Effect And Mechanism Of Micropillar Arrayed Topographic Substrates On VGCC Functional Response In SH-SY5Y Cells

In order to explore the possibility of using micropillar arrayed topographic substrates for regulating the responsiveness of VGCC function and providing the necessary theoretical support for the construction of new drug screening platform targeting VGCC,In this paper,PLLA micropillar arrayed topographic substrates with diameter of 2 ?m and 4 ?m,spacing of 2 ?m and 7 ?m and height of 4 ?m were fabricated by micromachining techniques.Cell spreading,cytoskeleton and vinculin of SH-SY5 Y cells on different substrates were investigated by scanning electron microscopy and fluorescence quantification.Cell proliferation of SH-SY5 Y cells on different substrates were investigated by MTS.The effect of micropillar substrates on the responsiveness of VGCC were investigated using calcium ion dye Calcium Green-1AM and laser confocal technique.The expression of VGCC on mRNA and protein were investigated by qPCR technique and fluorescence quantitative technique.The method of measuring cell membrane potential by TMRM was improved and the membrane potential of SH-SY5 Y cells on different substrates were investigated.The results showed that the micropillar arrayed substrates had a significant effect on the cell morphology of SH-SY5 Y cells.When the cells were in the small micropillars,the cells were inclined to grow on the micropillar and the cell roundness increased.As the spacing between the micropillars increases,the cells were trapped in the micropillars.The thick micropillars prevent the spreading of the cells.The thin micropillars provide cells with adhesion points and promote cell spreading,and even some micropillars penetrate in cytoplasm or nucleus,but this change in cell morphology did not have a significant impact on cell proliferation.Compared with the cells on the flat substrate,the distribution of the focal adhesion and the cytoskeleton of the SH-SY5 Y cells on micropillar substrates was characterized by the concentration on the micropillars or around the micropillars.The micropillars destroyed the cytoskeletal ordering.The VGCC responsiveness were significantly different in the flat,2-2 and 2-7 substrates,and this effect was related to the cell spreading area.The larger the cell spreading area,the stronger the VGCC responsiveness.However,this difference was not associated with the expression of VGCC channels.The expression of VGCCs of cells with large spreading area tend to decrease.In this study,we modified the nonspecific adsorption method of TMRM in membrane potential measurement to improve the accuracy of the results.The measurement results show that although the micropillar substrates have a significant effect on the cell membrane potential,this change does not correspond to the VGCC responsiveness difference.The experimental results show that the use of micropillar substrate to regulate VGCC functional responsiveness is feasible.However,the construction of VGCC-based drug-screening platform based on micropillar substrate also depends on the precise design and mechanism of micropillar arrayed topographic substrates.