角膜内皮细胞高压力损伤机制的实验研究  

The mechanism of damage induced by high pressure in cultured corneal endothelial cells

作　　者：梁玲玲[1,2] 袁进[1] 陈家祺[1] 廖洪斐[2] 幸正茂[3] 周世有[1] 

机构地区：[1]中山大学中山眼科中心国家眼科学重点实验室,广州硕士研究生510060 [2]南昌大学第三附属医院 [3]南昌大学研究生院

出　　处：《眼科》2011年第3期155-159,共5页Ophthalmology in China

基　　金：国家自然科学基金(30801263)

摘　　要：目的探讨青光眼急性高眼压对角膜内皮细胞的损伤机制。设计实验性研究。研究对象体外培养的角膜内皮细胞。方法采用后弹力层撕除联合酶消化法获取角膜内皮细胞,免疫组化法鉴定细胞。实验分两组:A组:急性压力增高组,压力为6.67 kPa;B组:压力仿生培养,压力为2.0 kPa。倒置显微镜定期观察细胞形态及生长规律;HE染色观察细胞形态结构变化;台盼兰-茜素红染色观察高压对细胞的损伤作用;流式细胞术分析细胞活性;免疫荧光检测细胞胞浆中细胞色素C(CytC)的表达。主要指标角膜内皮细胞形态结构、凋亡率及胞浆中CytC的表达。结果获取的细胞经免疫法证实为角膜内皮细胞表型。两组细胞分别培养24 hr后,流式细胞术分析显示,高压力组的早、晚期细胞凋亡率分别为(16.40±0.95)%和(41.37±1.29)%;而正常压力组早、晚期细胞凋亡率分别为(1.07±0.40)%和(0.70±0.00)%,差异有统计学意义(P=0.000)。免疫荧光检测到高压力组角膜内皮细胞胞浆CytC呈阳性表达。结论高压力对角膜内皮细胞损伤呈时间敏感性。Objective To investigate the mechanism of corneal endothelial cell damage induced by acute high intraocular pressure. Design Experimental study. Participants Corneal endothelium cells cultured in vitro. Methods Corneal endothelium cells were acqured by tearing apart the descemet and digesting with trypsin, and identified by immunoassay. Then the cells were cultured in pressure provided by the pressure bionic system. Cells were divided into two groups： group A was acute stress increased group, cells were cultured under 6.67 kPa; group B was pressure bionic culture, ceils were cultured under 2.0 kPa pressure in vitro. Cells morphology and growth pattern were observed with inverted microscope; changes in cell morphology were determined with HE staining; cells damage induced by high pressure was observed with trypan blue - alizarin red staining. Cells activity was detected with flow cytometry; the expression of cell cytoplasm （CytC） was detected with immunofluorescence. Main Outcome Measures The changes of cell morphology, cell activity and the expression of CytC. Results The cells which were detected with immunoassay were proved as corneal endothelium cells. The apoptosis rate was analyzed after cultured for 24 hours in two groups： in the high-pressure group, ceils early and late apopto- sis rate was （16.40±0.95）% and （41.37±1.29）%, respectively; but in the normal pressure group, the early and late apoptotic rate was （1.07±0.40）% and （0.7±0.00）%, respectively（P=0.000）. The expression of CytC was positive by immunofluorescence assay in the corneal endothelial cells cultured under high pressure. Conclusion High pressure could induce damage of corneal endothelial cells in a manner of time-dependent, and apoptosis activation might be the main mechanism. （Ophthalmol CHN, 2011, 20： 155-159）

关 键 词：角膜内皮细胞 眼压 凋亡 

分 类 号：R775[医药卫生—眼科]