【作者】 权国波；

【导师】 章金刚； 韩颖；

【作者基本信息】 中国人民解放军军事医学科学院 ， 免疫学， 2007， 博士

【摘要】 冰冻干燥(冻干)保存人红细胞在临床输血和战伤救治方面具有重要意义。目前临床上主要的红细胞保存方法有4℃冷藏和80℃深低温保存。这些保存方法都有很好的效果，但又存在明显不足：4℃保存时间短，而且容易受到污染；-80℃保存可以将保存时间延长至10年以上，但需要笨重的超低温冰箱等专门储存设备，而且解冻后需要复杂的洗涤程序以去除甘油。对比之下，冻干保存的红细胞具有以下优势：便于室温保存，重量大大减轻，方便运输等。然而红细胞冻干保存并非易事，研究发现冻干后红细胞血红蛋白严重外漏，其主要原因是干燥过程对细胞膜产生严重损伤。有研究表明海藻糖能够提高细胞膜的耐干燥性。本实验以此为切入点，以小分子糖类(海藻糖和葡萄糖)和大分子物质作为主要保护剂进行人红细胞的冻干保存研究，为红细胞冻干保存的深入研究奠定基础。首先进行红细胞对海藻糖和葡萄糖吸收规律以及糖类吸收过程对红细胞结构和理化性质影响的研究，在简单扩散基础上，采用膜内外渗透压差异和磷脂相变结合的方法将小分子糖类导入细胞内。其次，采用葡萄糖、海藻糖、人血白蛋白和PVP作为主要保护剂对红细胞冻干保存的可行性进行了研究，并对冻干条件，如保护剂浓度、玻璃化程度、预冻温度、搁板温度以及再水化条件进行了优化筛选。结果表明：1．红细胞对糖类的吸收存在一定的规律性，即随着细胞外液糖浓度的增加、孵育温度的上升以及孵育时间的延长，红细胞对糖类的吸收效率呈上升的趋势；红细胞对葡萄糖的吸收效率明显高于海藻糖。另外在相同孵育条件下，海藻糖对红细胞溶血率和变形性造成的损伤明显高于葡萄糖，然而海藻糖维持PS非对称性分布和提高红细胞渗透耐受性的能力显著高于葡萄糖；另外海藻糖保护膜磷脂过氧化损伤的能力也要高于葡萄糖；在葡萄糖缓冲液中添加一定浓度的海藻糖，可以显著提高红细胞的渗透压耐受性、维持红细胞膜PS非对称性分布以及减少膜脂质过氧化损伤，同时葡萄糖在一定程度上也缓解了海藻糖对红细胞溶血率的不利影响。在葡萄糖引起的红细胞凋亡过程中，胱门蛋白酶—3和胱门蛋白酶—8没有活化，而亮抑酶肽却可以很好地抑制红细胞PS外翻。2．冻干红细胞保护液的筛选过程中，将海藻糖和葡萄糖导入红细胞可以显著降低冻干保存红细胞再水化后的溶血率，而且可以减少红细胞代谢功能的损失；DSC分析结果表明，随着保护液中PVP浓度的升高，红细胞的结晶起始温度随之下降，这表明保护液的玻璃化程度呈上升的趋势。然而随着玻璃化程度的升高，再水化后红细胞溶血率呈先下降后上升的趋势，这表明过高的玻璃化程度可能不利于红细胞的冻干保存；另外在保护液中添加人血白蛋白也可以对冻干红细胞提供保护作用。3．对冻干程序的研究表明，过低的预冻温度可以造成溶血率的增加，这主要是由于预冻过程中二次复温对红细胞造成冰晶损伤。当搁板温度低于—30℃时，再水化后红细胞溶血率在20％左右，各组之间差异不显著。4．再水化液中添加低浓度的大分子物质有利于再水化后红细胞的存活，而且较高的再水化温度也可以显著降低再水化后红细胞的溶血率。5．电镜观察结果表明再水化后80％的红细胞保持膜完整，但是仍有一些细胞呈棘形，而且也有部分红细胞发生血红蛋白泄漏。综上所述，通过本研究初步证明将小分子糖类和大分子物质作为主要保护剂冻干保存人红细胞是可行的。海藻糖在细胞的干燥保存方面具有不可比拟的优势，但是由于红细胞自身结构功能的特点从而导致海藻糖吸收过程中红细胞损伤的增加，而这些损伤直接导致冻干—再水化后红细胞损伤的增加，将海藻糖和葡萄糖结合起来，不但可以提高细胞内海藻糖和葡萄糖的浓度，而且海藻糖可以提高红细胞的渗透压耐受性、减少PS外翻和质膜过氧化损伤。下一步工作将集中于寻求一种安全有效的糖类导入方法，并对冻干过程对红细胞超微结构的损伤进行深入研究。更多还原

