压力仿生培养对兔角膜内皮细胞调控的研究

目的 探讨体外压力仿生培养系统下不同梯度压力对角膜内皮细胞形态和功能的调控作用。设计 实验性研究。研究对象 兔角膜内皮细胞。 方法 将体外培养的第一代兔角膜内皮细胞分为五组：A组为无压力常规培养（空白对照），B组为正常压力仿生培养（15 mmHg），C组为压力波动组，将压力设为15mmHg、25 mmHg、20 mmHg、10 mmHg，D组30 mmHg压力培养，E组50 mmHg压力培养。细胞均培养24h，免疫法鉴定原代角膜内皮细胞，HE染色和电镜观察细胞形态的改变，流式细胞术检测细胞活性。主要指标 角膜内皮细胞的形态、存活率。 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。五组细胞分别培养24h后，经HE染色和电镜检测发现正常压力微环境培养的角膜内皮细胞排列紧密，六边形细胞居多，细胞表面微绒毛丰富，细胞核染色质丰富，而高压力培养的角膜内皮细胞活性差，细胞间隙加大。经流式细胞术分析显示，正常压力组、30 mmHg组、压力波动组、50 mmHg组的角膜内皮细胞培养24 h后细胞存活率分别为（98.16±0.45）%、（78.83±1.65）%、（70.2±3.54）%、（41.33±0.25）%（P=0.016）。高压力培养组中随着压力的升高和持续时间延长，细胞活性显著下降。结论 正常压力微环境培养对角膜内皮细胞形态和功能具有正向调节作用，而高压力对角膜内皮细胞具有损伤性，并随时间延长而加重。（眼科，2017，26： 56-60）

Research on regulation of rabbit corneal endothelial cells with pressure bionic cultivation

Objective To investigate the morphological and functional of regulations under different pressures culture on rabbit corneal endothelial cells by pressure bionic culture system in vitro. Design Experimental study. Participants Rabbit corneal endothelial cells. Methods The rabbit corneal endothelial cells were divided into five groups: Group A: without pressure culture group, as a blank control group; Group B: normal pressure bionic culture group (15 mmHg); Group C: fluctuated pressures were 15 mmHg, 25 mmHg, 20 mmHg, 10 mmHg, each pressure was for 6 hours; Group D: the cells were exposed to 30 mmHg pressure in vitro; Group E: 50 mmHg pressure bionic culture group. The five groups were cultured for 24 h respectively. Cells origin was identified with neuronal-specific enolase immunoassay. Cellular change in the structure was observed with HE staining and electron microscopy (SEM) test. Cell activity was detected with flow cytometry. Main Outcome Measures Cell morphology and the survival rate of cell. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stromal cells. Cells were hexagon in the majority, arranged closely and were rich in microvilli and nuclear chromatin in the normal pressure bionic culture group. Survival rate of cell in normal pressure bionic culture group, fluctuated pressures group, 30 mmHg group, 50 mmHg group was (98.16±0.45)%, (78.83±1.65)%, (70.2±3.54)%, (41.33±0.25)%, respectively (P=0.016). However, in other high pressures groups, corneal endothelial intercellular space increased and the cell activity was lower than that of Group B by HE staining and flow cytometry. Conclusions The experiment confirmed that cultivative rabbit corneal endothelial cells morphology and function were positive adjusted with the normal pressure bionic cultivation, but high pressures would cause damage to corneal endothelial cells, and the damage would serious with extended over time. (Ophthalmol CHN, 2017, 26: 56-60)