Objective To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. Methods Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa( 14. 66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. Results NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexiv-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0. 1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (x2 =594. 0,P ＜0. 01 ). After cultured for 96 h,the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. Conclusions The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.%目的 探讨模拟前房压力对角膜内皮细胞形态及生物学功能的影响.方法 实验研究.采用后弹力层撕除联合酶消化法分离及纯化角膜内皮细胞,细胞悬液接种于培养板内,分为两组:A组采用常规的无压力培养;B组为压力仿生培养,压力设为2.0 kPa(14.66 mm Hg).倒置显微镜定期观察细胞形态及生长规律,神经元烯醇化酶免疫法鉴定细胞来源;苏木素-伊红染色及扫描和透射电镜分析细胞结构的变化,流式细胞术检测细胞活性.免疫荧光检测细胞间紧密连接蛋白(ZO-1)表达情况.结果 获取的细胞神经元烯醇化酶抗体表达阳性,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.两组细胞分别培养120～144 h后,压力仿生培养组见细胞扁平,胞质丰富,细胞排列紧密,呈铺路石状,六边形细胞居多;常规培养组细胞在形成单层的时间上与压力仿生培养组并无差异,但细胞以多角形为主,大小不一,细胞中颗粒样物质较多.扫描电镜下压力仿生培养组角膜内皮细胞形成连接紧密的单层,形态为多边形,表面微绒毛丰富,细胞间伸出足突相连,与底物贴附紧密,而常规培养组细胞脱片现象较明显.压力仿生培养组荧光标记的磷酯结合蛋白-碘化丙啶(Annexin V-FTTC/PI)细胞凋亡检测示细胞存活率为98.2%,早期凋亡率为0.7%,末期凋亡率为1.0%,死亡率为0.1%,而常规培养组细胞相对应上述检测值分别为92.2%,5.2%,2.3%及0.3%,经卡方检验两组比较差异存在统计学意义(x2=594.0,P＜0.01).培养96 h后压力培养组角膜内皮细胞间ZO-1蛋白表达明显高于对照组.结论 低压力对角膜内皮细胞生物学活性有正向调节作用,并且表现为时间敏感性,建立了全新的角膜内皮细胞压力仿生培养模式,为体外进行角膜内皮细胞的生理功能与损伤修复研究提供了新的平台.
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