AQUAPORINS, ICE FORMATION IN CELLS,/CRYOPRESERVATION

描述(由申请人提供)： 冷冻保存的细胞在健康相关领域发挥着越来越重要的直接和间接作用。其直接作用包括辅助生殖和在临床医学中使用冷冻保存的细胞和组织。间接的作用包括维持加速数量的胚胎和小鼠等模式动物精子的突变系。这些突变系对于揭示疾病的遗传基础至关重要。尽管这些细胞类型中的许多都可以成功冷冻，但目前仍有相当数量的细胞无法冷冻。此外，到目前为止，只有少数组织和更少的器官可以被冷冻保存。在冷冻过程中细胞损伤的一个主要原因是细胞内结冰的形成，而高存活率要求避免或将其降至最低。一个问题是，为避免这种情况而采取的通常措施可能会带来其他原因的伤害。细胞内结冰的一种理论是，外部冰与细胞表面接触，并通过细胞膜上预先存在的小孔生长，使细胞内部成核。许多细胞中一类重要的预先存在的毛孔是由水通道蛋白形成的。这些是大约十年前发现的跨膜蛋白，可以形成Angstrom大小的毛孔。一些水通道蛋白(如AQP-1)的孔只允许水通过；另一些(如AQP-3)允许水和甘油等小的非电解质通过。这项提议的一个主要的具体目标是确定这些气孔是否构成了外部冰的进入路线，以及随后在细胞内部形成(致命的)冰。这将通过比较来自小鼠和青蛙的正常卵母细胞和表达水通道蛋白的卵母细胞的冰核温度来检验。为了让外部冰通过质膜生长，它必须首先与质膜接触。研究人员将通过确定改变外部介质中冰的比例的影响来检验这一假设，从而改变细胞与冰在存在和不存在水通道蛋白的情况下接触的概率。另一个因素可能是外部冰的结晶形式。抗冻蛋白会影响这种形式，并已知会影响冰核温度。研究人员将与其他两项指标(水通道蛋白的存在和不存在以及冷冻部分的大小)一起测试它们的效果。最后，研究人员将利用上述信息来确定是否可以在不引入其他有害因素的情况下最大限度地减少细胞内冻结，从而为提高困难类型的细胞和组织的存活率提供新的方法。

DESCRIPTION (provided by applicant): Cryobiologically preserved cells are playing an increasingly important direct and indirect role in health-related areas. The direct roles include assisted reproduction and the use of cryopreserved cells and tissues in clinical medicine. The indirect role includes the maintenance of an accelerating number of mutant lines of embryos and sperm of model animals like the mouse. These mutant lines are vital to uncovering the genetic basis of diseases. Although many of these cell types can be successfully frozen, a sizeable number currently cannot. Furthermore, only a few tissues and fewer organs can be cryopreserved as of now. A major cause of injury in cells during freezing is the formation of intracellular ice, and high survivals demand that it be avoided or minimized. One problem is that the usual steps taken to avoid it may introduce injury from other causes. One theory of intracellular ice formation is that external ice makes contact with the cell surface and grows through pre-existing pores in the cell membrane to nucleate the cell interior. One important class of pre-existing pores in many cells are those formed by aquaporins. These are transmembrane proteins discovered about a decade ago that form Angstrom-size pores. The pores in some of the aquaporins (e.g. AQP-1) allow the passage of water only; others (e.g. AQP-3) allow the passage of water and small non-electrolytes like glycerol. A major specific aim of this proposal is to determine whether these pores constitute a route of entry for external ice, and consequent (lethal) ice formation in the cell interior. This will be tested by comparing the ice-nucleation temperatures of normal oocytes from mouse and the frog Xenopus with those of oocytes in which aquaporins have been expressed. In order for external ice to grow through the plasma membrane, it must first make contact with that membrane. The investigators will test this supposition by determining the effects of varying the fraction of ice in the external medium and, thus varying the probability of contact between cell and ice both in the presence and absence of aquaporins. Another factor may be the crystalline form of the external ice. Antifreeze proteins affect that form and are known to influence ice nucleation temperatures. The investigators will test their effect in combination with the other two (presence and absence of aquaporins and the magnitude of the frozen fraction). Finally, the investigators will use the above information to determine whether intracellular freezing can be minimized in ways that do not introduce other deleterious factors, thus providing novel approaches to enhancing the survival of difficult types of cells and tissues.