DETERMINATION OF HYPERTHERMIC CELL KILLING MECHANISMS

初步数据表明，高血压引起的增加， 细胞内游离钙浓度（（Ca+2）i）具有细胞毒性 并有助于最终的细胞杀伤。 高温致 （Ca+2）i和总细胞内钙的变化将是 分别用荧光钙染料indo 1和 原子吸收 钙细胞毒性将被诱导为 使用钙离子载体离子霉素的非加热细胞，使得 细胞杀伤水平与以下观察到的相似 体温过高 如果细胞内钙的变化与 处于等存活状态的加热和非加热细胞， 细胞内钙将被牵连作为，或其中之一， 细胞对高温的反应 不能繁殖 生存水平的差异将 表明钙细胞毒性作用程度（如果有的话 高温杀人 钙离子激动剂和拮抗剂， 钙调节功能将用于进一步建立 钙在高温细胞死亡中的作用，并阐明如何 钙介导其细胞毒性作用。 完整和透化 加热的电池将用于确定（Ca+2）i是否增加，因为 钙离子从细胞外介质中流入， 细胞内储存。 将使用相同的系统来确定 处理过的细胞如何试图重新调节钙， 程度. (Ca+2）i将在单个单元中测量，该单个单元还将 为了建立一个殖民地， 杀死细胞所需的（Ca+2）i的阈值增加。 变化 在内质网的形态和细胞质分布中， 将监测网状组织、线粒体和细胞骨架， 在这些相同的细胞中定量，以确定 细胞被改变的（Ca+2）i损伤，代表致死性损伤 网站. 回答本研究提出的问题将有助于 确定高温如何与细胞相互作用。 这反过来将 导致在临床中更有教育意义地应用这种模式。

Preliminary data suggests that hyperthermia-induced increases in intracellular free calcium concentrations ((Ca+2)i) are cytotoxic and contribute to the resultant cell killing. Hyperthermia induced changes in (Ca+2)i and total intracellular calcium will be measured, respectively, with the fluorescent calcium dye indo 1 and atomic absorption. Calcium cytotoxicity will be induced into nonheated cells using the calcium ionophore ionomycin, such that the cell killing level is similar to that observed following hyperthermia. If changes in intracellular calcium are similar in the heated and nonheated cells at isosurvival, altered intracellular calcium will be implicated as the, or one of the, cellular responses to hyperthermia that renders cells reproductively nonviable. A disparity in survival levels will indicate to what degree (if any) calcium cytotoxicity contributes to hyperthermic killing. Agonists and antagonists of calcium and calcium-regulated functions will be used to further establish the role of calcium in hyperthermic cell death and elucidate how calcium mediates its cytotoxic effects. Whole and permeabilized heated cells will be used to determine if (Ca+2)i increases because of a calcium influx from the extracellular medium or a release from intracellular stores. The same system will be used to determine how treated cells attempt to re-regulate calcium to homeostatic levels. (Ca+2)i will be measured in individual cell that will also be scored for the ability to form a colony in order to establish the threshold increase in (Ca+2)i required to kill a cell. Changes in the morphology and cytoplasmic distribution of the endoplasmic reticulum, mitochondria, and cytoskeleton will be monitored and quantified in these same cells to establish which aspects of the cell are damaged by altered (Ca+2)i and represent lethal lesion sites. Answers to the questions posed by this study will help determine how hyperthermia interacts with cells. This in turn will lead to a more educated application of this modality in the clinic.