【作者】 宋健民；

【导师】 谢晖；

【作者基本信息】 哈尔滨工业大学 ， 机械电子工程， 2020， 博士

【摘要】 随着对癌细胞探索的不断深入,仅从细胞层面对癌症发病原理、转染机制的探索已经很难满足当前科学研究的需求,亚细胞层面的探索为细胞特性研究提供了一种新思路。但是,对于细胞/亚细胞力学特性的研究,目前仍存在诸如测量方法针对性差、测量参数单一、测量精度低的难题。因此,本文采取细胞外部到细胞内部、静态测量到动态测量的研究路线,从原子力显微镜(Atomic force microscopy,AFM)探针技术入手,深入开展了细胞局部静态杨氏模量、细胞核静态杨氏模量、细胞动态复泊松比,以及亚细胞结构动态粘弹性谱测量方法的研究。弥补现有AFM细胞测量的不足,拓展了AFM技术在细胞表征领域的应用。开展了细胞静态杨氏模量测量的研究,分析细胞死亡过程的杨氏模量变化规律,提出了基于超软探针技术的细胞死亡过程力学描述方法。在研制的细胞活性保持器中,测量与统计分析癌细胞杨氏模量的变化,从力学角度定量描述了癌细胞凝胶化死亡过程,验证了所提出的方法的有效性与所构建原子力显微镜细胞测量平台的稳定性。开展了细胞核静态杨氏模量测量的研究,分析多形态细胞核与针尖的接触模型,提出了穿透细胞膜原位测量细胞核静态杨氏模量的方法。通过探针结构优化设计与仿真,开发了具有超大长径比纳米针尖的光纤探针。同时,研制了光学/荧光显微镜双视觉系统,判断细胞生长状态及确定针尖-细胞核相对位置,完成了多形态细胞核的静态杨氏模量测量,并分析细胞形态对细胞核杨氏模量的影响规律。开展了细胞动态复泊松比测量的研究,建立基于平面针尖的细胞动态复泊松比测量模型,提出了磁驱平行板式拉压-剪切同时加载测量细胞动态复泊松比的方法。制备了磁驱平行板探针,解决了动态力耦合问题。通过静态泊松比测试验证了所提出方法的可行性,并分析了频率变化以及压痕深度变化对复泊松比数值的影响规律,为进一步研究细胞/亚细胞结构的动态粘弹性提供了解决手段。开展了亚细胞结构动态粘弹性谱测量的研究,分析动态粘弹性测量原理,建立纳米针尖-亚细胞结构的动力学模型,提出了亚细胞结构动态粘弹性测量方法。研制了高频磁驱纳米针尖探针,通过实时计算振动探针原位穿透细胞过程中与深度位置相对应的动态粘弹性参数值,绘制出亚细胞结构的动态粘弹性谱,揭示了细胞生长状态对亚细胞结构粘弹性谱的影响规律。另外,提出了亚细胞结构粘弹性的多维机械表型评价方法,获得了三种示范细胞(He La,Si Ha,NIH3T3)的多维机械表型,解决了静态力学特性测量方法难以分辨同源癌细胞的难题,证明了所提出方法的可行性与潜在应用价值。综上所述,本文研究的细胞静态杨氏模量、细胞核静态杨氏模量、细胞动态复泊松比、亚细胞结构动态粘弹性谱的测量方法,为细胞/亚细胞结构力学特性研究提供了新方法与系统,在疾病诊断学、生物细胞学等领域具有重要的科研价值和应用前景。更多还原

【Abstract】 With the deepening research,the exploration of the pathogenesis and transfected mechanism from the cellular level has been difficult to meet the needs of current scientific research,and the exploration of the subcellular level provides a new way of thinking for the study of cell characteristics.However,there are still difficulties in the study of cellular/subcellular mechanical properties,such as poorly targeted measurement methods,single measurement parameters,and low measurement accuracy.So this work has carried out research on the Atomic force microscopy(AFM)measurement technology of cellular/subcellular structures.Take a research route from the extracellular level to the intracellular level and from quasi-static measurements to dynamic measurements.Intensive research has been carried out such as measuring static Young’s modulus of cancer cells,Young’s modulus of nucleus,complex Poisson’s ratio,and dynamic viscoelasticity spectra of subcellular structure.The results have successfully compensated for the shortcomings of existing cell measurements.A method for measuring the static Young’s modulus of cells was studied.Analyze the change law of the Young’s modulus of cell death,and propose a method for mechanical description of cell death based on ultra-soft probe technology.In the developed cell viability retainer,changes in the Young’s modulus of cancer cells were measured and statistically analyzed.The gelation and death process of cancer cells was quantitatively described from the perspective of mechanics,and the validity of the proposed method was verified and the AFM cell measurement platform was stability.A method for measuring the static Young’s modulus of the nucleus was studied.By analyzing the contact model of polymorphic nucleus and needle tip,a method of measuring the static Young’s modulus of the nucleus with needle-tip penetration into large-scale cells was proposed.Through the design and simulation of the probe structure,a fiber probe with ultra-large aspect ratio nano-tips was developed.At the same time,an optical/fluorescent microscope dual vision system is used to accurately determine the cell status and the relative position of the tip-nucleus.The measurement of the static Young’s modulus of the nucleus under different cell morphologies.The effect of cell morphology on the Young’s modulus of the cells was analyzed.The research on the measurement method of cell dynamic complex Poisson’s ratiowas carried out.A method for measuring the dynamic complex Poisson’s ratio of cells by parallel-plate tension-compression-shear simultaneous loading with magnetic drive was proposed.A dynamic Poisson’s ratio measurement model based on a parallel-plate tip was established.A magnetic drive parallel plate probe was prepared to solve the problem of dynamic force coupling.The feasibility of the proposed method was verified by static Poisson’s ratio test,and the effect of frequency change and indentation depth change on the value of complex Poisson’s ratio was analyzed.The research of dynamic viscoelasticity spectra measurement of subcellular structures was carried out.Based on the analysis of the principle of dynamic viscoelasticity,a dynamic mechanical model of the nano-needle tip-subcellular structure was constructed,and then a method of dynamic viscoelasticity measurement of subcellular structure was proposed.A high-frequency magnetic-drive nanoneedle probe was designed and developed.The dynamic viscoelasticity spectrum was used to describe the viscoelasticity of the whole process of the nanoneedle penetrating through the subcellular structure.At the same time,analyze the characteristics of dynamic viscoelastic spectra of subcellular structures and the effect of cell status on the spectra.In addition,a multidimensional mechanical phenotypic evaluation method is proposed,and multiple mechanical phenotypes of three exemplary cells(He La,Si Ha,NIH3T3)were obtained.It makes up for the shortcomings that the static mechanical characterization method cannot distinguish homologous cancer cells,and it fully prove the feasibility,practicability and potential application value of the method.In summary,measuring the static Young’s modulus of the cells,Young’s modulus of the nucleus,complex Poisson’s ratio,and dynamic viscoelasticity spectra of subcellular structures.The measurement of the mechanical properties of the subcellular structures provides a new method and a new system,and has important scientific research value and application prospects in the fields of disease diagnostics and biological cytology.更多还原