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

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。④羊膜上接种细胞的生长特征:24h细胞贴壁生长,1周左右融合成单层。气液界面培养,细胞透明饱满,均匀成片生长,持续培养2周,细胞呈复层生长。⑤组织工程化角膜上皮形态特征:苏木精-伊红染色,细胞呈多角形,在羊膜上融合成片。组织切片,细胞在羊膜上可达到四五层。透射电镜可见桥粒样结构。⑥自体组织工程化角膜上皮修复实验:自体角膜上皮移植组:移植区角膜上皮细胞覆盖,呈复层排列,基底细胞呈柱状,基底膜完整。基质内有炎性细胞和红细胞浸润。供体角膜未出现临床病理性损伤。羊膜移植组:移植区域表层有上皮细胞覆盖,层次紊乱,极性消失。基质内有炎性细胞和红细胞浸润。对照组:角膜上皮排列紊乱,基底膜不完整,基质内有中性白细胞和红细胞浸润。结论:①角膜干细胞在体外能够培养传代。②培养角膜干细胞在保存人羊膜上能够生长,经气液界面培养,构建出复层上皮组织,与正常角膜上皮组织近似。③自体工程化角膜上皮可用于修复角膜缘缺陷症。

AIM： 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 segment photography was done in 7,10,20,30 days after transplantation experiment. The tissue of the cornea was taken to observe under light microscopy by histological analysis. RESULTS： ①ln primary culture, most of the cells attached to the bottom and began to proliferate after being cultured for 48 hours. In 5 days, the cells continued proliferation rapidly, and in about 10 days, the cells were confluent into film gradually with a very smooth and regular perimeter. In subculture, the cells attached the bottom during the first 24-hour and were confluent in 10 days. ② The cells ranged mosaic structure and localized together with clear rim. The cells had large nucleus and distinct nucleoli. Most of them were regularly of same size as polygonal appearance. Some cells stained positive to cytokeratin by AE1 monoclonal antibody and negative for the PAS. ③Transmission electron microscope showed lots of microvilli on the surface of the cells and tight junctions between them. Desmosomal cell-cell contact was evident by scanning electron microscopy. ④ Most of the cells attached to the anmiotic membrane in 24 hours. A confluent culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 7 days. A multi-layered epithelium was obtained with the air-liquid interface culture for more than 2 weeks, and more desmosomal junctions between the cells were found. ⑤The cultivatod epithelial sheets had 5 or 6 layers of stratified, well-differentiated cells. Histological examination revealed that the cultivated epithelial cells were similar in appearance to those of in vivo normal corneal epithelium. ⑥Corneas that were grafted with the cultivated corneal epithelial cells on an AM carrier were clear and were epithelialized 20 and 30 days after surgery. CONCLUSION： We have generated cultures of rabbit corneal epithelial cells from limbal tissues on AM. We have successfully carried out autologous transplantation of these cultivated corneal epithelial cells onto the ocular surfaces of rabbit eyes. We believe that autologous transplantation of cultivated corneal epithelium is a feasible method for ocular surface recon- struction.