【Abstract】 Lyophilization of human red blood cells has great implication on clinical transfusion and therapy of war wound. At present, the main methods to preserve human red blood cells include 4℃storage and cryopreservation at -80℃. The protective effect of these methods is excellent, but there are some significant shortages in these methods. The storage time at 4℃is short and easy contamination by microbes can occur. Although cryopreservation at -80℃can greatly prolong the storage time to about 10 years, this method needs a heavy ultra-low-temperature refrigerator or other storage equipments. In addition, after thawing, glycerol needs removal through complicated washing process. Compared with the conventional methods, lyophilization has the following advantages: room temperature storage, less weight, easy transportation.However, lyophilization may be difficult. Serious leakage of hemoglobin in red blood cells can occur during lyophilization and rehydration. Lyophilization seriously injures the membrane. Trehalose can increase the ability of membrane to tolerate drying. In this study, small molecular sugars (trehalose and glucose) and polymer were used as main lyoprotectants to lyophilize human red blood cells. At first, the sugar loading regularity and the effect of sugar loading process on structure and physico-chemical property of red blood cells were studied. Small molecular sugars were loaded into red blood cells through a combination of osmotic imbalance and phospholipids phase transition. Then glucose, trehalose, human serum albumin, and PVP were used as main protectants to evaluate the feasibility of lyophilization of red blood cells. The lyophilization conditions including the protectant concentration, vitrification degree, freezing temperature, shelf temperature, and the rehydration conditions were also optimized. The data showed: 1 the uptake of sugars in red blood cells has certain regularity. With increase of extracellular sugar concentration, elevation of incubatin temperatures, and prolonging of incubation time, the sugar loading efficiency was increased steadily. But the glucose loading efficiency was significantly more than the trehalose loading efficiency. At the same incubation condition, the injuries of trehalose on hemolysis and deformability of red blood cells were significantly more than that of glucose, but the ability of trehalose to maintain the asymmetrical distribution of membrane PS and increase osmotolerance of red blood cells was significantly higher than that of glucose. In addition, trehalose can decrease the peroxidative damage of membrane phospholipids during sugar loading process. Addition of trehalose in the glucose buffer can significantly increase osmotolerance of red blood cells, maintain the asymmetrical distribution of PS and decrease peroxidative damage. Moreover, glucose can mitigate hemolysis during trehalose loading process. The caspase-3 and caspase-8 have not been activated during apoptosis of human red blood cells induced by high glucose. In addition, leupeptin can efficiently inhibit PS exposure of red blood cells induced by high glucose. 2 during the optimization of the lyoprotectants, loading both trehalose and glucose into cytoplasma can significantly decreases the percent hemolysis and loss of metabolic function of red blood cells after lyophilization and rehydration. The DSC data showed with increase of the PVP concentrations, the onset temperature of crystal was decreased, which showed the vitrification degree of protective solutions was increased. With the increase of the vitrification degree, the percent hemolysis after rehydration was firstly decreased and then increased, which showed excessive vitrification might not be suitable for lyophilization. Moreover, human serum albumin also can provide protection for lyophilized red blood cells. 3 the ultra-low freezing temperatures can lead to increase of hemolysis of lyophilized red blood cells owing to second warming during prefreezing. When the shelf temperatures were lower than -30°C, the percent hemolysis of cells after lyophilization and rehydration was approximately 20% and there were not significant difference among the treating groups. 4 adding low concentrations of polymers in the rehydration solutions can increase survival of lyophilized red blood cells. In addition, higher temperature also can significantly decrease hemolysis after rehydration. 5 Approximately 80% lyophilized cells observed by TEM have intact membrane, but some cells still had an echinocytic shape and the hemoglobin of some cells had partially leaked away.In conclusion, this study showed it is feasible to lyophilize human red blood cells using small molecular sugars and polymers as main lyoprotectants. Although trehalose has supervier advantages on dried cells, the self characteristics of red blood cells lead to serious injuries during trehalose loading process. Sugar loading process causes serious cell injuries which in turn manifest themselves during subsequent lyophilization and rehydration. But combination of glucose and trehalose can increase intracellular sugar concentration and osmotolerance, inhibit PS exposure, and decrease peroxidative injuries. The future work will focus on finding a safe and efficient sugar loading process and deeply study the effect of lyophilization on ultrastructure of red blood cells.更多还原