自体组织工程化角膜上皮修复角膜缘缺陷症  

Autologous tissue engineered corneal epitheled in reconstruc-tion of ocular surface

作　　者：王亚冬[1] 袁南荣[1] 刘超[2] 刘翠萍[2] 张寄南[3] 杜青[3] 马宏泰[4] 郑肇巽[4] 黄鹏[5] 张建桥[5] 

机构地区：[1]南京医科大学附属第一临床医学院眼科,江苏省南京市210029 [2]南京医科大学附属第一临床医学院内分泌科实验室,江苏省南京市210029 [3]南京医科大学附属第一临床医学院中心实验室,江苏省南京市210029 [4]南京医科大学附属第一临床医学院病理科,江苏省南京市210029 [5]南京医科大学附属第一临床医学院动物实验中心,江苏省南京市210029

出　　处：《中国临床康复》2005年第46期65-68,T0003,共5页Chinese Journal of Clinical Rehabilitation

基　　金：江苏省教委自然科学基金资助项目(736FA0105)~~

摘　　要：目的:用组织工程方法构建角膜上皮组织,观察自体工程化角膜上皮修复角膜缘缺陷症的可行性。方法:①实验于2000-01/2003-12在南京医科大学第一临床医学院中心实验室和内分泌科实验室完成。选用新西兰白兔15只,雌雄不拘。用手术方法做成单眼角膜缘缺陷动物模型,为移植实验的受体眼。另一只眼为供体眼。15只兔被随机分为3组:自体角膜上皮移植,羊膜移植组,对照组。②自体角膜上皮移植组:取供体眼角膜缘上皮组织约3mm3,体外培养。对原代培养和传代培养的角膜干细胞进行形态学观察,采用糖原PAS反应鉴定细胞糖原染色;采用免疫组化法鉴定细胞角蛋白AE1表达;对培养细胞进行透射电镜和扫描电镜观察。在去除上皮的保存人羊膜组织上,接种培养细胞,气液界面培养2周构建复层上皮组织。倒置显微镜观察细胞形态和生长特征;组织固定,包埋,切片,染色,观察细胞生长状态和蛋白表达。透射电镜观察超微结构。5只眼成模后4周,受体眼去除角膜浅层新生血管膜,行自体培养角膜上皮移植术。羊膜移植组:5只眼成模后4周,行羊膜移植修复角膜表面;对照组:5只眼成模后不做处理,为模型对照组。③移植实验后7,10,20,30d,分别行裂隙灯显微镜检查,角膜荧光素染色,眼前部照相。并分别处死各组动物,取手术区域角膜组织,甲醛固定,石蜡包埋,病理切片,染色,镜检观察。结果:①培养细胞的生长状态:原代培养48h细胞贴壁,15d左右形成单层。传代培养次日细胞贴壁,10d形成良好的单层。细胞胞体饱满,呈多角形,大小基本一致。②培养细胞的形态学特征:苏木精-伊红染色,细胞呈多角形,核呈长圆形,细胞大小一致,排列整齐。AE1单克隆抗体染色阳性,PAS染色阴性。③培养细胞的超微结构:透射电镜可见培养细胞之间有桥粒连接,缝隙连接。扫描电镜可见细胞表面有微绒毛结构。④AIM： To construct a cornea epithelium by means of tissue engineering, and investigate the feasibility of autologous engineered corneal epithelium in the reconstruction of the corneal surface. METHODS： The experiment was carried out in the Central Laboratory and Laboratory of Endocrinology of the First Clinical Medical College of Nanjing Medical University between January 2000 and December 2003. Fifteen New Zealand white rabbits of beth genders were made into models of unilateral limbal dysfunction by surgery, which were the recipient corneas to receive the transplantation experiment. The animals were divided into three groups randomly：Group A （tissue engineering cornea epithelium autograft transplantation）, Group B （anmiotic membrane transplantation）, Group C （model control）. Group A： With the aid of a surgical microscope, 3-by-3-mm biopsy samples were taken from the rabbit＇s limbus and transported to the laboratory for culture. The growth characteristics of the cultured cells were observed. The cells were stained positive to cytokeratin by AE1 monoclonal antibody and negative for the PAS. The characteristics of the cultured ceils were also observed under transmission electron microscope and scanning electron microscopy. Tissue-engineered epithelial-cell sheets were obtained ex vivo by culturing the cells on amniotic membrane with the air-liquid interface culture for two weeks. Samples were fixed and processed with use of standard histologic procedures. The cell sheets on the anmiotic membrane were examined by both light and electron microscopy. At 4 weeks after the ocular surface injury, the surfaces of the recipient conjunctivalized cornea of the rabbit were surgically reconstructed by transplanting tissue-engineered epithelial-cell sheets. Group B： 5 eyes underwent anmiotic membrane transplanting to reconstruct the corneal surfaces. Group C： 5 eyes obtained no treatment being used as a model control. Corneal epithelial fluorescein staining with Slit lamp examination and anterior eye

关 键 词：移植 自体 角膜缘/细胞学 干细胞/移植 羊膜/细胞学 

分 类 号：R394.2[医药卫生—医学遗传学]