【作者】 陈聪；

【导师】 李维仲；

【作者基本信息】 大连理工大学 ， 制冷与低温工程， 2009， 博士

【摘要】 胞内冰的生成和生长对细胞冷冻损伤的研究有着至关重要的意义。要充分了解胞内冰对细胞的伤害作用,除了冰晶的生成和生长,还需要研究胞内冰与冷冻保护剂(cryoprotective agent,CPA)、胞内蛋白和细胞膜的相互作用,而作为这一系列研究的基础,必须首先分析冷冻保护剂水溶液的结构和动力。以甘油为CPA,小鼠卵母细胞为模型细胞,修正了Karlsson胞内冰生长模型,并利用分子动力学方法对CPA—水二元溶液和CPA—水—氯化钠三元溶液进行了模拟。提出了一种求解变组元体系成核温度的方法,并联合细胞脱水方程,研究了胞内溶液均相成核温度随时间的变化。发现甘油—水—氯化钠三元溶液均相成核温度的下降值ΔT_h与平衡凝固点的下降值ΔT_m呈现如下的关系:ΔT_h=1.17ΔT_m。利用该关系和软冲突修正方程,修正了Karlsson扩散控制胞内冰生长模型。并利用修正后的模型预测了模型细胞玻璃化过程中的临界冷冻速率和临界浓度。预测值与实验值吻合较好,且修正后模型的预测值明显要优于Karlsson模型的预测值。进一步研究表明,当最终的晶体体积分数大于0.1时,必须考虑软冲突对胞内冰生长的影响。分析了CPA—水二元体系和CPA—水—氯化钠三元体系中氢键的结构和动力。发现在二元和三元体系中仅存在O—H…O类型的氢键。水中O原子(OH2)的氢键能力明显强于甘油分子中O原子(O1、O2和O3)的氢键能力。平均每个OH2原子参与的氢键数目随甘油浓度的增加而减少,而平均每个O1、O2和O3原子参与的氢键数目随甘油浓度的变化没有明显的变化规律。水分子中H原子的氢键能力略小于甘油分子中H原子的氢键能力,但与O原子不同,H原子的这种差别相当小。平均每个H原子参与的氢键数目随甘油浓度的变化几乎不变。分子的统计结果显示:随甘油浓度的增加,平均每个甘油分子参与的氢键数目有降低的趋势,但是考虑到偏差,差别并不明显;随甘油浓度的增加,平均每个水分子参与的氢键数目缓慢减少。动力分析结果表明:随甘油浓度的增大,氢键截断自相关函数的松弛时间和生存周期均呈现增大的趋势;水分子之间氢键相互作用的周期小于甘油分子与水分子之间氢键作用的周期,表明甘油分子和水分子之间的氢键比水分子之间的氢键更牢固。甘油的存在对Na~+离子和Cl~-离子的水合结构并没有产生太大的影响。根据氢键结构和动力的分析结果,从微观角度分析了CPA降低水溶液的冰点。加入CPA后,平均每个水分子参与的氢键数目减少以及CPA分子与水分子之间氢键相互作用的周期大于水分子之间氢键作用的周期是CPA降低水溶液冰点的微观机理。为了进一步从量的角度探求CPA降低水溶液的冰点与溶液微观结构的关系,提出了一种新的参数——氢键受体数目与供体数目比。研究表明:在较低的CPA浓度下,溶液平衡融化温度T_m与水分子氢键受体数目与供体数目比φ_w呈线性关系;随CPA浓度的增大,T_m与φ_m的线性关系逐渐破裂,而T_m与甘油分子氢键受体数目与供体数目比φ_g呈线性关系。φ_w与T_m和φ_g与T_m的线性关系成立的浓度范围与采用的偏差容忍度有关。采用2K的偏差容忍度时,在所有氢键准则和温度下,二元和三元溶液中均存在某个浓度x_g~*:当x_g≤x_g~*时,T_m与φ_m呈线性关系;当x_g≥x_g~*时,T_m与φ_g呈线性关系。对于二元溶液,x_g~*=0.15,对于三元溶液,0.13＜x_g~*＜0.15。更多还原

