Sequential development of intercellular junctions in bioengineered human corneas
Sequential development of intercellular junctions in bioengineered human corneas
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DOI：
10.1002/term.178
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发表时间：
2009-08-01
影响因子：
3.3
通讯作者：
Alaminos, M.
中科院分区：
文献类型：
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作者：
Gonzalez-Andrades, M.;Garzon, I.;Alaminos, M.
我们已经进行了一个连续的研究细胞间连接的形成和分化的人角膜替代品组成的人工角膜基质和角膜上皮，开发的组织工程。为了产生这些人工人类角膜，我们开发了一种角膜基质替代物，使用纤维蛋白和琼脂糖支架，将人类角膜细胞浸入其中，然后在上面培养人类角膜上皮。电子显微镜和免疫荧光分析显示，人工角膜与一个或两个上皮细胞层没有显示出任何形成的细胞间连接。相反，在多层成熟角膜替代物中发现了几种类型的细胞-细胞连接，尤其是桥粒。同时，编码斑珠蛋白3（PKG 3）、桥粒芯糖蛋白3（DSG 3）和桥粒斑蛋白（DSP）、闭锁小带1（ZO-1）和2（ZO-2）以及连接蛋白37（Cx 37）的基因的表达在多层人工角膜中比在未成熟人工角膜中更高，如微阵列和免疫荧光所示。虽然ZO-1，ZO-2和Cx 37蛋白的表达是均匀的，PKG 3，DSG 3和DSP的表达仅限于最顶端的细胞层在人工角膜浸没在培养基中的所有时间，而表达较高的中间细胞层，类似于正常的人类对照角膜，当角膜替代品提交到空气-液体培养技术。这些结果表明，提交给空气-液体培养技术的培养的角膜替代物倾向于形成与人类天然角膜上皮非常相似的发育良好的上皮，这表明这些人工角膜最终可用于临床或体外目的。版权所有（C）2009约翰威利父子有限公司
We have carried out a sequential study of intercellular junction formation and differentiation on human corneal substitutes consisting of an artificial corneal stroma and a corneal epithelium, developed by tissue engineering. To generate these artificial human corneas, we developed a corneal stroma substitute, using fibrin and agarose scaffolds with human keratocytes immersed within, then cultured the human corneal epithelium on top. Electron microscopy and immunofluorescence analyses revealed that artificial corneas with one or two epithelial cell layers did not show any formation of intercellular junctions. in contrast, several types of cell-cell junction, especially desmosomes, were found in multilayered mature corneal substitutes. Concomitantly, the expression of genes encoding for plakoglobin 3 (PKG3), desmoglein 3 (DSG3) and desmoplakin (DSP), zonula occludens 1 (ZO-1) and 2 (ZO-2) and connexin 37 (Cx37) was higher in multilayered artificial corneas than in immature artificial corneas, as shown by both microarray and immunofluorescence. Although expression of ZO-1, ZO-2 and Cx37 proteins was homogeneous, PKG3, DSG3 and DSP expression was restricted to the most apical cell layers in artificial corneas submerged in culture medium at all times, whereas expression was higher in intermediate cell layers, similar to normal human control corneas, when corneal substitutes are submitted to air-liquid culture techniques. These results suggest that cultured corneal substitutes submitted to air-liquid culture technique tend to form a well-developed epithelium that is very similar to the epithelium of human native corneas, suggesting that these artificial corneas could eventually be used for clinical or in vitro purposes. Copyright (C) 2009 John Wiley & Sons, Ltd.