几类细胞的生物力学实验研究
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摘要
细胞(cell)是生命的实体和生命的基本单位,几乎所有的有机体都是由细胞和细胞的产物所组成。对细胞力学的研究是近十几年来生物力学领域中迅速发展起来的一个前沿课题。众所周知,细胞的形态结构及其功能,细胞的生长、发育、成熟、癌变、增殖、衰老以及死亡,细胞的分化及其调控机理,都和力学有着密切的关系。高技术发展到今天,不但使人们可以看到微观层次的结构形态,而且可以在这一微观层次上操作、控制和测量材料变化过程。如,高倍光学显微镜和显微操作仪的应用,使人们抓住细胞,并测量细胞的物理特性,如红细胞、白细胞、内皮细胞变形的研究,生物大分子与细胞黏附力的研究,存在于细胞内、细胞之间的力学—生化耦合。
细胞力学实验技术的发展推动了细胞力学的研究,同时也提出了许多新的问题。诸如,细胞实验装置的力学问题的理论分析,在不同实验条件下细胞力学模型的应用等。这些既有复杂又有相当具体的问题是生物力学工作者必须面对,并逐一加以解决的。作者对几类细胞的生物力学实验的几个方面问题做了一些理论和实验研究,取得了一些成果。如,微管吸吮技术和内含不可压缩流体弹性球壳模型在动物卵母细胞透明带杨氏模量研究中的应用等。具体的工作为如下几方面:
1.从细胞的一种力学模型—液滴模型(droplet model)研究、说明了一类细胞在微管准静吸吮过程出现的失稳问题。采用一定的数—力学近似,导出了细胞准静吸吮过程中,失稳点和失稳临界吸附压强与细胞液滴模型参数、微管内半径的关系式。并与Derganic J等的严格数字计算解比较,在实际应用范围内是符合得相当好的。
2.传统的、较为简单易行的底部附着拉伸法实验已广泛应用于力学刺激引发的细胞多方面响应研究,而目前对细胞在实验过程的应变张量缺乏定量的理论分析。我们根据细胞加载实验中,成骨类细胞黏附在基底(substrate)的几何形态特征,定量地分析了细胞应变张量与实验装置加载参数的关系,给出了细胞铺展面足够大时,细胞应变张量趋于均匀的极限值——特征张量。引入一个场变量来描述一般情况下细胞应变张量的非均匀性,并导出参数的变化范围与细胞形态几何参数的关系。专家评价:该研究填补了一项空白,理论分析是可靠的、可行的,对今后的这类实验有一定的指导意义。
3.应变加载设备对细胞拉伸加载的同时,由于培养液的来回流动,细胞会受到流体剪切力的作用。这作用与细胞加载应变相比在不同的加载设备中是否可忽
Cells are the entities and the basic units of life, almost organic bodies are composed of cells and their matrix. Cellular mechanics is an important branch of biological mechanics and has developed rapidly in decades to be a front field of investigation. It is well known that cellular configuration, function, cellular growth, cellular maturity, cacinoma, cellular proliferation, decrepitude, as well as death and diferentiation, are associated with mechanics. Nowaday advanced technologies enable people not only to observate the cellular conformation in microcosmic level but also to manipulate, control and meassure their material change process. For example, with use of microscope and micro-manipulating device people can get hold of cells and measure their physical traits. Such as the metabolic study of red cells, leukocytes endothelial cells, study of adhesive force between biological big molecules and cells, and mechano-chemical coupling that exist commonly inside or between cells.The improvement of experimental techniques promotes cellular biomechanics development. On the other espect, it brings about new problems. For instance, the mechanical analysis of some question occurred in experiment; the application of cellular mechanical models in special experimental technique. Such problems are complex or practical, which we biomechanists have to face and endevour to settle one by one. Author has studied some problems in biomechanical experiments for some kinds of cells, and achieved some outcomes. This dissertation introduce concrete theoritical and experimental work on cellular mechanics in follows:1 .It is generally realized phenomenon in suction experiment of some kind of cells that if the aspiration pressure is larger than a certain threshold, cell flows continuously into the pipette. The point of the threshold aspiration pressure at which the cell can still be held in a stable equilibrium is called the critical point of aspiration. Here we represent a theoretical analysis of the equilibrium behavior and stability of cell by liquid drop model. In the method of analysis, the areal change due to a small movement AL of the portion of the membrane in pipette is given to the first approximation.The threshold pressure and the critical point are shown as simple formulae of the model parameters and inner relative radius of pipette . The results derived from formulas are consistent with rigorous ones by numerical computation in the approximate range.2.Traditional substrate attached cell loading experiments have been applied broadly to
studies of cellular response to mechanical stimulation in many aspects. Wheras analysis on strain of the cells in such experiment has not been in satisfaction. Based on the morphological character of osteoblast-like cells spreading on substrate in tensile loading experiments, the uniformity of cellular strain tensor is discussed with cellular solid model and droplet model. Characteristic strain tensors of cells are presented for uniaxial or biaxial tensile loading, which are the limit ones when cells flatten enough. A field variable is inducted to describe the variation of strain tensor in normal case, and its deviation can be scaled by a geometrical parameter of cells. This article provides a quantitative strain description in tensile loading experiments for osteoblasts as well as other cells. Experts comment on our study: a study to fill up a blankness in cellular biomechanics; the theoretic analysis is leliable, practical and also instructional to such experiments.3. In vitro cell loading experiments are used to investigate stimulation of strain to cellular proliferation. The flowing conditions of culture fluid in loading systems has been little known, so people can not detect the influence of strain to cellular proliferation exactly because shear flow can enhance cell proliferation either. This is a problem need to be solved in such experiments. Based on the working principle and circle loading parameters, we simplify Navier-Stokes formula to describe the flow of culture fluid on substrates of