人红细胞冻干前负载海藻糖最佳化研究

为更好的实现海藻糖在红细胞冻干保存中的保护作用,关键是克服质膜对海藻糖的非渗透性,使胞质内海藻糖达到有效浓度。本研究的目的是通过对人红细胞负载海藻糖的规律性研究,筛选出海藻糖负载的最佳负载条件并评价海藻糖负载对红细胞各项理化指标的影响。在不同孵育温度(4、22和37℃)、孵育时间(0、2、4、6、8、10小时)、不同负载缓冲液浓度(0、200、400、600、800、1000mmol/L)条件下检测新鲜红细胞对海藻糖的成功负载量及红细胞各项理化指标;在固定负载条件下,对新鲜红细胞和4℃保存72小时红细胞海藻糖负载、游离血红蛋白(FHb)、血红蛋白(Hb)和红细胞平均体积(MCV)进行了比较。结果表明:红细胞对海藻糖的负载与孵育温度、时间及负载缓冲液海藻糖浓度密切相关。随着温度的升高、时间的延长和负载缓冲液海藻糖浓度的增加,红细胞对海藻糖的摄取量也随之增加。在海藻糖负载最佳条件下,新鲜红细胞和4℃保存72小时红细胞的胞内海藻糖浓度、FHb浓度分别为65.505±6.314mmol/L、66.2±5.002mmol/L和6.567±2.568g/L、16.168±3.922g/L。结论:红细胞负载海藻糖的最佳条件是采用新鲜红细胞,在37℃条件下、海藻糖浓度为800mmol/L的负载缓冲液中孵育8小时,这一条件可使胞内海藻糖达到有效浓度,并保持红细胞细胞理化性质稳定和膜完整性。

Optimization of Trehalose Loading in Red Blood Cells before Freeze-drying

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.