AQUAPORINS, ICE FORMATION IN CELLS,/CRYOPRESERVATION

描述（由申请人提供）： 低温生物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像小鼠这样的模型动物的胚胎和精子的突变系的加速数量。 这些突变株系对于揭示疾病的遗传基础至关重要。 虽然这些细胞类型中的许多可以成功冷冻，但目前有相当大的数量不能。 此外，到目前为止，只有少数组织和更少的器官可以冷冻保存。 冷冻过程中细胞损伤的一个主要原因是细胞内冰的形成，高存活率要求避免或尽量减少它。 一个问题是，为避免这种情况而采取的通常措施可能会引起其他原因造成的伤害。 细胞内冰形成的一种理论是外部冰与细胞表面接触，并通过细胞膜中预先存在的孔生长，使细胞内部成核。 许多细胞中预先存在的一类重要孔是由水通道蛋白形成的孔。 这些是大约十年前发现的跨膜蛋白，形成了埃大小的孔。 一些水通道蛋白（例如AQP-1）中的孔仅允许水通过;其他水通道蛋白（例如AQP-3）允许水和小的非电解质（如甘油）通过。 这个建议的一个主要的具体目标是确定这些孔是否构成外部冰的进入路线，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蛙爪蟾的正常卵母细胞的冰成核温度与已经表达水通道蛋白的卵母细胞的冰成核温度来测试。 为了让外部的冰穿过质膜生长，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分数的影响来测试这一假设，从而改变细胞和冰之间在存在和不存在水通道蛋白的情况下的接触概率。另一个因素可能是外部冰的结晶形式。 抗冻蛋白影响这种形式，并且已知会影响冰成核温度。 研究人员将测试它们与其他两种（水通道蛋白的存在和不存在以及冷冻部分的大小）的组合的效果。 最后，研究人员将使用上述信息来确定是否可以以不引入其他有害因素的方式最小化细胞内冷冻，从而提供新的方法来提高困难类型的细胞和组织的存活率。

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.