uniaxial/biaxial flat tensile loading systems and four point bending system. With approximative formula, the distributions of velocity field and shear flow to cells are gained. Results show: shear flows are zero in the middle of substrate for all systems, and they get larger proportionally to distance from middle and elongation magnitude; the shear flow on the substrate of four point bending system is much greater than those of others. This shear flow in four point bending system, confirmed by Owan, I., et al.with OPN mRNA increase in their experiment, could cause more influence to osteoblast-like cells than that caused by strain. But they could not provide the wallshear flow in quantity. We estimate the average magnitude of shear stress to cells in their device, and the results are consistant with other experiments'datas about shear flow. Our study makes it possible to differentiate the stimulation of strain to cell proliferation and that of shear flow or the anabolic effect of this two mechanical operation in loading experiments with the devices mentioned above.4.1n suction experiment of baffalo ovum in vitro cultured using micropipette with its
inner diameter varring from the double thikness of zona pellucida to one third of baffalo ovum diameter, elastic shell model with incompressible liquid inside is adopted to study the mechanical properties of their zona pellucida. Through mechanical analysis, a formula was derived to calculate the Young's modulus of Zona Pellucida from quasi-static measurements. At temperature of 24°C~26°C, 20 mature but with no polocyte cells were tested. The mean Young's modulus value E is 2.2x104 Pa, with the deviation within 30%. The study will provide a effective method for gaining the E of zona pellucida of oocytes in animals, approaching biological function in mechanical aspect, variation of physical or chemical trait in their process.5. Using Flexercell-4000T? Unit, the effects of cyclic mechanical stretch on human adenocarcinoma cell line A549 are studied for time within 4h. The results showed that short time mechanical stimulation could enhance the proliferation rate of A549 cells that subjected to 20% elongation of biaxial stretch at frequency 30 cycles/min. The proliferation responding ratio M(%) vary with the loading time: M(%)=18% for 0.5 h, M(%)=22% for lh and M(%)=5% for 4h. There seems to be a peak of M(%) at cycle number about 1800 cycles. This feature is similar to the human bone derived cells. Further experiment demonstrates that intermittent loading is an effective way to result in high proliferation of the A549 cells for long time. On contrary, prior studies confirmed that continuous loading for long time restrict A549 cells'proliferation.
细胞力学实验技术的发展推动了细胞力学的研究,同时也提出了许多新的问题。诸如,细胞实验装置的力学问题的理论分析,在不同实验条件下细胞力学模型的应用等。这些既有复杂又有相当具体的问题是生物力学工作者必须面对,并逐一加以解决的。作者对几类细胞的生物力学实验的几个方面问题做了一些理论和实验研究,取得了一些成果。如,微管吸吮技术和内含不可压缩流体弹性球壳模型在动物卵母细胞透明带杨氏模量研究中的应用等。具体的工作为如下几方面:
1.从细胞的一种力学模型—液滴模型(droplet model)研究、说明了一类细胞在微管准静吸吮过程出现的失稳问题。采用一定的数—力学近似,导出了细胞准静吸吮过程中,失稳点和失稳临界吸附压强与细胞液滴模型参数、微管内半径的关系式。并与Derganic J等的严格数字计算解比较,在实际应用范围内是符合得相当好的。
2.传统的、较为简单易行的底部附着拉伸法实验已广泛应用于力学刺激引发的细胞多方面响应研究,而目前对细胞在实验过程的应变张量缺乏定量的理论分析。我们根据细胞加载实验中,成骨类细胞黏附在基底(substrate)的几何形态特征,定量地分析了细胞应变张量与实验装置加载参数的关系,给出了细胞铺展面足够大时,细胞应变张量趋于均匀的极限值——特征张量。引入一个场变量来描述一般情况下细胞应变张量的非均匀性,并导出参数的变化范围与细胞形态几何参数的关系。专家评价:该研究填补了一项空白,理论分析是可靠的、可行的,对今后的这类实验有一定的指导意义。
3.应变加载设备对细胞拉伸加载的同时,由于培养液的来回流动,细胞会受到流体剪切力的作用。这作用与细胞加载应变相比在不同的加载设备中是否可忽
Cells are the entities and the basic units of life, almost organic bodies are composed of cells and their matrix. Cellular mechanics is an important branch of biological mechanics and has developed rapidly in decades to be a front field of investigation. It is well known that cellular configuration, function, cellular growth, cellular maturity, cacinoma, cellular proliferation, decrepitude, as well as death and diferentiation, are associated with mechanics. Nowaday advanced technologies enable people not only to observate the cellular conformation in microcosmic level but also to manipulate, control and meassure their material change process. For example, with use of microscope and micro-manipulating device people can get hold of cells and measure their physical traits. Such as the metabolic study of red cells, leukocytes endothelial cells, study of adhesive force between biological big molecules and cells, and mechano-chemical coupling that exist commonly inside or between cells.The improvement of experimental techniques promotes cellular biomechanics development. On the other espect, it brings about new problems. For instance, the mechanical analysis of some question occurred in experiment; the application of cellular mechanical models in special experimental technique. Such problems are complex or practical, which we biomechanists have to face and endevour to settle one by one. Author has studied some problems in biomechanical experiments for some kinds of cells, and achieved some outcomes. This dissertation introduce concrete theoritical and experimental work on cellular mechanics in follows:1 .It is generally realized phenomenon in suction experiment of some kind of cells that if the