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Osmotic Injury of Cells by the Solution Effect

溶液效应对细胞的渗透损伤

基本信息

批准号：
13650226
负责人：
TAKAMATSU Hiroshi
金额：
$ 2.3万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

To develop a fundamental understanding of cell damage due to the hypertonic solution during freezing, we have developed a new perfusion technique that isolates the effect of exposure to hypertonic solutions from other effects associated with ice crystals. This technique employs two speed-variable syringe pumps to give arbitrary change in the solution tonicity around the cells in a new perfusion chamber.Experiments were performed with a human prostatic adenocarcinoma cell line PC-3. First, the cells were exposed suddenly to a hypertonic HEPES buffered NaCl solution for variety of time between 3 and 40 minutes, and then returned to the isotonic condition. The post-hypertonic viability gradually decreased with increasing the time of exposure in 2. OM NaCl solution, while at 3.8 M viability decreased to 50 % after exposure of only 3 minutes and further decreased with increasing the time of exposure. Next, three patterns of change in tonicity were applied to the cells : the NaCl concentration was linearly increased with time and suddenly decreased, suddenly increased and linearly decreased, and linearly increased and decreased. In each experiment the cells are kept in the highest concentration, 2.0 or 3.8 M, for 12 minutes. No statistical difference was found between the viability obtained with sudden changes and linear changes with the rate of 1.0 M/min. At 0.1 M/min, however, more cells survived after the linear increase of NaCl concentration compared with the sudden increase. The viability was however independent of the decrease manner in concentration. The result that showed time-dependent survival indicates that the cell might be damaged chemically in part. On the other hand, the fact that the cell damage depends not on the decrease manner but the increase manner in concentration at lower rate is interesting and important. It may suggest that the cell is injured mechanically during osmotic shrinking.

为了从根本上了解冷冻过程中高渗溶液对细胞的损伤，我们开发了一种新的灌注技术，将暴露于高渗溶液的影响与冰晶相关的其他影响隔离开来。该技术采用两个变速注射泵在一个新的灌注室中任意改变细胞周围的溶液张力。首先，将细胞突然暴露于高渗HEPES缓冲的NaCl溶液中3至40分钟的各种时间，然后恢复到等渗条件。随着暴露时间的延长，2。OM NaCl溶液中，而在3.8 M下，仅暴露3分钟后，存活率下降至50%，并随着暴露时间的增加而进一步下降。接下来，将张力变化的三种模式应用于细胞：NaCl浓度随时间线性增加并突然降低，突然增加并线性降低，以及线性增加和降低。在每个实验中，将细胞保持在最高浓度2.0或3.8 M下12分钟。没有发现统计学差异之间的生存率与突然变化和线性变化的速率为1.0 M/min。在0.1 M/min，然而，更多的细胞存活后，线性增加的NaCl浓度相比，突然增加。然而，活力与浓度降低方式无关。显示时间依赖性存活的结果表明细胞可能部分受到化学损伤。另一方面，细胞损伤不依赖于浓度的降低方式而是依赖于浓度在较低速率下的增加方式的事实是有趣且重要的。这可能表明细胞在渗透收缩过程中受到机械损伤。