冻干红细胞残余水含量的测量、分布及其代谢影响研究

发布时间：2018-06-20 17:34

本文选题：红细胞 + 冻干保存　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的本实验研究基于课题组先前的基础,探索冻干红细胞残余水分的最佳测定方法,探索冻干条件如温度、干燥时间对红细胞残余水含量的影响,同时研究了残余水的分布和其红细胞代谢的影响,为进一步深入探索残余水在红细胞冷冻干燥保存方面的影响提供实验和理论基础。方法对同一程序冻干的红细胞分别使用Karl-Fisher萃取法结合滴定法(Karl-Fisher法)、热重分析法、核磁共振法测定冻干红细胞残余水,分析各组间结果差异;使用同一程序同时冻干等量的含保护剂的血影细胞、含保护剂的红细胞、无保护剂的血影细胞、无保护剂的红细胞,分别测定其残余水含量并进行对比分析;控制冻干条件(时间、温度、真空度)制备不同残余水含量的冻干红细胞,用Karl-Fisher法测定其残余水含量,室温放置保存,分别在冻干之前,冻干完成时、完成1个月后、完成3个月后、完成6个月后测定其ATP活性、SOD活力,分析不同残余水含量组之间代谢数据的变化和关联情况。结果本研究使用含12%甘油浓度(w/v)的保护剂,使用三种不同方法测定冻干红细胞、冻干血影细胞、及水分标准品的残余水含量。Karl-Fisher法测得冻干红细胞水分含量为(3.37±0.05)%,无保护剂冻干红细胞水分含量为(1.22±0.09)%,水分标准品水分含量为(5.14±0.13)%;热重分析法测得冻干品水分含量为(12.40±0.56)%,无保护剂冻干品水分含量为(4.60±0.78)%,水分标准品水分含量为(5.28±0.16)%;核磁共振法测得冻干品水分含量为(0.78±0.09)%,无保护剂冻干品水分含量为(0.89±0.12)%,水分标准品水分含量为(4.99±0.18)%。除水分标准品外,三组间比较结果差异具统计学意义(P0.05)。使用方差分析多样本间LSD检验,冻干血影细胞组和冻干红细胞组比较均为P0.05,差异没有统计学意义。血影细胞和红细胞的残余水分量无差异。Karl-Fisher法能够很好地反应含保护剂及不含保护剂冻干红细胞的残余水分含量,热重分析法适用于无甘油保护剂的冻干红细胞水分测定,核磁共振法测定值过低。通过控制冻干条件制备残余水含量不同的六组冻干红细胞,由低到高分别为2.80%,5.12%,9.10%,10.77%,11.73%,12.45%。冻干后完成一周、1个月、3个月、6个月后复水、洗涤,测得ATP含量和SOD活性,同时与冻干前红细胞的测定值进行比较。各组间ATP值和SOD活力均随时间有所下降,残余水含量在2.80%和5.12%的两组样本功能随着时间下降趋势开始逐渐平缓。结论对于含甘油保护剂的冻干红细胞,Karl-Fisher法、热重法和核磁共振法均可以测定其残余水含量,测定绝对值之间存在显著差异。热重分析法测得结果偏高,核磁共振法测得结果偏低;冻干红细胞残余水与冻干血影细胞残余水含量差异无统计学意义,提示冻干红细胞残余水分布于红细胞膜上;不同残余水含量组冻干红细胞的ATP含量和SOD活性水平随着保存时间的延长均产生不同程度的下降趋势,残余水含量2.80%和5.12%组的下降趋势较其他组别在保存后期趋于平稳,提示将残余水含量控制在此区间将会有效提高红细胞冻干保存的时间。
[Abstract]:Objective to explore the optimal determination method of residual water in freeze-dried red cells based on the previous basis of the project group, explore the effect of freeze drying conditions such as temperature and drying time on the residual water content of red cells, and study the distribution of residual water and the effect of its red cell metabolism, in order to further explore the residual water freezing in red blood cells. The effects of drying preservation provide experimental and theoretical basis. Methods the residual water of freeze-dried red cells was determined by Karl-Fisher extraction combined with titration (Karl-Fisher), thermogravimetric analysis and nuclear magnetic resonance (NMR), respectively. The blood shadow cells, the red blood cells containing the protectant, the unprotected blood shadow cells and the unprotected red cells, the residual water content was measured and compared. The frozen dry red cells with different residual water content were prepared by controlling the freeze drying conditions (time, temperature and vacuum), and the residual water content was determined by Karl-Fisher method, and stored at room temperature. Do not have frozen dry before the completion of freeze dry, completed 1 months after completion of the completion of 3 months, completed 6 months after the completion of the determination of its ATP activity, SOD activity, analysis of different residual water content of the metabolic data between the changes and association. Results this study used 12% glycerol concentration (w/v) protection agent, the use of three different methods to determine freeze-dried red blood cells, freeze-dried blood The water content of the frozen dry red cells was (3.37 + 0.05)%, the water content of the freeze-dried red cells was (1.22 + 0.09)%, and the water content of the water standard was (5.14 + 0.13)%, and the moisture content of the freeze-dried freeze-dried products was (12.40 + 0.56)% (12.40 + 0.56)%, and the moisture content of the freeze-dried freeze-dried products without protectant was measured by.Karl-Fisher method. The content of the water content was (4.60 + 0.78)%, the water content of the water standard was (5.28 + 0.16)%, the water content of the freeze-dried products was (0.78 + 0.09)%, the water content of the freeze-dried freeze-dried products was (0.89 + 0.12)%, and the water content of the water standard was (4.99 + 0.18)%. The difference of the results between the three groups except the water standard was statistically significant (P0.05). The variance analysis varied the LSD test. The difference between the freeze-dried blood shadow cell group and the freeze-dried red cell group was P0.05, the difference was not statistically significant. There was no difference between the residual water components of the blood shadow cell and the red cell, and the.Karl-Fisher method could react well to the residual water content of the protective agent and the freeze-free red blood cell, and the thermogravimetric analysis was suitable. Six groups of frozen dry red cells with different residual water content were prepared by controlling freeze-drying conditions, from low to high to 2.80%, 5.12%, 9.10%, 10.77%, 11.73%, and 12.45%. were completed for one week, 1 months, 3 months and 6 months after the freeze drying, and the ATP content was measured. The activity of SOD was compared with the determination of red blood cells before freeze-drying. The ATP value and SOD activity of each group decreased with time. The two groups of samples with residual water content in 2.80% and 5.12% began to gradually decrease with time. Conclusion for lyophilized red cells with glycerol protectants, Karl-Fisher, thermogravimetry, and nuclear magnetic Co The residual water in the lyophilized red cell and the lyophilized blood shadow cells were low, and the residual water of the freeze-dried red cells and the lyophilized blood shadow cells had no statistical significance, suggesting that the residual water of the lyophilized red cells was distributed on the red cell membrane; The ATP content and SOD activity level of the frozen dry red cells in the residual water content decreased with the prolongation of the preservation time. The decrease trend of the residual water content 2.80% and 5.12% groups was more stable than the other groups in the later period of preservation, suggesting that the residual water content control in this interval would effectively improve the preservation of red blood cells. Time.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.52

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