基底刚度对细胞骨架与细胞弹性模量的调节作用

发布时间：2018-04-15 22:42

  本文选题：基底刚度 + 肌球蛋白Ⅱ　； 参考：《太原理工大学》2017年硕士论文

【摘要】：细胞外基质是一个复杂的大分子网络,具有独特的物理特性、生物化学特性和力学特性,这些性质能够直接或间接地影响细胞的生物学行为,成为正常组织发育必不可少的因素。当细胞外基质的代谢发生异常时,细胞外基质的组织结构和力学特性发生改变,同时伴随一些疾病的发生。例如组织的纤维化、癌细胞的增殖、迁移、上皮间质转换等。由此可见细胞所处的微环境刚度在癌症的产生、迁移和恶化过程中起重要作用,研究癌细胞如何感知周围组织刚度以及对硬度的响应规律,能够为应对癌症的产生机制提供有价值的参考,并协助改进治疗方案。本文主要研究不同刚度的基底对于细胞生物力学特性的影响。采用具有良好生物兼容性的水溶性聚合物——聚丙烯酰胺水凝胶,作为模拟细胞外环境的培养基底。通过调节丙烯酰胺和甲叉双丙烯酰胺的比例,制备不同刚度的弹性基底。把处于对数生长期的宫颈癌细胞接种到1KPa、10KPa、150KPa的聚丙烯酰胺水凝胶基底上,进行24-48小时的培养,对培养后的宫颈癌细胞进行以下实验,观测基底硬度对细胞生物力学行为特性的影响:(1)测量细胞骨架中分子马达蛋白——肌球蛋白Ⅱ的分布与运动速率。将宫颈癌细胞稳定转染荧光肌球蛋白Ⅱ,利用激光共聚焦显微镜测定三种刚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;运用荧光漂白恢复技术,测定荧光肌球蛋白Ⅱ在细胞末端的恢复速率。(2)测量肌动蛋白纤维在整个细胞内的分布情况。使用Phalloidin-Tetramethylrhodamine B Isothiocyanate对宫颈癌细胞肌动蛋白纤维进行荧光染色,测定三种刚度上培养的宫颈癌细胞肌动蛋白纤维的分布。(3)采用微管吸吮技术,结合半无限体模型,测定单个宫颈癌细胞在铺展状态下的细胞弹性模量。结果显示,不同硬度基底上生长的宫颈癌细胞呈现出肌球蛋白Ⅱ和肌动蛋白纤维的分布差异。在软基底上生长的宫颈癌细胞,细胞边缘肌球蛋白Ⅱ和肌动蛋白纤维分布较少,肌球蛋白Ⅱ的运动速率较慢,细胞弹性模量降低;在较硬基底上生长的宫颈癌细胞,肌球蛋白Ⅱ和肌动蛋白蛋白纤维呈现出明显的细胞边缘聚集状态,细胞弹性模量也明显高于在较软两组基底上生长的细胞。实验表明,基底硬度会明显改变肌球蛋白Ⅱ、肌动蛋白等重要的细胞骨架蛋白区域性作用的效果,对细胞的弹性模量造成影响。影响癌细胞在不同微环境下的细胞状态。
[Abstract]:Extracellular matrix (ECM) is a complex network of macromolecules with unique physical, biochemical and mechanical properties that directly or indirectly affect the biological behavior of cells.To become an essential factor in the development of normal tissues.When the metabolism of extracellular matrix is abnormal, the tissue structure and mechanical properties of extracellular matrix change, and some diseases occur.Such as tissue fibrosis, cancer cell proliferation, migration, epithelial interstitial transition and so on.It can be seen that the microenvironmental stiffness of the cell plays an important role in the generation, migration and deterioration of cancer, and studies how cancer cells perceive the stiffness of the surrounding tissue and its response to hardness.It can provide valuable reference for the mechanism of cancer response and help to improve the treatment plan.In this paper, the effects of different stiffness substrates on the biomechanical properties of cells are studied.Polyacrylamide hydrogel, a water-soluble polymer with good biocompatibility, was used as the culture substrate to simulate the extracellular environment.The elastic substrates with different stiffness were prepared by adjusting the ratio of acrylamide to methacrylate.The cervical cancer cells in logarithmic growth phase were inoculated on the polyacrylamide hydrogel substrate of 1 KPA 10 KPA and 150 KPA, and cultured for 24-48 hours. The following experiments were carried out on the cultured cervical cancer cells.The effect of substrate hardness on the biomechanical behavior of cells was observed. (1) the distribution and movement rate of molecular motor protein-myosin 鈪，

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