ANTIFREEZE PROTEIN MODULATES CELL-SURVIVAL DURING CRYOPRESERVATION - MEDIATION THROUGH INFLUENCE ON ICE CRYSTAL-GROWTH
ANTIFREEZE PROTEIN MODULATES CELL-SURVIVAL DURING CRYOPRESERVATION - MEDIATION THROUGH INFLUENCE ON ICE CRYSTAL-GROWTH
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DOI：
10.1073/pnas.89.19.8953
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发表时间：
1992-10-01
影响因子：
11.1
通讯作者：
HANSEN, TN
中科院分区：
文献类型：
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作者：
CARPENTER, JF;HANSEN, TN
抗冻蛋白(AFP)在抑制冰冻溶液中重结晶方面非常有效。Knight和Duman[Knight，C.A.&Duman，J.G.(1986)低温生物学23,256-263]提出，这可能是蛋白质在耐冻生物中的一个重要功能。我们已经在体外测试了这一建议，通过表征AFP对低温保存细胞恢复的影响，这些细胞通常可以在降温中存活下来，但随后会在升温过程中被冰晶生长破坏。较低浓度(例如，5-150微克/毫升)的甲胎蛋白可提高在羟乙基淀粉溶液中冷冻保存的红细胞的存活率。这种影响在以次优速度升温的样品中最为明显，也就是说，在这种情况下，冰的再结晶会被夸大。冷冻显微镜显示，在升温周期的后期，甲胎蛋白抑制了细胞外区域的冰重结晶。提高红细胞存活率的甲胎蛋白浓度可部分抑制重结晶。相对较高浓度的甲胎蛋白(例如1.54 mg/ml)在抑制细胞外重结晶方面要有效得多。然而，注意到细胞周围广泛的冰生长，以及随之而来的细胞损伤。甲胎蛋白诱导的冰生长的机制尚不清楚。我们认为，在AFP诱导的细胞保存增强和AFP诱导的细胞损伤增强之间存在一种微妙的平衡，这种平衡取决于对冰重结晶的抑制程度和细胞周围冰的优先生长。我们的结论是，在适当的条件下，AFP在自然界中的一项拟议功能可以被模拟，并可能应用于具有生物医学意义的材料的冷冻保存。
Antifreeze proteins (AFPs) are extremely efficient at inhibiting ice recrystallization in frozen solutions. Knight and Duman [Knight, C. A. & Duman, J. G. (1986) Cryobiology 23, 256-263] have proposed that this may be an important function of the proteins in freeze-tolerant organisms. We have tested this proposal in vitro by characterizing the influence of AFP on the recovery of cryopreserved cells, which often can survive cooling and yet subsequently be damaged by ice crystal growth during warming. Relatively low concentrations (e.g., 5-150 mug/ml) of winter flounder (Pseudopleuronectes americanus) AFP enhance survival of red blood cells cryopreserved in hydroxyethyl starch solutions. This effect is most apparent in samples warmed at suboptimal rates, i.e., where ice recrystallization would be exaggerated. Cryomicroscopy demonstrates that AFP inhibits ice recrystallization in the extracellular regions during the latter stages of the warming cycle. AFP concentrations that enhance survival of red cells confer partial inhibition of recrystallization. Relatively high concentrations of AFP (e.g., 1.54 mg/ml) are much more effective at inhibiting extracellular recrystallization. However, extensive growth of ice around the cell, and concomitant cell damage, is noted. The mechanism for this AFP-induced ice growth is unknown. We propose that there is a delicate balance between AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell damage and that this balance hinges on the degrees of inhibition of ice recrystallization and of preferential growth of ice around the cells. We conclude that, under appropriate conditions, one of the proposed functions of AFPs in nature can be emulated, and perhaps have application, in cryopreservation of materials of biomedical interest.