基于微流控模型初探细胞外基质粘弹性对HepG2转移过程的影响

发布时间：2019-03-31 19:59

【摘要】：癌细胞的转移是一个复杂、多步骤且随时空级联动态变化的过程，是超过90%的癌症患者死亡的原因。癌细胞转移实质上也是癌细胞与其周围微环境相互作用的过程，受其周围微环境中多种生物化学和生物物理学因素的影响。基质力学作为一个重要的微环境因素而备受关注。基质力学性质对细胞行为的影响，目前研究较多的是基质的“硬度”对细胞行为、结构和功能的调节。在胞外基质力学对癌细胞转移的影响方面已取得的研究结果表明，基质的力学特性对癌细胞转移过程具有与生化因子同等重要的作用：近来的大量研究结果都显示不仅细胞的行为、结构和功能受到细胞外基质硬度的调节，而且基质硬度对于大量生理功能或是病理功能的发挥都起着重要的作用。但是细胞外基质是一种粘弹性材料，除了通常意义上的“软-硬”的概念外，，基质的粘性分量，即描述其与细胞相互作用的能量损耗部分，势必也应对癌细胞的迁移产生影响。另外，前期的研究中细胞主要培养在二维平面上并且采用的基底材料也多为人工合成材料。进行体内研究虽更真实但又会影响因素众多且难于控制、难于直接观察。所以为了在接近生理病理状态的立体微环境中时空可控的考察天然基质粘弹性对癌细胞转移这一级联动态的多步骤过程的影响。本研究首先用旋转流变仪对三种不同浓度的胶原凝胶粘弹性进行了定量分析。结果表明胶原凝胶的粘弹性随着胶原凝胶中胶原蛋白浓度的增加而增大。从而确定了通过胶原浓度调节胶原这种天然细胞外基质材料粘弹性的可行性。然后在Carlos P. Huang等人的研究工作基础上结合胶原凝胶技术构建了一种针对癌细胞转移的微流控系统，即微流控癌细胞转移研究模型，该模型能完整地实现以下功能：（1）实现肿瘤细胞的立体培养；（2）能在细胞外基质中形成生化因子浓度梯度分布，并且能定量分析；（3）可以调节细胞生长的细胞外基质材料的理化特性，实时定量跟踪考察细胞与胞外基质材料相互作用过程中的动态变化。最后本研究利用构建的微流控癌细胞转移实验平台，基于第一部分的研究结果，调节癌细胞生长的胞外基质的粘弹性，利用免疫荧光和实时观测的方法考察了不同粘弹性的胞外基质对HepG2细胞的形态和活性、骨架装配、侵袭和迁移的影响。细胞生物学研究结果显示在本研究所考察的范围内，HepG2在软的、粘性低的胶原凝胶中的活性比硬的、粘性高的胶原中的活性低；在硬的、粘性高的胶原中细胞骨架较软的、粘性低的胶原凝胶中；而迁移和侵袭速度随着胶原粘性和弹性的升高而降低。这些结果表明，本研究成功构建了一个能够为细胞培养提供模拟体内生理或病理的微环境并且能够实现对癌细胞对其周围微环境响应实时监测的微流控癌细胞转移模型。生物学的研究结果表明，胶原的粘弹性对HepG2的形态和活性、骨架装配以及侵袭和迁移能力有一定的影响。
[Abstract]:The metastasis of cancer cells is a complex, multi-step and time-to-empty cascade, and is the cause of the death of more than 90% of the cancer patients. The metastasis of cancer cells is also a process of the interaction between the cancer cells and its surrounding microenvironment, which is influenced by a variety of biochemical and biophysical factors in the surrounding microenvironment. The matrix mechanics is regarded as an important micro-environmental factor. Note: The effect of the mechanical properties of the matrix on the behavior of the cells is more than that of the "hardness" of the matrix on the behavior, structure and function of the cells. The results of the study on the effect of extracellular matrix mechanics on the metastasis of cancer cells have shown that the mechanical properties of the matrix have the same important role as the biochemical factors in the transfer of cancer cells: recent studies have shown not only the lines of the cells, The structure and function are regulated by the hardness of the extracellular matrix, and the hardness of the matrix plays an important role in the function of a large number of physiological functions or pathological functions. The extracellular matrix, however, is a viscoelastic material, in addition to the concept of "soft-hard" in the general sense, the viscous component of the matrix, that is, the energy loss component that describes its interaction with the cells, is bound to also produce a shadow on the migration of the cancer cells. In addition, in the previous study, the cells were mainly cultured on the two-dimensional plane and the substrate material used was also a synthetic material. In-vivo studies, though more realistic but also have a large number of factors and are difficult to control, are difficult to view directly Therefore, in order to study the multi-step process of the dynamic multi-step transfer of the natural matrix viscoelastic to the cancer cells in a three-dimensional micro-environment, which is close to the physiological and pathological state, In this study, the viscoelasticity of three different concentrations of collagen gel was determined by a rotary rheometer. The results show that the viscoelasticity of the collagen gel increases with the collagen concentration in the collagen gel. Thereby determining the viscoelastic properties of the natural extracellular matrix material that modulate the collagen through the collagen concentration. Feasibility. Then, on the basis of the research of Carlos P. Huang et al., a micro-flow control system for metastasis of cancer cells, that is, a microflow control cancer cell transfer study model, is constructed, which can completely realize the following functions: (1) the development of tumor cells (2) a biochemical factor concentration gradient distribution can be formed in the extracellular matrix, and the quantitative analysis can be performed; (3) the physical and chemical properties of the extracellular matrix material capable of regulating the growth of the cells can be adjusted, and the activity of the cell and the extracellular matrix material during the interaction process can be quantitatively and quantitatively tracked in real time. Based on the results of the first part, the viscoelasticity of the extracellular matrix in the growth of the cancer cells was adjusted based on the results of the first part, and the morphology of the extracellular matrix on the HepG2 cells was investigated by means of immunofluorescence and real-time observation. and activity, framework assembly, invasion, and migration The results of the cell biology show that, within the scope of this study, the activity of HepG2 in a soft, viscous, low-viscosity collagen gel is lower than that of hard, sticky, high-viscosity collagen; in hard, highly viscous, high-viscosity collagen, the cytoskeleton is soft, low in viscosity, in that gel; and the speed of migration and invasion increases with the increase in the viscosity and elasticity of the collagen The results show that the present study successfully constructed a micro-environment that can provide a micro-environment for cell culture to simulate in vivo physiological or pathological conditions and can realize the real-time monitoring of the surrounding microenvironment of cancer cells. The results of the study of biology indicate that the viscoelasticity of collagen has a good effect on the morphology and activity of HepG2, the assembly of skeleton and the ability of invasion and migration.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.0

【相似文献】