricate three-dimensional poly-L-lactide(PLLA)microwell patterns. Human neuroblastoma(SH-SY5Y)cells were integrated with the PLLA patterns, and then cell growth behaviors and distribution were evaluated. Voltage-gated calcium channels(VGCCs) response of SH-SY5Y cells cultured in microwells and on flat PLLA substrates was detected by confocal microscopy with a calcium fluorescent indicator( Calcium Green-1 ,AM). It was found that SH-SY5Y cells distributed on patterns's top surface showed two-dimensional(2-D)adhesion behavior,while those cells attached to the microwell's side-wall and bottom surface showed three-dimensional ( 3-D ) and boundary two-dimensional ( B2-D ) adhesion behaviors, respectively. In response to 50 mmol/L high K+ depolarization, the percentage of 2-D cells on PLLA flat substrates with positive VGCCs response was 91. 2% , while this percentage was 75% and 81% ,for 3-D and B2-D cells,respectively, significantly lower than that for 2-D cells. These results suggested that cell adhesion behaviors might be critical factors in affecting VGCCs responsiveness of SH-SY5Y cells. Microwell patterns could be an effective way for engineering 3-D microenvironment for neural cell culture and establishing 3-D cell-based biosensing platforms.%以紫外光光刻、硅蚀刻及软光刻技术制备了聚乳酸( poly-L-lactide,PLLA)三维微小凹图式,考察入神经母细胞瘤细胞( SH-SY5Y)在微小凹结构上的生长与分布行为.以共聚焦显微技术结合钙离子荧光染料Calcium Green-1,AM评价SH-SY5Y细胞在PLLA三维微小凹图式和平面基底上电压依从式钙离子通道(VGCCs)的功能响应性.实验发现,分布在微小凹顶部的细胞为二维生长方式,凹内侧壁的为三维生长方式,凹内底部的为边界二维生长方式;以50 mmol/L高钾溶液去极化刺激发现,三维及边界二维细胞VGCCs阳性响应比率为75%和81%,较平面培养的91.2%明显降低.上述结果表明,PLLA三维微结构及细胞生长行为是影响SH-SY5Y细胞VGCCs功能响应性的重要因素,微小凹图式是模拟神经细胞三维生长环境进而构建三维细胞生物传感器的有效途径.