基于原子力显微镜的细胞若干力学特性的定量研究

发布时间：2018-06-16 11:50

本文选题：AFM力检测 + 细胞力学特性　；参考：《哈尔滨工业大学》2015年博士论文

【摘要】：癌症是当今最常见的发病和死亡原因之一,寻找有效的癌症早期诊断方法和治疗手段是医学界的研究热点。癌细胞是癌症的病源,传统生物学主要采用定性非定量方法对其进行研究,存在诸多不足。对细胞生命过程进行基于力学特性的定量研究被认为是癌症早期诊断和治疗的突破性手段,研究细胞的力学特性变化,能较准确的定量表征细胞的内部组织变化以及细胞的病变程度。原子力显微镜(AFM)具有高精度的力分辨率、精确稳定的操作和适用于生理环境的特点,是研究细胞力学特性的有力工具。由于力学特性范围较广,本文着重选择对细胞生命过程有重要影响的若干力学特性进行研究。癌症是正常细胞变异后有丝分裂失常而产生无限增殖并破坏健康组织的疾病,研究细胞有丝分裂的力学特性是研究癌细胞病变机理的重要切入点。将AFM搭载在倒置显微镜上,实现对不同细胞系有丝分裂时期细胞平衡力和中截面面积的定量测量,通过建立理论模型计算出细胞的内压强和体积。实验过程中将传统的AFM平面悬臂梁探针改进为楔形悬臂梁探针,优化实验方法,简化实验模型。研究结果表明有丝分裂时期细胞的平衡力、体积和内压强随细胞系的不同而差异显著,为细胞水平上的癌症早期诊断提供参考数据。细胞刚度是区分癌细胞与正常细胞的标识之一,也是医学研究中癌症病理研究的重要切入点。利用AFM的力成像模式对生化反应过程中细胞的形貌变化和刚度变化进行实时定量研究。结合活细胞的物理特性,对前人建立的细胞刚度测量理论进行分析,将力-位移曲线压入线的斜率定义为细胞刚度,通过软件分析对实验所得数据进行有效筛选和处理,得到悬浮的人肝细胞血红蛋白生成素配体(ephrin-A1)对前列腺癌细胞PC3的外部形貌、内部骨架和细胞刚度的改变规律,结果表明在骨架重组过程中细胞刚度未见影响,为细胞刚度的定量研究提供新的参考信息。细胞粘附特性的改变是癌细胞脱离原发组织向健康组织转移的先决条件,定量研究细胞粘附力学作用的分子基础是研究癌细胞转移特性的重要手段。利用单细胞力谱技术在生理条件下定量研究细胞与外基质蛋白之间的粘附力学作用。通过分析单细胞力谱技术的力学原理,提出整合素-外基质蛋白分子连接断裂力与单细胞粘附力的关系模型。结合分子连接解离的力学原理,测量并分析在不同的操作参数下单细胞粘附特性和细胞表面单分子粘附特性的变化规律,揭示细胞对外基质粘附力学作用的分子基础,为深入认识癌症的基本生物学特性提供了理论依据。单细胞力谱实验操作的复杂性导致实验效率不高,为提高实验效率,研制具有聚二甲基硅氧烷(PDMS)分区模型的样品基底,分别利用AFM扫描刮除法和光漂白法对分区模型进行性能检测,成功将这种带有分区模型的样品基底应用于单细胞力谱实验。实现了在单细胞力谱实验中同一细胞对四种不同外基质蛋白的粘附力的测量,提高实验效率和可靠性,节约实验成本,为该实验技术的工程应用打下基础。癌细胞的粘附连接调节与癌症的扩散和转移密切相关。用单细胞力谱技术代替传统生物技术,以测量粘附力的方式发现并定量研究ephrin-A1诱导的前列腺癌细胞PC3与I型胶原蛋白的粘附连接增强现象,得到粘附连接增强的交互对话(crosstalk)信号,实现对细胞内生物信号的定量研究。前列腺癌常易发生骨转移,PC3细胞是前列腺癌骨转移后的细胞,而I型胶原蛋白是人体骨骼的主要组成成份,因此可知ephrin-A1的作用可以改变前列腺癌细胞PC3的骨转移特性。
[Abstract]:Cancer is one of the most common causes of disease and death today. To find effective methods and methods for early diagnosis and treatment of cancer is a hot topic in the medical field. Cancer cells are the source of cancer. The traditional biology mainly uses qualitative and non quantitative methods to study it, and there are many not enough. Quantitative research is considered as a breakthrough method for early diagnosis and treatment of cancer. The study of changes in mechanical properties of cells can accurately quantify the internal tissue changes of cells and the degree of cell lesions. Atomic force microscopy (AFM) has a high precision of force resolution, precise and stable operation and the characteristics of the physiological environment. A powerful tool for studying the mechanical properties of cells. Because of the wide range of mechanical properties, this paper focuses on the study of several mechanical properties that have important effects on cell life processes. Cancer is a disease that produces infinite proliferation and destroys healthy tissue after normal cell mutation, and studies the mechanical properties of cell mitosis. It is an important point to study the mechanism of cancer cell lesion. AFM is carried on an inverted microscope to measure the balance force and the area of the middle section of cell lines in the mitosis of different cell lines. The internal pressure and volume of the cells are calculated by establishing a theoretical model. In the experiment, the traditional AFM plane cantilever beam probe is improved to be a wedge. The results show that the balance force, volume and pressure of cells in the mitotic period vary significantly with the cell lines, and provide reference data for the early diagnosis of cancer at the cell level. Cell stiffness is one of the