软基底介导细胞骨架蛋白与力学特性的改变  

Changes in cytoskeleton proteins and mechanical properties of the cells on soft substrates

作　　者：侯添 安美文 王立 Hou Tian;An Mei-wen;Wang Li(School of Mechanics,Taiyuan University of Technology,Taiyuan 030024,Shanxi Province,China)

机构地区：[1]太原理工大学力学学院,山西省太原市030024

出　　处：《中国组织工程研究》2018年第22期3461-3466,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(11372208)~~

摘　　要：背景:前期研究发现在不同硬度基底上生长的细胞,细胞调控受基底硬度的影响,但抑制肌球蛋白的活性会阻碍基底硬度对细胞的调控作用,然而关于基底硬度对于肌球蛋白的影响及在这个过程中对于肌球蛋白的量化描述较少。目的:量化肌球蛋白Ⅱ分布及运动速率、肌动蛋白纤维分布与基底硬度的关系。方法:采用聚丙烯酰胺凝胶制备硬度分别为1,10,150 kPa的基底,将稳定表达转染荧光肌球蛋白Ⅱ的宫颈癌Hela细胞分别接种于不同硬度的基底上12 h,检测不同硬度基底上生长的宫颈癌细胞荧光肌球蛋白Ⅱ沿细胞长轴的荧光分布,利用荧光漂白恢复技术测定荧光肌球蛋白Ⅱ的运动速率,荧光染色观察肌动蛋白纤维的分布,利用微管吸吮技术测定单个宫颈癌细胞在铺展状态下的细胞膜弹性模量。结果与结论:(1)硬度为150 kPa基底上细胞的边缘荧光肌球蛋白Ⅱ强度显著高于其他部分;在1 kPa和10 kPa基底上生长的细胞,荧光肌球蛋白Ⅱ在边缘的分布呈现出一定回落现象;(2)硬度为150 kPa基底上细胞末端荧光肌球蛋白Ⅱ的恢复速率最大,显著快于1 kPa和10 kPa基底上细胞的恢复速率;(3)3种硬度基底上生长的细胞肌动蛋白纤维整体荧光强度近似;(4)硬度为150 kPa基底上Hela细胞细胞膜的弹性模量显著高于1 kPa和10 kPa基底上的细胞;(5)结果表明,基底硬度会明显改变肌球蛋白、肌动蛋白等重要细胞骨架蛋白区域性作用的效果,影响癌细胞在不同微环境下的细胞状态。BACKGROUND： Preliminary studies have shown that substrate hardness is directly involved in cell regulation, but this effect can be hindered via suppression of myosin expression. Therefore, the effect of substrate hardnesson myosin expression needs to be further quantified. OBJECTIVE： To quantify the effect of substrate hardness on the distribution and movement rate of myosin II as well as on the distribution of actin fibers. METHODS： Polyacrylamide gels were used to prepare substrates with hardness of 1, 10, 150 kPa. Cervical cancer were Hela cells stably transfected with fluorescent myosin II, and these transfected cells were incubated onto the substrates of different hardness. The fluorescence distribution of fluorescent myosin II along the long axis of Hela cells on the substrates of different hardness was measured. The movement rate of fluorescent myosin II was determined by fluorescence photobleaching recovery technique. F-actin distribution was observed using fluorescent staining. The elastic modulus of single cervical cancer cells spreading on different substrates was determined using micropipette aspiration technique. RESULTS AND CONCLUSION： For the cells growing on the 150 kPa substrate, the highest intensity of fluorescent myosin II was located on the cell edge. For the cells on the substrates of 1 and 10 kPa, there was a reduced distribution of fluorescent myosin II on the cell edge. For the cells on the 150 kPa substrate, the recovery rate of fluorescent myosin II was significantly faster than that on the substrates of 1 and 10 kPa. Actin fibers in the cells growing on the substrates of 1, 10 and 150 kPa shared similarity in the overall fluorescence intensity. The elastic modulus of Hela cells on the substrate of 150 kPa was significantly higher than that on 1 kPa and 10 kPa substrates. Experimental results show that substrate hardness can significantly alter the regional effects of cytoskeletal proteins such as myosin and actin, and affect the cell status of cancer cells in different microenv

关 键 词：肌球蛋白类 肌动蛋白类:弹性模量 组织工程 细胞骨架 肌球蛋白 肌动蛋白纤维 细胞外基质 聚丙烯酰胺凝胶 基底硬度 弹性模量 人宫颈癌细胞 生物材料 

分 类 号：R318[医药卫生—生物医学工程]