聚乳酸微柱阵列型拓扑结构基底上SH-SY5Y细胞VEGF和IL-8的分泌及表达  

Secretion and expression of vascular endothelial growth factor and interleukin-8 by SH-SY5Y human neuroblastoma cells

作　　者：范志刚[1] 林雨[1] 黄岂平[1] 罗美蓉[1] 田青华[1] 钟冬火[1] 冯全义[1] 吴泽志[1] 

机构地区：[1]重庆大学生物工程学院生物流变科学与技术教育部重点实验室,重庆400044

出　　处：《生物工程学报》2013年第11期1629-1643,共15页Chinese Journal of Biotechnology

基　　金：高等学校博士学科点专项科研基金资助课题(No.20100191110032);重庆市科技攻关重点项目(No.CSTC2009AB5202);国家自然科学基金(No.30870570);教育部国家外专局生物力学与组织修复学科创新引智基地(111计划)(No.B0623);重庆大学大型仪器设备开放基金(No.2011121535)资助~~

摘　　要：以紫外光光刻、硅蚀刻及复制模塑技术制备了聚乳酸(PLLA)微柱阵列型拓扑结构基底,考察了SH-SY5Y人神经母细胞瘤细胞在拓扑结构基底上的生长及血管内皮生长因子(VEGF)和白细胞介素-8(IL-8)的分泌及表达。细胞的形态及铺展采用扫描电子显微图像进行分析,细胞在拓扑结构基底上生长24 h后的VEGF及IL-8分泌量采用ELISA进行检测,VEGF及IL-8在mRNA水平的表达量以实时定量PCR进行评价。实验中成功制备了微柱名义直径为2μm和4μm、微柱名义间距为2μm和7μm的4种拓扑结构基底。研究发现,SH-SY5Y细胞在2-2μm(微柱名义直径-名义间距)、4-2μm、4-7μm拓扑结构基底上的VEGF和/或IL-8分泌量和表达较之PLLA平面基底上相应值出现上调,而在2-7μm拓扑结构基底上VEGF及IL-8二者均表现出表达和分泌量大幅度和明显的上调。与在PLLA平面基底上相比,SH-SY5Y细胞在拓扑结构基底上表现出细胞形态(铺展面积及圆度)的明显变化,细胞VEGF及IL-8分泌量和表达的上调伴随细胞铺展面积的明显降低。结果表明微柱阵列型拓扑结构是影响SH-SY5Y细胞VEGF、IL-8分泌及表达的重要微环境因素,VEGF和IL-8可能构成SH-SY5Y细胞在拓扑结构基底上生长的重要分泌物标志。To establish vascular endothelial growth factor （VEGF） and interleukin-8 （IL-8） as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide （PLLA） micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay （ELISA） and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 ~tm and 4 pro, and a nominal pillar spacing of 2/~m and 7 #m. We found that the secretion and mRNA expression of VEGF and^or IL-8 by SH-SY5Y cells on 2-2 gm （pillar diameter-spacing）, 4-2 gm and 4-7 gm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 gm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, ceils on topographic substrates showed significant changes in morphology （spreading area, perimeter and roundness）, and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SYSY cells.

关 键 词：复制模塑 ELISA 实时定量PCR 人神经母细胞瘤细胞 分泌物标志 

分 类 号：Q25[生物学—细胞生物学]