BIOCHEMICAL MECHANISMS IN TOXIC CELL DEATH

细胞杀伤是心肌等缺血性疾病的一个重要方面 脑梗塞和中风。在这些疾病中，动脉闭塞可以防止 或减少器官的血液供应，使氧气供应中断 受影响的细胞。当替代能源耗尽时，受影响的 细胞进入不可逆转的损伤状态，最终死亡。 在发生重大细胞死亡之前重建血液供应 导致细胞迅速死亡，而不是恢复。有几条证据 说明血液中导致这种现象的物质是钙离子， 它沿着陡峭的电化学梯度进入细胞； 缺乏能量的细胞无法泵出钙离子，从而导致 引起细胞死亡的细胞内钙离子升高。高架 在培养细胞中，细胞内钙离子浓度可以用 二价阳离子载体A23187。关于这方面的初步研究 实验室表明，细胞内升高引发的细胞死亡 Ca2+是一个活跃的过程，至少涉及3个可识别的生化过程 步骤，第一个看起来是磷脂酶。该计划的目的是 建议的研究是为了了解这一过程的生物化学，即 阐明所涉及的步骤，并尽可能说明 参与每一步的酶。这项研究的长期目标是 开发这一过程中关键步骤的抑制剂。这些抑制剂可能 可想而知被开发成药物来防止细胞死亡与 缺血性疾病。 为研究细胞死亡的生物化学而选择的模型系统是 培养中的永久细胞系进行可行的突变选择等 遗传方法。细胞活性将通过物理(例如 台盼蓝排除)和遗传(如增殖)标准。 阐明这种机制的实验方法包括：(1)选择 以及缺乏酶的突变(变异)细胞系的特征 个别步骤所需；(2)伤者的进一步特征 根据生化标准确定步骤的细胞和对 药理探针；(3)磷脂酶的特性 与第一步相关联的活动。

Cell killing is an important aspect of ischemic diseases such as myocardial infarction and stroke. In these diseases occlusion of an artery prevents or reduces blood supply to an organ, interrupting the oxygen supply to affected cells. When alternate energy sources are depleted the affected cells enter a state of irreversible injury and ultimately die. Reestablishing the blood supply before significant cell death occurs initiates rapid cell death rather than recovery. Several lines of evidence indicate that the agent in blood responsible for this phenomenon is Ca2+, which enters the cells down a steep electrochemical gradient; the energy-deprived cells are unable to pump the Ca2+ back out resulting in elevated intracellular Ca2+ which triggers cell death. Elevated intracellular Ca2+ concentrations can be modeled in cultured cells using the divalent cation ionophore A23187. Preliminary studies in this laboratory indicate that cell death triggered by elevated intracellular Ca2+ is an active process involving at least 3 identifiable biochemical steps, the first which appears to be a phospholipase. The aim of the proposed research is to understand the biochemistry of this process, i.e. to elucidate the steps involved and to characterize as far as practical the enzymes involved with each step. The long term goal of the research is to develop inhibitors of key steps in the process. These inhibitors could conceivably be developed into drugs to prevent cell death associated with ischemic diseases. The model system chosen for studying the biochemistry of cell death is a permanent cell line in culture making feasible mutant selection and other genetic approaches. Cell viability will be assessed by physical (e.g. trypan blue exclusion) and genetic (e.g. proliferation) criteria. Experimental approaches to elucidating the mechanism include (1) selection and characterization of mutant (variant) cell lines deficient in enzymes required for individual steps; (2) further characterization of the injured cells at the identified steps by biochemical criteria and response to pharmacological probes; and (3) characterization of the phospholipase activity associated with the first step.