Xu Bin, Fan Ting-Jun, Yang Hong-Shou, Sun Ai, Zhao Jun, Ma Xi-Ya, Hu Xiu-Zhong
Key Laboratory for Corneal Tissue Engineering, Ocean University of China, Qingdao 266003, Shandong Province, China.
Int J Ophthalmol. 2012;5(3):281-5. doi: 10.3980/j.issn.2222-3959.2012.03.06. Epub 2012 Jun 18.
To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium (TE-HCEP) with seeder cells from an untransfected HCEP cell line.
The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line, and scaffold carriers of denuded amniotic membrane (dAM) in air-liquid interface culture for 3, 5, 7 and 9 days, respectively. The specimens were examined with hematoxylin-eosin (HE) staining of paraffin-section, immunocytochemical staining, scanning and transmission electron microscopy.
During in vitro reconstruction of TE-HCEP, HCEP cells formed a 3-4, 6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3, 5 and 7 days, respectively. But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9. And the cells maintained positive expression of marker proteins (keratin 3 and keratin 12), cell-junction proteins (zonula occludens-1, E-cadherin, connexin 43 and integrin β1) and membrane transport protein of Na(+)-K(+) ATPase. The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5.
The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo. The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells, including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation. The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.
用未转染的人角膜上皮细胞系（HCEP）的种子细胞展示体外重建的组织工程化人角膜上皮（TE-HCEP）的形态和结构。
用未转染的HCEP细胞系的种子细胞体外重建TE-HCEP，并将脱细胞羊膜（dAM）支架载体置于气液界面培养3、5、7和9天。对标本进行石蜡切片苏木精-伊红（HE）染色、免疫细胞化学染色、扫描和透射电子显微镜检查。
在TE-HCEP的体外重建过程中，HCEP细胞在气液界面培养3、5和7天时，分别在dAM上形成了3 - 4层、6 - 7层和8 - 10层类似HCEP的结构。但在第9天时，细胞减少到5 - 6层，复层上皮结构变得松散。细胞维持标记蛋白（角蛋白3和角蛋白12）、细胞连接蛋白（紧密连接蛋白-1、E-钙黏蛋白、连接蛋白43和整合素β1）以及Na(+)-K(+)ATPase膜转运蛋白的阳性表达。TE-HCEP中的HCEP细胞在第5天时顶端表面富含微绒毛，并建立了大量细胞-细胞和细胞-dAM连接。
重建的TE-HCEP的形态和结构与体内HCEP相似。重建的TE-HCEP中的HCEP细胞保持了HCEP细胞的特性，包括形成细胞间和细胞-细胞外基质连接的能力以及进行膜转运的能力。未转染的HCEP细胞和dAM有望用于重建等效的HCEP，用于临床角膜上皮移植。
