红细胞携氧功能随孵育时间动态变化的研究

血红蛋白氧合与脱氧的周期循环在体内驱动红细胞结构与功能调节中发挥着重要作用，已引起广泛关注;但是对体外培养的红细胞在单个活态水平上进行携氧功能随孵育时间动态变化研究未见报道。本研究应用显微激光共聚焦拉曼散射技术，采集不同时间的活态红细胞拉曼光谱，分析表征血红蛋白携氧能力特征峰(1 636和1 562 cm-1)和构象敏感的酰胺Ⅲ谱带(1 240～1 300 cm-1)随时间变化规律;同时结合扫描电镜观察相应RBC形态。结果发现，在0～24 h内，结构稳定、变构协同功能正常的血红蛋白发生着携氧量增加与减少和构象R态与T态交替变化，与此同时扫描电镜下双凹圆盘型红细胞也发生“伸长”与“收缩”交替变化。本结论不仅为体外研究红细胞携氧功能提供来自单个活细胞内的多水平特征参数，同时也为体外开展针对红细胞筛选活性组分、评价药物药效和毒副作用研究开拓思路。

Study on RBC Oxygen-Carrying Function with the Incubation Time

The cycle of Hemoglobin oxygenation and deoxidation plays an important role in driving structure and regulating function of red blood cell in vivo, has attracted wide attention. But it has not yet been reported about any studies on the oxygen-carrying function of individual living RBC in vitro with the incubation time. This study involved that using confocal Raman scanning microscopic technique, collecting the Raman spectra of living erythrocyte cultured in vitro at different time, analysing the peaks (1 636, 1 562 cm-1) with characterization of hemoglobin oxygen carrying capacity and the amide Ⅲ band (1 240～1 300 cm-1) with sensitivity to conformation for understanding those changes both of hemoglobin oxygen carrying capacity and protein conformation over time. Meanwhile, its corresponding surface micromorphology was observed via scanning electron microscopy (SEM). The results indicated that the during 24 h hemoglobin with stable structure and normal allosteric collaborative function occurred alternately with the oxygen uptake of increase and decrease and conformation R state and T state, while double concave disk red blood cells observed under SEM also alternately between stretch and shrink. The conclusion not only provides a multi-level characteristic parameter from a single living cell for the study of RBC oxygen-carrying function in vitro, but also the potential research ideas for the screening of the active component, the evaluation of drug efficacy and toxicity for RBC in vitro.