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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。
Objective To investigate the effect of different gradient pressures on the morphology and function of corneal endothelial cells under in vitro biomimetic culture system. Design experimental research. Research object rabbit corneal endothelial cells. Methods The first generation rabbit corneal endothelial cells cultured in vitro were divided into five groups: group A was normal pressure-free culture (blank control), group B was normal pressure bionic culture (15 mmHg), group C was pressure-fluctuation group, Set to 15 mmHg, 25 mmHg, 20 mmHg, 10 mmHg, D group 30 mmHg pressure training, E group 50 mmHg pressure culture. The cells were cultured for 24 hours. The primary corneal endothelial cells were identified by immunohistochemistry. The cell morphology was observed by HE staining and electron microscopy. The cell viability was detected by flow cytometry. The main indicators of corneal endothelial cell morphology, survival rate. Results All cells obtained were confirmed as corneal endothelial cell phenotypes without corneal epithelial cells and stromal cell contamination. After cultured for 24h in each group, corneal endothelial cells cultured in normal pressure microenvironment were closely arranged, mostly hexagonal cells, abundant microvilli on the cell surface and abundant chromatin in the nucleus, while high pressure cultured cornea Endothelial cell activity is poor, increased cell gap. Flow cytometry analysis showed that the cell viability of the corneal endothelial cells in normal pressure group, 30 mmHg group, pressure fluctuation group and 50 mmHg group were (98.16 ± 0.45)%, (78.83 ± 1.65)%, (70.2 ± 3.54)%, (41.33 ± 0.25)% (P = 0.016). The cell viability decreased significantly with the increase of pressure and the prolongation of duration in high pressure culture group. Conclusions Normal pressure microenvironment has a positive effect on the morphology and function of corneal endothelial cells. However, high pressure can damage corneal endothelial cells and aggravate them with time.