FUNDAMENTAL STUDY ON PROSTATE CRYOSURGERY -VIABILITY OF DEFORMED CELLS-

冷冻外科是利用冷冻来破坏不正常的组织，然后将这些组织留在原位，由身体吸收或吸收。尽管冷冻手术比传统的外科手术组织切除有许多优势，但由于过去早期治愈率出人意料地低，最近日本很少有病例报道。由于冻结区的实时监测是过去的关键问题，现在利用现代成像方法已经具备了能力，因此，发展合适的冷冻手术方案是普及冷冻外科的重要问题之一。为评估该议定书提供方法学的理论和科学背景是在细胞水平上对冻害机制的研究。最近的研究表明，细胞在缓慢冷冻过程中，除了化学损伤外，还可能受到冰晶压缩引起的机械损伤。这项研究的目标是对变形对细胞的破坏有一个定量的理解。…开发了更多新的实验方法来研究细胞活力与变形之间的关系。在这种方法中，将一滴带有精确已知直径的微型玻璃微珠的细胞悬浮液放置在玻璃板上，并将顶部盖片推向底部玻璃基板。一个特制的工作台被用来在压缩过程中保持两个玻璃表面的平行，并确保在精确控制的温度下完成该过程。玻璃微珠随机分布在整个溶液中，其作用类似于具有精确已知尺寸的间隔物。细胞在两个平面之间变形，从玻璃珠的直径可以知道这些表面之间的距离。将带有压缩细胞的玻璃板放置在显微镜的视路下观察。变形细胞的活力用台盼蓝染料排斥实验来测量，并由细胞总数与染上染料的细胞的比率来定义。实验是用人前列腺癌PC-3细胞株进行的。细胞几乎呈球形，平均直径约18微米。在0℃和23℃，以及3 2.3、16.2、11.4、8.8、5.9、3.5微米6个不同的标称间隙下，测量变形细胞的活力。来自32.3微米间隙实验的数据用作对照。测得的0℃和23℃的存活率与间隙大小的关系是一致的。在11.4微米的间隙中，80%以上的细胞在变形中存活，当间隙尺寸减小到3.5微米时，40%到50%的细胞被破坏，90%的细胞被破坏。这些结果表明，当细胞被挤压到其原始直径的30%时，大约一半的细胞被破坏。如果假设细胞膜均匀膨胀，这相当于细胞膜表面积增加50%。0℃和23℃的生存能力本质上是相同的，这一事实非常有趣。23℃时细胞高于细胞膜脂质相变温度，0℃时细胞低于膜脂相变温度。因此，观察到变形细胞的活性与温度无关，这可能表明变形损伤与脂膜的机械性能无关。因此，冷冻细胞压缩过程中的一种可能的损伤机制可能与细胞膜的变形有关。较少

Cryosurgery is the use of freezing to destroy abnormal tissue, which is then left in situ to be sloughed or resorbed by the body. Despite a number of advantage of cryosurgery over traditional surgical tissue removal, few cases are reported in Japan recently because of unexpected low cure rate in early days in the past. Since the real-time monitoring of frozen region, that was the critical problem in the past, is now capable by using modern imaging method, one of the most important problems to make cryosurgery popular is to develop appropreate cryosurgical protocols. Theoretical and scientific background that provides a methodology to assess the protocol is the study on the mechansm of freezing damage in cell level. Recent studies show that during slow freezing of cells, in addition to chemical damage, the cells may be also injured by ice crystal compression induced mechanical damage. The goal of this study is to develop a quantitative understanding of cell destruction by deformation.A … More new experimental procedure was developed to study the relation between the cell viability and deformation. In this method a drop of cell suspension with micro-glass beads with a precisely known diameter is placed on a glass plate, and the top cover slip is pushed toward the bottom glass substrate. A specially made stage is used to keep the two glass surfaces parallel during the compression process and to ensure that the procedures is done at a precisely controlled temperatures. The glass beads, which are randomly distributed throughout the solution, behave as spacers with precisely known dimensions. The cells become deformed between two flat surfaces and the distance between these surfaces is known from the diameter of the glass beads. The glass plates with the compressed cells are placed under the ocular path of microscope and viewed. The viability of the deformed cells is measured with a trypan blue dye exclusion assay and is defined by the ratio between the total number of cells and those that have taken up the dye.Experiments were performed with a human prostatic adenocarcinoma cell line PC-3. The cell is almost spherical with mean diameter of about 18 micron. The viability of the deformed cells was measured at 0 ℃ and 23 ℃, and six different nominal gaps : 32.3, 16.2, 11.4, 8.8, 5.9, 3.5 microns. The data from the 32.3 micron gap experiments served for controls. The relation between measured viability and gap size for 0 ℃ and 23 ℃ are identical. While more than 80 % of cells survive deformation in a gap of 11.4 micron, 40 to 50 % are destructed at 5.9 micron and 90 % are destroyed when the gap size is reduced to 3.5 micron. These results show that about half of cells are destroyed when cells are squeezed to 30 % of their original diameter. If uniform expansion of cell membrane is assumed, this corresponds to 50 % increase in the cell membrane surface area. The fact that the viability at 0 ℃ and 23 ℃ is essentially identical is extremely interesting. At 23 ℃ the cells are above the cell membrane lipid phase transition temperature and at 0 ℃ they are below the lipid phase transition temperature. The observation that the deformed cell viability is independent on temperature may therefore suggest that deformation damage is not related to the mechanical properties of the lipid membrane. Therefore a possible mechanism of damage during freezing cell compression may be related to the deformation of the cytoskelton. Less