角膜内皮细胞体外原代培养及生物学鉴定  

Primary culture of corneal endothelial cells in vitro and biological identification

作　　者：祁冰[1] 侯光辉[1] 季青山[2] 崔裕波[3] 吴静[3] 

机构地区：[1]暨南大学附属第三医院珠海市人民医院眼科中心,珠海519000 [2]安徽医科大学附属省立医院眼科,合肥230001 [3]暨南大学医学院眼科研究所,广州510630

出　　处：《中华实验眼科杂志》2014年第10期881-885,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：珠海市科技重点项目（2013D0401990017、PB20051014）

摘　　要：背景角膜盲是中国主要的致盲眼病之一。随着组织工程技术的进步和发展，组织工程移植膜的构建将为角膜病的治疗开辟新的途径，而选择和培养理想的种子细胞是工程化角膜的基础。目的采用胰蛋白酶消化法体外分离培养角膜内皮细胞（CECs）并进行常规培养，观察并鉴定细胞体外生长的生物学特性，为实验研究和工程化角膜的构建提供种子细胞。方法3月龄新西兰白兔12只，摘除新鲜眼球，光学显微镜下将兔角膜带有内皮细胞的后弹力层面完整分离，胰蛋白酶消化、纯化后得到CECs，进行体外培养。倒置显微镜下观察细胞的形态变化；锥虫蓝染色法计算培养细胞的存活率并绘制细胞生长曲线；采用CM—Dil染色法进行细胞标记传代；采用苏木精一伊红染色法观察培养细胞的形态，并用免疫组织化学染色法观察神经元烯醇化酶（NSE）抗体的表达，对细胞进行细胞表型鉴定；扫描电子显微镜下观察培养细胞的表面结构；采用免疫荧光染色法观察细胞质紧密连接蛋白1（ZO-1）的表达。结果揭取带有内皮细胞的后弹力层联合酶消化法可快速获得大量纯化的CECs，且快速贴壁生长并增生，体外培养5～7d即融合形成单层，呈铺路石样排列，培养细胞的存活率为95％；CM—Dil染色显示培养的CECs呈现红色环状的荧光颗粒，标记率达90％以上，连续传代后仍显示红色荧光。苏木精一伊红染色显示培养细胞呈多角形，核圆，细胞质中NSE呈棕黄色染色。扫描电子显微镜下可见细胞表面有丰富的微绒毛，细胞间足突相互连接，细胞体积大而扁平；荧光显微镜下可见细胞间ZO-1呈绿色荧光。结论通过完整分离角膜后弹力层联合胰蛋白酶消化法可体外培养、纯化和传代兔CECs，传2～3代的细胞具有CECs的生物学特性，有望为组织工程化角膜内皮移植膜的构建提供优质�Background Corneal blindness is one of the major blinding eye diseases in China. With the development and progress of tissue engineering technology, tissue-engineered corneas offers a new approach to the treatment of corneal diseases. To select and cultivate ideal seed cells is a foundation of construction of tissueengineered corneas. Objective This study was to evaluate the efficiency of stripe off the Deseemet membrane with endothelium plus enzymic digestion in the isolation of corneal endothelial cells and analyze the bionomies of rabbit corneal endothelial cells （CECs） in vitro. Methods Descemet membrane was stripped from fresh cornea of New Zealand rabbit under the dissection microscope. Descemet membrane with endothelium was incubated in trypsin and EDTA solution at 37 ℃ and then purified for CECs subculture in vitro. The morphology of the cultured cells was observed under the inverted microscope and marked by CM-Dil dye solution. Then the shape of the ceils was observed by hematoxylin and eosin staining and the cells were identified for the expression of neuron specific enolase （NSE） using immunoehemistry. The viability of the cells were evaluated by trypan blue staining. The surface structure of the cells were examined under the scanning electron microscope. Intercellular zonula occludens-1 （ZO-1） was identified by immunofluorecsence staining. Results A large number of purified CECs were obtained from Descemetmembrane with endothelium through enzymic digestion. Cultured cells grew well and formed monolayer 5-7 days later with the cobblestone stone-like arrangement. The survival rate of the cells was 95%. CECs presented with the red annular fluorescence for CM-Dil with the labeling rate 〉90%. NSE was positively expressed in the cytoplasm. Polygon CECs were seen by hematoxylin and eosin staining and showed the brown staining. Abundant microvilli on the cellular surface and interconnected foot process were seen under the scanning electron microscope. ZO-1 showed the green fluorescence. Conclu

关 键 词：角膜内皮细胞 后弹力层撕除联合酶消化 细胞培养 细胞标记 

分 类 号：R772.2[医药卫生—眼科]