波动的压力对兔角膜内皮细胞的影响

目的　观察波动的压力对角膜内皮细胞的影响。方法　将体外培养的第一代兔角膜内皮细胞分为两组:Ａ组为压力波动组（压力设置为:１５ｍｍＨｇ～２５ｍｍＨｇ～２０ｍｍＨｇ～１０ｍｍＨｇ，每个压力持续６ｈ；１ｋＰａ＝７．５ｍｍＨｇ）；Ｂ组为３０ｍｍＨｇ压力组；Ｃ组为无压力组。三组细胞分别培养２４ｈ。免疫细胞化学染色法鉴定原代角膜内皮细胞形态；台盼蓝-茜素红联合染色检测细胞活性，ＨＥ染色观察细胞形态；Ｗｅｓｔｅｒｎ-ｂｌｏｔｔｉｎｇ检测细胞中Ｂｃｌ-２和Ｐ５３蛋白的表达水平。结果　获取的所有细胞证实为角膜内皮细胞表型，无角膜上皮细胞及基质细胞污染。三组细胞分别培养２４ｈ后，经台盼蓝-茜素红染色和ＨＥ染色证实:两个压力培养组的细胞活性较无压力培养组明显下降，其中压力波动组的细胞活性低于３０ｍｍＨｇ压力组。同时无压力组、３０ｍｍＨｇ压力组和压力波动组细胞中Ｐ５３蛋白的相对表达量分别为０．１５０±０．００５、０．２５３±０．０１４、０．６７０±０．０１９，差异有统计学意义（Ｐ＜０．０５）。结论　证实了高压力及非生理性波动的压力对角膜内皮细胞均具有损伤作用。

Effects of fluctuated pressure on corneal endothelial cells of rabbits

Objective To investigate the effects of fluctuated pressure bionic culture on rabbit corneal endothelial cells. Methods The rabbit corneal endothelial cells were divided into three groups , Group A : Fluctuated pressures were 15 mmHg - 25 mmHg - 20 mmHg - 10 mmHg ( I kPa = 7. 5 mmHg) , each pressure was for 6 hours; Group B:The cells were exposed t0 30 mmHg pressure in vitro; Group C : Without pressure culture group, as a blank control group, the three groups were cultured for 24 hours , respectively. Cells origin were identified by neuronal-specific enolase immunoassay, the cellular change in the structure were observed by HE staining , the cell activity was detected by trypan blue-alizarin red staining , and the expression level of Bcl-2 and P53 protein were detected by Western-blotting. Results NSE antibody of the primary corneal endothelial cells were positive without corneal epithelial cells and corneal stromal cells. Cells were cultured for 24 hours respectively, cell activity in two pressure groups was more decreased than that of group C by HE staining and blue staining with trypan blue-alizarin red staining ,the cell activity of the fluctuated group was lower than that of 30 mmHg goup. In the no pressure goup ,pressure fluctuated group and 30 mmHg group , the relative amount of P53 protein expression were 0. 150 +0. 005 ,0. 253 + 0. 014 ,0. 670 + 0. 019 ,respectively ,the relative amount of P53 protein expression was statistically significant (P < 0. 05 ) . Conclusion The experiment confirm that high pressure and abnormal fluctuated pressure will cause damage to corneal endothelial cells.