细胞低温保存跨膜传质机理及保护剂溶液冻结特性研究

发布时间：2018-10-10 15:06

【摘要】：低温生物学是热科学、工程学和生物学与医学之间的边缘交叉学科。低温保存技术和低温医疗技术是低温生物学的两个最重要的应用。其中,低温保存技术对于胚胎干细胞、血液和珍稀动植物种质资源的长期保存及人体器官移植等均具有重要意义。制约低温保存效果的最关键因素是冷冻保存对生物材料产生的低温损伤,如冰晶损伤和溶质损伤等。使用低温保护剂能够有效抑制低温损伤,从而大幅提高生物材料的冻存后复活率。因此,典型的程序化慢速冷冻保存过程包括低温保护剂导入、程序化降温、液氮温度下长期保存、复温和保护剂洗脱五个环节。在每一个环节中,生物细胞和组织均会发生复杂的物理、化学和生物反应,这些反应是由细胞内外温度、压力、溶液组成、蛋白质活性及新陈代谢率的变化引起的。所以,预测上述物化参数的变化,分析低温损伤机理,探索提高冻存后复活率的有效方法,对于低温生物学的发展和应用具有深远意义,也是低温工程领域的研究人员面临的重要课题。本文应用物理化学、热力学和传质学理论研究了程序化慢速冷冻保存细胞过程中的跨膜传质情况,应用差示扫描量热技术和分子动力学模拟手段分别对醇和二甲基亚砜水溶液的冻结特性进行了量热分析和分子机理探究。首先,本文以保护剂溶液相图为基础数据建立了适用于非理想溶液的细胞脱水模型。该模型的预测结果表明,传统的理想溶液脱水模型低估了冷冻过程中细胞的胞内水量。而后,本文从Gibbs自由能理论出发建立了计算冷冻和复温过程中水和保护剂跨膜传递量的热力学模型,该模型针对真实溶液环境建立,避免了传统模型对细胞膜的理想半透膜假设。本文应用该模型预测了ICR小鼠精子细胞和人角膜基质细胞在冷冻过程中的体积变化。另外,本文利用差示扫描量热仪测定了甲醇／氯化钠／水和1,2-丙二醇／氯化钠／水三元体系的相图,不仅进一步扩展了新模型的适用范围,而且证明了在有限浓度范围内利用甲醇／水、1,2-丙二醇／水和氯化钠／水体系的二元相图合成相应三元相图的可行性。本文利用差示扫描量热仪测定了冻结醇和二甲基亚砜溶液的未冻水量,建立了保护剂溶液中冰的融化潜热与溶液初始浓度的定量关系。量热分析结果表明,随着溶液初始浓度的增大,冻结溶液中未冻水量增多。针对宏观实验结果,本文还利用分子动力学模拟手段统计计算了乙二醇、甘油和二甲基亚砜溶液的氢键特性和水的自扩散系数,发现未冻水量与保护剂-水氢键的比例存在显著的正相关关系,从而证明保护剂分子与水分子间氢键是冻结溶液中存在未冻水的直接原因。本文的研究结果能够更准确地预测冷冻保存过程中细胞的生物物理反应,计算胞内水和保护剂含量的变化；扩展了低温保护剂溶液的热力学数据；从宏观和微观层次上解释了醇和二甲基亚砜的低温保护机理,为确定合理的低温保存方案提供依据。
[Abstract]:Low-temperature biology is the interdisciplinary subject of thermal science, engineering and biology and medicine. Cryopreservation and low-temperature medical technology are two most important applications of low-temperature biology. The cryopreservation technique is of great significance to the long-term preservation of embryonic stem cells, blood and rare animal and plant germplasm resources and human organ transplantation. The most critical factors which restrict the effect of low temperature preservation are freezing preservation of low temperature damage to biological materials, such as ice crystal damage and solute damage. By using the low-temperature protective agent, the low-temperature injury can be effectively inhibited, thereby greatly improving the freeze-thaw recovery rate of the biological material. Therefore, the typical programmed slow freezing preservation process includes the introduction of cryoprotectant, programmed cooling, long-term preservation at liquid nitrogen temperature, and the elution of complex mild protective agent. In each link, biological cells and tissues undergo complex physical, chemical and biological reactions that are caused by changes in temperature, pressure, solution composition, protein activity, and metabolic rate within and outside the cell. Therefore, the change of the above-mentioned physical and chemical parameters is predicted, the mechanism of low temperature damage is analyzed, and the effective method for improving the recovery rate after freezing storage has profound significance for the development and application of low temperature biology, and is also an important topic for researchers in the field of low temperature engineering. In this paper, physical chemistry, thermodynamics and mass transfer theory were used to study the trans-membrane mass transfer in programmed slow freezing storage cells. In this paper, the thermal analysis and molecular mechanism of the freezing properties of alcohol and water solution were carried out by means of differential scanning calorimetry and molecular dynamics simulation. First of all, this paper establishes the cell dehydration suitable for non-ideal solution based on the phase diagram of the protective agent solution. The prediction of the model shows that the traditional ideal solution dehydration model underestimates the intracellular concentration of cells in the freezing process. Based on the theory of free energy, this paper establishes a thermodynamic model for calculating the trans-membrane transfer of water and protective agent in the process of freezing and rewarming. The model is based on the real solution environment, which avoids the ideal semi-permeable membrane of the traditional model to the cell membrane. This model is used to predict the volume of sperm cells and human corneal stromal cells in ICR mice during freezing. In addition, the phase diagram of methanol/ sodium chloride/ water and 1,2-propanediol/ sodium chloride/ water ternary system is determined by using the differential scanning calorimeter, which not only expands the scope of application of the new model, but also proves that methanol can be used in the limited concentration range. The binary phase diagram of water, 1,2-propanediol/ water and sodium chloride/ water system is used to synthesize the corresponding ternary phase diagram. In this paper, the unfrozen water quantity of frozen alcohol and alcohol solution was determined by using the differential scanning calorimeter, and the latent heat of melting ice and the initial concentration of the solution were established in the protective agent solution. The quantitative relationship and the quantitative thermal analysis show that, as the initial concentration of solution increases, the frozen solution does not In the light of macroscopic experimental results, the hydrogen bond characteristics and self-diffusion coefficient of water were calculated by means of molecular dynamics simulation, and the ratio of unfrozen water to protective agent-water hydrogen bond was found to be significant. It is proved that the hydrogen bond between the protective agent molecule and the water molecule is the frozen water in the frozen solution. The results of this paper can predict the biophysical responses of cells in frozen storage more accurately, calculate the changes of intracellular water and protective agent content, and extend the cryoprotectant. The mechanism of low temperature protection of alcohol and alcohol is explained from the macroscopic and microscopic levels to determine the reasonable low temperature.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.52

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