MANIFESTATIONS OF CELL-DAMAGE AFTER FREEZING AND THAWING
MANIFESTATIONS OF CELL-DAMAGE AFTER FREEZING AND THAWING
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DOI：
10.1016/0011-2240(88)90024-7
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发表时间：
1988-06-01
期刊：
影响因子：
2.7
通讯作者：
WALTERSON, M
中科院分区：
文献类型：
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作者：
MCGANN, LE;YANG, HY;WALTERSON, M
尽管质膜通常被认为是冷冻损伤的主要部位，但细胞在冷冻和解冻过程中遭受的原发损伤的性质尚不清楚。这项研究旨在通过监测细胞和质膜的几项功能测试来调查解冻后立即造成的损害的性质。仓鼠成纤维细胞、人淋巴细胞和人粒细胞在没有冷冻保护剂的情况下，在-1度降温的情况下受到分级冻融应力。C/分钟，温度在-10到-40度之间。C，然后直接在37℃的水中加热。或迅速冷却至零下196度。在快速变暖之前。用四甲基偶氮唑盐(3-[4，5-dimethylthiazol-2-yl]2，5-diphenyl-tetrazolium)比色法、二乙酸荧光素(FDA)法、集落生长试验和高渗液渗透压测定法评价细胞线粒体功能。细胞在-1℃降温后表现为渗透压计。C/min对低温没有反应，这表明质膜不是缓慢冷却过程中遭受损伤的主要部位。这些结果还表明，FDA测试不是测量细胞膜的完整性，而是反映荧光素离开细胞的通道的通透性。在细胞内可能冻结的条件下冷却后，较少的细胞可以渗透反应，这意味着质膜直接受到导致细胞内冻结的条件的破坏。提出了有核哺乳动物细胞冷冻损伤的一般模型，在该模型中，在低温脱水条件下冷却的细胞中，溶酶体的破坏构成主要损伤。
The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. The study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymph ocytes, and human granulocytes were subjected to a graded freeze-thawed stress in the absence of cryoprotective compound by cooling at -1.degree. C/min to a temperature between -10 and -40.degree. C, and then either warmed directly in water at 37.degree. C or cooled rapidly to -196.degree. C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1.degree. C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.