AQUAPORINS, ICE FORMATION IN CELLS,/CRYOPRESERVATION

水通道蛋白、细胞中的冰形成、/冷冻保存

基本信息

批准号：
6788807
负责人：
PETER MAZUR
金额：
$ 36.39万
依托单位：
UNIVERSITY OF TENNESSEE KNOXVILLE
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-08 至 2007-05-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：在与健康相关区域中，冷冻生物学保存的细胞在越来越重要的直接和间接作用中起着越来越重要的直接和间接作用。直接作用包括辅助生殖以及在临床医学中使用冷冻保存细胞和组织。间接作用包括维持胚胎的突变线和模型动物（如小鼠）的精子的维持。这些突变线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可以成功地冻结，但目前却不能进行大量数字。此外，到目前为止，只能冷冻保存几个组织和更少的器官。冻结过程中细胞损伤的主要原因是形成细胞内冰，高存活率要求避免或最小化。一个问题是，避免它采取的通常步骤可能会引起其他原因的伤害。细胞内冰形成的一种理论是，外部冰与细胞表面接触，并通过细胞膜中现有的毛孔生长，以对细胞内部成核。在许多细胞中，一类重要的毛孔是由水通道蛋白形成的细胞。这些是大约十年前发现的跨膜蛋白，它们形成了作为孔的孔。一些水通道蛋白（例如AQP-1）中的孔仅允许水通过。其他（例如AQP-3）允许水和小型非电解质（如甘油）通过。该提案的主要特定目的是确定这些孔是否构成外部冰的进入途径，并在细胞内部构成（致命的）冰形成。通过比较小鼠和青蛙武士的正常卵母细胞的冰 - 核温度与表达水通道蛋白的卵母细胞的冰 - 核细胞的冰 - 核温度。为了使外部冰通过质膜生长，必须首先与该膜接触。研究人员将通过确定在外部培养基中改变冰分的影响，从而改变细胞和冰之间的接触概率，从而在存在和不存在水aquaporin的情况下，从而改变这种假设。另一个因素可能是外冰的结晶形式。防冻蛋白会影响该形式，并已知会影响冰核温度。研究人员将与其他两个（存在水通道蛋白和冷冻部分的大小）结合测试其效果。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冻结，从而提供了新的方法来增强困难细胞和组织的困难类型的存活。