中文摘要：

背景如何选择高质量、生物学特性更接近在体生理状态的角膜内皮种子细胞是组织工程角膜研究的基础。角膜内皮细胞（CECs）的培养、鉴定及生物学特性检测是优选角膜内皮种子细胞的瓶颈问题。目的建立兔CECs原代培养及鉴定的方法，检测传代后细胞的生物学特性。方法从30只新西兰大白兔的角膜组织完整撕除后弹力层及CECs层，采用胰蛋白酶消化法进行原代培养，观察培养细胞的生长状态。分别从形态学、基因水平和蛋白水平鉴定细胞：茜素红染色法观察细胞形态，并与新鲜角膜组织的内皮细胞进行对比；逆转录聚合酶链反应（RT—PCR）法检测IVα2型胶原（COL4A2）、血管内皮生长因子受体2（FLK1）、Na^-K^＋ATP酶α亚单位（ATPIA1）、水通道蛋白1（AQP1）、电压依丛性阴离子通道（VDACs）等相对特异性基因；免疫细胞化学法观察神经元特异性烯醇化酶（NSE）、Na^-K^＋ATP酶及紧密连接蛋白ZO-1的表达。采用MTT比色法测定传代细胞的增生活性变化，采用超微量ATP酶试剂盒及免疫荧光法分别测定传代细胞Na^-K^＋ATP酶活性及其表达量的变化。结果原代培养的兔CECs多在24h内贴壁，2—3d达融合，形态呈六边形。随着传代代数的增加，细胞形态逐渐发生改变，第2代、第3代细胞可见空泡样变。原代培养的细胞茜素红染色可见清晰的细胞轮廓，与在体的CECs形态相似。RT—PCR法检测可见COL4A2、FLKl、ATP1Al、AQP1、VDACs等基因在培养细胞中表达。免疫荧光染色结果显示NSE、Na^-K^＋ATP酶、ZO-1在培养细胞中呈阳性表达。MTT检测结果显示，传代后细胞的增生活性逐渐下降，尤其第2代、第3代细胞下降明显。定量检测结果显示随着传代代数的增加，兔CECs的Na^-K^＋ATP酶活性逐渐降低，各代细胞的Na^-K^＋ATP酶活性总体比较的差异有统计学意义（F=77．174，P=0．000）。结论?

英文摘要：

Background How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein,to establish a good culture method and effective identification method of corneal endothelial cells （CECs） is the key. Objective Present study was to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged ceils. Methods Rabbits CECs were isolated from Descemet＇ s membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0. 25% trypsin-0. 02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2 （ COIAA2 ） , vascular endothelial growth factor receptor 2 （ FLKI ） , Na^＋-K^＋ ATPase alpha 1 subunit （ ATP1 A1 ） , aquaporin 1 （ AQP1 ） , voltage-dependent anion channels （VDACs） by reverse transcription-polymerase chain reaction （RT-PCR）. The expression and distribution of neurone specific enolase （NSE） , Na^＋-K^＋ATPase, zonula occludens-1 （ ZO-1 ） were also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay,and Na^-K^＋ATPase activity of different generations cells was detected by ATPase kit. Results Majority of the primarily cultured cells adhered in 24 hours,infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well-defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of COL4A2, FLK1, ATP1A1 ,AQP1 and VDACs were positively expressed in the cells. However,the expression of CK12 was a