FUNDAMENTAL STUDY ON PROSTATE CRYOSURGERY -VIABILITY OF DEFORMED CELLS-

冷冻手术是使用冷冻来破坏异常组织，然后将其留在原位以被身体脱落或吸收。尽管冷冻手术比传统的外科手术切除组织有许多优点，但由于过去早期的治愈率出乎意料的低，最近在日本报道的病例很少。由于冷冻区域的实时监测是过去的关键问题，现在已经能够利用现代成像方法，因此，使冷冻手术普及的最重要的问题之一是制定合适的冷冻手术方案。从细胞水平对冷冻损伤机制的研究为评价该方案提供了理论和科学背景。近年来的研究表明，细胞在缓慢冷冻过程中，除了受到化学损伤外，还可能受到冰晶压缩引起的机械损伤。本研究的目的是发展一个定量的了解细胞破坏的变形。 ...更多信息 发展了一种新的实验方法来研究细胞活力与变形之间的关系。在该方法中，将一滴具有精确已知直径的微玻璃珠的细胞悬浮液置于玻璃板上，并将顶部盖玻片推向底部玻璃基板。一个特制的平台用于在压缩过程中保持两个玻璃表面平行，并确保在精确控制的温度下完成该过程。玻璃珠随机分布在整个溶液中，表现为具有精确已知尺寸的间隔物。细胞在两个平坦表面之间变形，并且这些表面之间的距离从玻璃珠的直径已知。将具有压缩细胞的玻璃板置于显微镜的目镜路径下并观察。用台盼蓝染料排除试验测量变形细胞的存活率，并通过细胞总数与吸收染料的细胞总数之间的比率来定义。细胞几乎是球形的，平均直径约为18微米。在0 ℃和23 ℃以及6种不同标称间隙（32.3、16.2、11.4、8.8、5.9、3.5 μ m）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。0 ℃和23 ℃下测得的存活率与间隙大小之间的关系相同。虽然超过80%的细胞在11.4微米的间隙中经受变形，但在5.9微米处40至50%被破坏，并且当差距尺寸减小到3.5微米时90%被破坏。这些结果表明，当细胞被挤压到其原始直径的30%时，约一半的细胞被破坏。如果假设细胞膜均匀膨胀，则这对应于细胞膜表面积增加50%。0 ℃和23 ℃下的活力基本相同，这一事实非常有趣。在23 ℃时，细胞高于细胞膜脂质相变温度，而在0 ℃时，细胞低于脂质相变温度。变形的细胞活力是独立于温度的观察，因此可能表明，变形损伤是不相关的脂质膜的机械性能。因此，冷冻压缩过程中细胞损伤的可能机制可能与细胞膜的变形有关。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