identification of cancer cells and normal cells, and is also a medicine. An important breakthrough point in the research of cancer pathology in the study. Using the force imaging model of AFM, the change of cell morphology and stiffness in the biochemical reaction process is studied in real time. The theory of cell stiffness measurement established by the predecessors is analyzed in combination with the physical characteristics of living cells. The slope of the force displacement curve is defined as the cell rigidity. Degree, through the software analysis, the experimental data were effectively screened and processed to obtain the external morphology, the internal skeleton and the cell stiffness of the suspended human hepatocyte hemoglobin ligand (ephrin-A1) on the prostate cancer cell PC3. The results showed that the cell stiffness was not affected during the skeleton regrouping, and it was the cell stiffness. Quantitative research provides new reference information. Changes in cell adhesion characteristics are the preconditions for cancer cells to transfer from the primary tissue to healthy tissues. The molecular basis for quantitative study of cell adhesion mechanics is an important means to study the metastatic characteristics of cancer cells. By analyzing the mechanical principle of the single cell force spectrum technique, the relationship model of the adhesion force of the integrin - External matrix protein molecules connected to the single cell adhesion was proposed, and the single cell adhesion properties and the cell surface single - cell surface were measured and analyzed in combination with the mechanics principle of molecular connection dissociation. The change law of molecular adhesion characteristics reveals the molecular basis of the adhesion mechanics of the extracellular matrix, which provides a theoretical basis for understanding the basic biological characteristics of cancer. The complexity of the single cell force spectrum experiment results in the low efficiency of the experiment. In order to improve the efficiency of the experiment, the polymethylsiloxane (PDMS) zoning model is developed. The sample substrate was tested by AFM scan curettage and photobleaching respectively. The sample substrate with the zoning model was applied to the single cell force spectrum experiment. The measurement of the adhesion of the same cells to four different extracellular matrix proteins in the single cell force spectrum experiment was realized to improve the efficiency and reliability of the experiment. Sex, saving the experimental cost, laying the foundation for the engineering application of the experimental technology. The adhesion regulation of cancer cells is closely related to the diffusion and metastasis of cancer. Using the single cell force spectrum technique instead of traditional biological technology, the ephrin-A1 induced prostate cancer cells PC3 and I collagen are detected and quantified by the method of measuring adhesion force. PC3 cells are the cells of bone metastasis of prostate cancer, and I type collagen is the main component of human skeleton, so that the effect of ephrin-A1 can be seen. To change the bone metastases of PC3 in prostate cancer cells.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TH742

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