量热法和微灌流法研究细胞膜的渗透特性

发布时间：2018-03-02 18:51

本文选题：细胞膜渗透特性　切入点：DSC　出处：《合肥工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：细胞低温保存主要由以下几个步骤组成:添加低温保护剂(CPA);低温冷冻;低温保存;复温;低温保护剂洗脱。细胞的渗透参数可以预测细胞在冷冻、复温过程以及保护剂添加、去除过程中的体积变化,进而使细胞的低温保存效益最优化。细胞膜的渗透特性参数的测量是低温生物学领域的基础性工作,广泛使用的方法包括热分析方法和微灌流方法。热分析方法通过差示扫描量热仪(Differential Scanning Calorimeter,DSC)采集的热信号间接获取细胞体积数据,适用于大样本测量。目前热分析方法主要有慢-快-慢(Slow-Fast-Slow,SFS)冷冻法,和一次慢冻(Single Slow,SS)法。分别采用这两种方法测量细胞的渗透特性,结果显示两种方法的结果比较相近,且都属于合理范围。使用测得的参数预测细胞的最佳冷却速率,并模拟其他降温速率下的细胞体积变化,验证实验显示模拟和实验结果非常吻合,所测参数可以有效预测细胞的冷冻响应。通过比较两种热分析方法发现,虽然SS方法的温度程序更加简单,但细胞体积在不同降温速率下的变化却无明显区别;SFS方法不需使用多个压积的样品,实验流程和数据分析更简便。为了与热分析方法比较,采用通过图像分析直接获取细胞体积数据、适用于小样本测量的微灌流法研究细胞渗透特性。使用灌流系统分别进行一步和三步添加保护剂(Cryoprotective agents,CPA)实验,基于一步添加得到的渗透参数,模拟三步添加过程中的体积响应。结果显示,CPA会显著减小水渗透率;与一步添加相比,三步添加过程中水和保护剂分子的跨膜运输速率更快,且对CPA分子的促进作用更明显;基于一步添加参数预测的细胞收缩体积更小、体积响应的波动范围更大。
[Abstract]:Cryopreservation consists of the following steps: adding cryopreservation agent (CPA); cryopreservation; cryopreservation; rewarming; low temperature protective agent elution. In order to optimize the efficiency of cryopreservation, the measurement of membrane permeability parameters is the basic work in the field of cryogenic biology. The widely used methods include thermal analysis and microirrigation. Thermal analysis indirectly acquires cell volume data through thermal signals collected by differential Scanning calorimeter. It is suitable for large sample measurement. At present, the main thermal analysis methods are slow-fast-slow Slow-Fast-Slow-SlowSs freezing method, and one-time slow freezing single Slow-SlowSol method. The two methods are used to measure the osmotic properties of cells, and the results show that the results of the two methods are quite similar. Using the measured parameters to predict the optimum cooling rate of the cell and simulate the cell volume change under other cooling rates, it is verified that the simulation results are in good agreement with the experimental results. The parameters measured can effectively predict the cryopreservation response of cells. By comparing the two thermal analysis methods, it is found that although the temperature program of SS method is simpler, However, the changes of cell volume at different cooling rates have no obvious difference. The SFS method does not need to use multiple compacted samples, and the experimental procedure and data analysis are more convenient. In order to compare with the thermal analysis method, Cell volume data were obtained directly by image analysis, and the microperfusion method, which was suitable for small sample measurement, was used to study the cell permeability. The perfusion system was used to carry out one-step and three-step protective agents Cryoprotectants CPA experiments, respectively. Based on the osmotic parameters obtained by one-step addition, the volume response of the three-step addition process is simulated. The results show that the water permeability can be significantly reduced by CPA, and the transmembrane transport rate of water and protectant molecules in the three-step addition process is faster than that in the one-step addition process. The effect on CPA molecule is more obvious, and the cell contraction volume is smaller and the fluctuation range of volume response is larger based on the one-step parameter prediction.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q2-33

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