基底/细胞系统刚度对人脐静脉内皮细胞增殖、凋亡及形态的影响

Effect of substrate stiffness on proliferation, apoptosis and morphology of vascular endothelial cells in vitro

目的:探讨基底刚度变化对人脐静脉内皮细胞(HUVECs)形态、增殖率和凋亡程度的影响.方法:采用双丙烯酰胺与丙烯酰胺构建不同硬度的聚丙烯酰胺基底(基底刚度为3.6kPa、10.7kPa及33.7kPa),将分离的人脐静脉内皮细胞(HUVECs)分别接种在不同基底刚度基底膜中连续培养96h;采用实时无标记细胞分析仪(RTCA)实时监测细胞72小时内的生长情况,并用CCK法检测三种细胞增殖速率;采用TUNEL染色分析三种不同基底刚度培养96h时细胞的凋亡情况;通过骨架蛋白Actin的免疫荧光染色比较细胞的形态,并比较各自骨架蛋白Actin和Tubulin的表达.结果:随着基底刚度的增加,细胞生长指数明显升高,细胞的增殖速率明显增强;基底刚度3.6kPa下HUVECs细胞96h 凋亡率均高于10.7kPa及33.7kPa;基底刚度10.7kPa及33.7kPa下HUVECs细胞96h 凋亡率无显著差异;共聚焦显微镜下,随着基底刚度的增加,HUVECs形态发生相应的变化,细胞由椭圆形逐渐向梭形转变,而细胞骨架蛋白的表达量也随之显著升高.结论:人脐静脉内皮细胞在不同基底刚度的体外培养中细胞的增殖、凋亡及形态均发生改变,较大的基底刚度更利于内皮细胞骨架分布、细胞增殖,同时降低细胞凋亡率.

Objective To explore the effect of substrate stiffness on proliferation, apoptosis and morphology of human umbilical vein endothelial cells (HUVECs) in vitro. Methods HUVECs are isolated and cultured for 96h on substrates of 3.6 kPa, 10.7 kPa and 33.7 kPa that built by bisacrylamide and acrylamide compounds. Cell growth status was monitored by RTCA (Real-Time Cellular Analysis) within 72h. At the same time, cell proliferation activity was detected using the cell counting kit-8 and survival curves were plotted. The apoptosis of these three kinds of cells was determined by one-step TUNEL staining. Cells morphology and their cytoskeleton were stained with fluorescent antibody and observed under a confocal microscope. The expressions of cytoskeletal proteins were detected by Western Blot. Results With the enhancement of base stiffness, the cell growth index and proliferation rate of HUVECs at different time points was higher significantly. Apoptosis rate of HUVECs at 96 h under baseline stiffness of 3.6 kPa was higher than 10.7 kPa and 33.7 kPa;though there was no significant difference in apoptosis rate under the under base stiffness between 10.7 kPa and 33.7 kPa. As the substrate stiffness increased, the changed of HUVECs under confocal microscopy was observed accordingly. That is to say, higher stiffness of dishes promoted cells to grow from elliptical to fusiform and expressed morphology increased cytoskeletal proteins. Conclusion The substrate stiffness plays an important role in the culture of HUVECs in vitro, which can change the proliferation, apoptosis rate and morphology of cells. Higher stiffness of dishes promoted cell proliferation and cytoskeletal distribution and inhibited cellular apoptosis.