一氧化氮对悬浮红细胞保存质量影响及糖酵解代谢关系的研究

发布时间：2018-01-04 12:24

本文关键词：一氧化氮对悬浮红细胞保存质量影响及糖酵解代谢关系的研究　出处：《北京协和医学院》2015年硕士论文　论文类型：学位论文

【摘要】：红细胞(red blood cell, RBC)输注效果与红细胞保存质量有着密切的关系。针对红细胞保存质量难以再提高的问题,有研究结果显示,给保存期长的红细胞补充一氧化氮(nitric oxide, NO)可以明显改善红细胞的变形性及输注后效果。本实验室在前期研究的基础上,推测NO可能通过某些特定细胞群的作用来提高整体红细胞保存质量。目的：研究外源性NO药物供体硝普钠(sodium nitroprusside, SNP)和前体L-精氨酸(L-arginie, L-arg)对红细胞不同保存时期质量的影响,且探究NO与红细胞糖酵解代谢的关系以此来阐明NO作用于红细胞的部分机理。方法：密度梯度离心法分离已孵育1h不同浓度的SNP和L-arg的红细胞,通过检测红细胞变形性和渗透脆性来分别确立SNP和L-arg的最佳添加浓度。在不同保存时期添加最佳浓度的NO的红细胞经密度梯度分离后,检测分层后各层红细胞数目比例、丙酮酸激酶、变形性、渗透脆性等指标。D.L甘油醛(D.L glyceraldehyde, DL-GA)是目前公认的最理想的糖酵解抑制剂,它能特异性抑制丙酮酸激酶(pyruvate kinase,PK)和己糖激酶(hexokinase, HK)活性从而达到抗糖酵解的目的。将保存末期的红细胞内分为6组,1组为对照组,2组为D.L甘油醛,3组为SNP,4组为D.L甘油醛加SNP,5组为L-arg,6组为D.L甘油醛加L-arg,孵育2h后密度梯度分离后洗涤2次所得的各层红细胞及未分层红细胞,分别检测变形性、PK、HK活性。结果：保存初期和中期的红细胞在添加10l,M的SNP或L-arg后,其变形性和渗透脆性改善效果明显,末期最佳浓度则为100μM。保存末期的红细胞数目比例在加入NO后2,3层红细胞增加,各层红细胞的天龄也在加入NO后有明显降低,其他指标均发现第4层细胞和保存末期红细胞在加入NO后有明显改善,其他红细胞没有明显的改变或规律性。保存末期的SNP或L-arg能明显提高各层红细胞的变形性、PK和HK活性,在此基础上添加D.L甘油醛能够降低上述指标。结论：保存初期和中期的红细胞SNP和L-arg的最佳浓度均为10μM,末期最佳浓度为NO仅对老化或保存损伤红细胞的保存质量有明显改善,对年轻红细胞没有规律性作用。NO能通过促进红细胞的糖酵解代谢从而提高红细胞的保存质量。
[Abstract]:Red blood cells (red blood cell, RBC) and the effect of infusion of red blood cell preservation quality are closely related. The red blood cell preservation quality difficult to increase the problem, some researches show that for long period of preservation of the red blood cells of nitric oxide (nitric oxide, NO) could significantly improve the erythrocyte deformability and transport after the injection effect. The laboratory on the basis of previous studies, suggesting that NO may through some specific cell populations to improve the overall quality of preservation of red blood cells. Objective: To study the effect of exogenous NO donor drugs (sodium nitroprusside, SNP) and precursor L- arginine (L-arginie, L-arg) on the quality of red cells to save time, and the relationship of NO and explore the mechanism of erythrocyte glycolysis metabolism in order to clarify the role of NO in the red blood cells. Methods: density gradient centrifugation had been incubated with different concentrations of 1H SNP and L-arg red blood cells The cell, and osmotic fragility to establish SNP and L-arg respectively, the optimal concentration of deformation through the detection of red blood cells. The best adding concentration of NO in different preservation period of red blood cells by density gradient separation, detection of stratified layers of RBC ratio, pyruvate kinase, deformability, osmotic fragility index of glyceraldehyde (.D.L D.L glyceraldehyde, DL-GA) is currently recognized as the most ideal inhibitor of glycolysis, which can specifically inhibit pyruvate kinase (pyruvate kinase, PK) and hexokinase (hexokinase, HK) activity so as to achieve the Antiglycolytic. Will the end of storage in the red blood cells were divided into 6 groups, 1 group the 2 group was D.L group, L-glyceraldehyde, 3 groups of SNP, the 4 group was D.L glyceraldehyde plus SNP, 5 were L-arg, 6 were D.L glyceraldehyde L-arg incubated 2H density gradient separation after washing 2 times the red blood cells and red blood cells were detected without stratification, deformation PK, the activity of HK. Results: the preservation of the early and mid red blood cells in the addition of 10L, M SNP or L-arg, the deformation and permeability improvement effect is obvious, the best concentration was increased at the end of the 2,3 layer 100 M. red blood cell preservation at the end of the red blood cell number ratio after joining NO, each layer of red the day old cells also decreased significantly after joining NO, the other indexes were found in fourth cell layers and preservation of red blood cells was significantly improved in the end after joining the NO, the other red cells did not change or obvious regularity. The end of storage of SNP or L-arg can significantly improve the deformation of each layer of red blood cells, and PK the activity of HK, based on the addition of D.L can reduce the index of glyceraldehyde. Conclusion: the optimum concentration of preservation of the early and mid red blood cells SNP and L-arg were 10 M, at the end of the best concentration of NO on aging or preservation injury of red blood cell preservation quality has improved significantly, for years Light erythrocytes have no regular effect on.NO by promoting the glycolysis and metabolism of red blood cells to improve the quality of red blood cells.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.1

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