A NEW MODEL FOR RAPID STRETCH-INDUCED INJURY OF CELLS IN CULTURE - CHARACTERIZATION OF THE MODEL USING ASTROCYTES
A NEW MODEL FOR RAPID STRETCH-INDUCED INJURY OF CELLS IN CULTURE - CHARACTERIZATION OF THE MODEL USING ASTROCYTES
复制标题
DOI：
10.1089/neu.1995.12.325
复制
发表时间：
1995-06-01
影响因子：
4.2
通讯作者：
POVLISHOCK, JT
中科院分区：
文献类型：
--
作者：
ELLIS, EF;MCKINNEY, JS;POVLISHOCK, JT
本研究的目的是开发一种简单的，可重复的模型，用于检查拉伸诱导损伤对脑组织培养细胞的形态，生理和生化后果。将来自1- 2日龄大鼠的大鼠皮质星形胶质细胞在具有2 mm厚柔性硅橡胶底部的市售25 mm直径组织培养威尔斯孔中培养至汇合。使用细胞损伤控制器来产生封闭系统并施加已知幅度（psi）和持续时间（毫秒）的快速正压。膜的变形以及在膜上生长的细胞的拉伸与气压脉冲的幅度和持续时间成比例。通过光学和电子显微镜定性评估细胞损伤的程度，并通过荧光染料碘化丙啶的核摄取进行定量评估，碘化丙啶从具有完整膜的细胞中排除。乳酸脱氢酶（LDH）酶释放进行了测定。发现细胞损伤与硅橡胶膜变形的程度成正比。增加细胞拉伸引起线粒体肿胀和空泡化以及神经胶质丝的破坏。拉伸也导致增加的染料吸收，与最大的染料吸收发生在50毫秒的压力脉冲持续时间，而在较长的时间段（秒）内产生的变形导致很少的染料吸收。随着损伤后存活率的增加，较少的细胞摄取染料，这意味着细胞修复。LDH释放也成正比的幅度的细胞拉伸，最大释放发生在2小时内的损伤。总之，我们已经开发了一种简单的，可重复的模型，以产生梯度，应变相关的损伤培养细胞。我们的持续实验表明，该模型可用于研究损伤的生物化学和生理学，以及作为一种工具来检查治疗剂的疗效。
The purpose of this study was to develop a simple, reproducible model for examining the morphologic, physiologic, and biochemical consequences of stretch-induced injury on tissue-cultured cells of brain origin. Rat cortical astrocytes from 1- to 2-day-old rats were cultured to confluency in commercially available 25-mm-diameter tissue culture wells with a 2-mm-thick flexible silastic bottom. A cell injury controller was used to produce a closed system and exert a rapid positive pressure of known amplitude (psi) and duration (msec). The deformation of the membrane, and thus the stretch of the cells growing on the membrane, was proportional to the amplitude and duration of the air pressure pulse. Extent of cell injury was qualitatively assessed by light and electron microscopy and quantitatively assessed by nuclear uptake of the fluorescent dye propidium iodide, which is excluded from cells with intact membranes. Lactate dehydrogenase (LDH) enzyme release was measured spectrophotometrically. Cell injury was found to be proportional to the extent of the silastic membrane deformation. Increasing cell stretch caused mitochondrial swelling and vacuolization as well as disruption of glial filaments. Stretching also caused increased dye uptake, with maximum dye uptake occurring with a 50 msec pressure pulse duration, whereas deformations produced over longer periods of time (seconds) caused little dye uptake. With increasing postinjury survival fewer cells took up dye, implying cell repair. LDH release was also proportional to the amplitude of cell stretch, with maximum release occurring within 2 h of injury. In summary we have developed a simple, reproducible model to produce graded, strain-related injuries in cultured cells. Our continuing experiments suggest that this model can be used to study the biochemistry and physiology of injury as well as serve as a tool to examine the efficacy of therapeutic agents.