【Abstract】 Intracellular ice formation(IIF) and ice crystal growth are of great importance in research on cell freezing injury.To understand the exact mechanism of IIF injury,besides IIF and ice crystal growth,the interactions between intracellular ice and cryoprotective agent (CPA),intracellular proteins and cell membrane must be investigated.As a starting work,the structure and dynamics of CPA aqueous solutions must be studied.In the present paper, glycerol is selected as a CPA and mouse oocyte is chosen as a model cell.Karlsson’s intracellular ice growth model has been modified and CPA-water binary and CPA-water-sodium chloride ternary solutions have been studied using molecular dynamics simulation methods.A method has been introduced to calculate nucleation temperature of systems with variable compositions.Coupling with the cell dehydration equation,the homogeneous nucleation temperature of intracellular solution has been calculated as a function of time.It has been found that for glycerol-water-sodium chloride ternary solution,the depression of homogeneous nucleation temperature is linearly related with the depression of equilibrium melting point with a factor of 1.17.Then,the relation,coupling with soft impingement effect modification,has been used to modify Karlsson’s intracellular ice growth model.Using the modified model,the critical cooling rates and critical CPA concentrations for model cell vitrification have been predicted.The results show that the predicted values of the modified model are in good agreement with the values in literature and are better than the predicted values of Karlsson’s model.Further investigation shows that soft impingement effects must be considered when the final intracellular ice volume fraction is larger than 0.1.The hydrogen bonding structure and dynamics in CPA-water binary and CPA-water-sodium chloride ternary solutions have been analyzed.Only O-H...O hydrogen bond exists in binary and ternary solutions.The hydrogen bonding ability of oxygen atom in water molecules(OH2) is larger than that of oxygen atoms in glycerol molecules(O1,O2 and O3).The mean number of hydrogen bonds per OH2 atom decreases as glycerol concentration increases.However,the mean numbers of hydrogen bonds per O1,O2 and O3 atom show no tendency as glycerol concentration increases.The hydrogen bonding abilities of hydrogen atoms in water molecules are slightly smaller than that of hydrogen atoms in glycerol molecules.Unlike oxygen atoms,the difference between hydrogen bonding abilities of hydrogen atoms in water and glycerol molecules are quite small.The mean number of hydrogen bonds per hydrogen atom doesn’t change with glycerol concentration.The mean number of hydrogen bonds per glycerol molecule shows a tendency of decrease as glycerol concentration increases,but the tendency is not obvious,considering the errors.The mean number of hydrogen bonds per water molecule decreases as glycerol concentration increases. The results of hydrogen bonding dynamics analysis show that the relaxation time of hydrogen bonding intermittent autocorrelation function and hydrogen bonding lifetime show a tendency of increase as glycerol concentration increases and the lifetime of hydrogen bonds between water molecules is shorter than that of hydrogen bonds between glycerol and water molecules indicating that hydrogen bonds between glycerol and water molecules are stronger than that between water molecules.The hydration structures of sodium cation and chlorine anion are not affected by glycerol molecules relative to other aqueous solutions.The depression of freezing point caused by CPA has been analyzed.The reason is that as CPA is added in,the mean number of hydrogen bonds per water molecule decreases and the lifetime of hydrogen bonds between glycerol and water molecules is larger than that of hydrogen bonds between water molecules.To explore the relation between freezing point depression caused by CPA and the hydrogen bonding network in quantity,a new parameter the ratio between hydrogen bonding acceptor to donor numbers has been introduced.It has been shown that,at low CPA cocentrations,the equilibrium melting temperature T_m is linearly related with the ratio between hydrogen bonding acceptor to donor numbers for water moleculesφ_w.As CPA concentration increases,the linarity betweenφ_w and T_m breaks and T_m becomes linearly related with the ratio between hydrogen bonding acceptor to donor numbers for glycerol moleculesφ_g.The concentration range in whichφ_w(φ_g) is linearly related with T_m depends on the temperature tolerance.When a tolerance of 2K is used,under all hydrogen bonding criteria and at all temperatures,a concentration x_g~* exists:when x_g≤x_g~*, T_m is linearly related withφ_w and when x_g≥x_g~*,T_m is linearly related withφ_g.For binary solutions,x_g~*=0.15 and for ternary solutions,0.13＜x_g~*＜0.15.更多还原