Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting
Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting
复制标题
DOI：
10.1016/s0014-4835(03)00033-2
复制
发表时间：
2003-06-01
影响因子：
3.4
通讯作者：
Kinoshita, S
中科院分区：
文献类型：
--
作者：
Ban, Y;Cooper, LJ;Kinoshita, S
目的。评估空气提升技术在羊膜（AM）上培养角膜缘上皮细胞用于眼表重建的有用性。具有良好屏障功能的培养片应该更适合此目的。在角膜上皮中，紧密连接M)在屏障功能中发挥着至关重要的作用。 TJ 复合物包括完整的跨膜蛋白occludin 和claudins，以及一些膜相关蛋白，例如ZO-1。在本文中，我们研究了TJ相关蛋白的屏障功能和表达。方法。从供体角膜获得并在无细胞 AM 上培养的角膜缘上皮分为两组。这些是非气升（Non-AL）组，其连续浸没在培养基中，以及气升（AL）组，其在培养基中浸没3周，然后通过降低培养基水平暴露于空气。通过电子显微镜测定形态和对辣根过氧化物酶（HRP）的渗透性。通过免疫印迹和逆转录聚合酶链反应评估紧密连接 (TJ) 相关蛋白和 mRNA 表达变化。结果。两组培养物均形成4-5层厚、分层良好的上皮。 AL 培养物具有紧密堆积的上皮细胞，所有 HRP/二氨基联苯胺 (DAB) 反应产物均积聚在浅表细胞的顶端表面。相比之下，非 AL 培养物的上皮细胞排列更松散，细胞间隙更大。 HRP/DAB反应产物渗透细胞间隙至3-4细胞层的深度。从统计学上看，各组之间表面细胞的细胞间隙和桥粒计数存在显着差异。对于 AL，TJ 相关蛋白定位于侧膜的顶端部分。 AL没有改变TJ相关蛋白和mRNA的量，而claudin亚型表达变得更加一致并且更接近体内角膜上皮的表达。结论。 AL技术减少了表层细胞的细胞间隙，促进屏障功能的形成。它可用于培养用于眼表重建的角膜上皮细胞。 (C) 2003 Elsevier Science Ltd. 保留所有权利。
Purpose. To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions M) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins.Methods. Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and rnRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction.Results. The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium.Conclusions. The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction. (C) 2003 Elsevier Science Ltd. All rights reserved.