aspiration pressure is larger than a certain threshold, cell flows continuously into the pipette. The point of the threshold aspiration pressure at which the cell can still be held in a stable equilibrium is called the critical point of aspiration. Here we represent a theoretical analysis of the equilibrium behavior and stability of cell by liquid drop model. In the method of analysis, the areal change due to a small movement AL of the portion of the membrane in pipette is given to the first approximation.The threshold pressure and the critical point are shown as simple formulae of the model parameters and inner relative radius of pipette . The results derived from formulas are consistent with rigorous ones by numerical computation in the approximate range.2.Traditional substrate attached cell loading experiments have been applied broadly to
studies of cellular response to mechanical stimulation in many aspects. Wheras analysis on strain of the cells in such experiment has not been in satisfaction. Based on the morphological character of osteoblast-like cells spreading on substrate in tensile loading experiments, the uniformity of cellular strain tensor is discussed with cellular solid model and droplet model. Characteristic strain tensors of cells are presented for uniaxial or biaxial tensile loading, which are the limit ones when cells flatten enough. A field variable is inducted to describe the variation of strain tensor in normal case, and its deviation can be scaled by a geometrical parameter of cells. This article provides a quantitative strain description in tensile loading experiments for osteoblasts as well as other cells. Experts comment on our study: a study to fill up a blankness in cellular biomechanics; the theoretic analysis is leliable, practical and also instructional to such experiments.3. In vitro cell loading experiments are used to investigate stimulation of strain to cellular proliferation. The flowing conditions of culture fluid in loading systems has been little known, so people can not detect the influence of strain to cellular proliferation exactly because shear flow can enhance cell proliferation either. This is a problem need to be solved in such experiments. Based on the working principle and circle loading parameters, we simplify Navier-Stokes formula to describe the flow of culture fluid on substrates of uniaxial/biaxial flat tensile loading systems and four point bending system. With approximative formula, the distributions of velocity field and shear flow to cells are gained. Results show: shear flows are zero in the middle of substrate for all systems, and they get larger proportionally to distance from middle and elongation magnitude; the shear flow on the substrate of four point bending system is much greater than those of others. This shear flow in four point bending system, confirmed by Owan, I., et al.with OPN mRNA increase in their experiment, could cause more influence to osteoblast-like cells than that caused by strain. But they could not provide the wallshear flow in quantity. We estimate the average magnitude of shear stress to cells in their device, and the results are consistant with other experiments'datas about shear flow. Our study makes it possible to differentiate the stimulation of strain to cell proliferation and that of shear flow or the anabolic effect of this two mechanical operation in loading experiments with the devices mentioned above.4.1n suction experiment of baffalo ovum in vitro cultured using micropipette with its
inner diameter varring from the double thikness of zona pellucida to one third of baffalo ovum diameter, elastic shell model with incompressible liquid inside is adopted to study the mechanical properties of their zona pellucida. Through mechanical analysis, a formula was derived to calculate the Young's modulus of Zona Pellucida from quasi-static measurements. At temperature of 24°C~26°C, 20 mature but with no polocyte cells were tested. The mean Young's modulus value E is 2.2x104 Pa, with the deviation within 30%. The study will provide a effective method for gaining the E of zona pellucida of oocytes in animals, approaching biological function in mechanical aspect, variation of physical or chemical trait in their process.5. Using Flexercell-4000T? Unit, the effects of cyclic mechanical stretch on human adenocarcinoma cell line A549 are studied for time within 4h. The results showed that short time mechanical stimulation could enhance the proliferation rate of A549 cells that subjected to 20% elongation of biaxial stretch at frequency 30 cycles/min. The proliferation responding ratio M(%) vary with the loading time: M(%)=18% for 0.5 h, M(%)=22% for lh and M(%)=5% for 4h. There seems to be a peak of M(%) at cycle number about 1800 cycles. This feature is similar to the human bone derived cells. Further experiment demonstrates that intermittent loading is an effective way to result in high proliferation of the A549 cells for long time. On contrary, prior studies confirmed that continuous loading for long time restrict A549 cells'proliferation.
引文
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