Effect of intermittent shear stress on corneal epithelial cells using an in vitro flow culture model
Effect of intermittent shear stress on corneal epithelial cells using an in vitro flow culture model
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DOI：
10.1016/j.jtos.2018.04.005
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发表时间：
2018-07-01
期刊：
影响因子：
6.4
通讯作者：
Paulsen, Friedrich
中科院分区：
文献类型：
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作者：
Hampel, Ulrike;Garreis, Fabian;Paulsen, Friedrich
目的：本研究的目的是建立并评估在模拟眼表泪膜运动的可调节培养基流量下培养人端粒酶永生化角膜上皮（hTCEpi）细胞的体外模型。方法：使用IBIDI泵系统，在单向、连续或振荡、不连续的培养基流下培养细胞。细胞表面和细胞骨架的结构进行了研究，通过扫描电子显微镜和免疫荧光。实时荧光定量PCR检测上皮钙粘蛋白、闭合蛋白、紧密连接蛋白、桥粒斑蛋白、桥胶蛋白和粘蛋白的基因表达。免疫印迹分析桥粒斑蛋白、TJP、闭合蛋白和e-钙粘蛋白的蛋白表达，免疫荧光定位。虎红染色用于评估粘蛋白（MUC）屏障完整性。结果：介质流诱导的切应力显著改变了hTCEpi细胞的形态。细胞在不连续剪切力作用下呈现典型的扁平、多边形的复层鳞状上皮浅层细胞形态。细胞表面在剪切应力下表现出较少的隆起和较少的细胞外间隙。振荡培养液中E-cadherin、occludin和TJP的mRNA表达增加。桥粒斑蛋白和occludin蛋白在振荡剪切应力下上调。应力纤维的形成与流动方向不一致。MUC 1、MUC 4和MUC 16蛋白在所有培养条件下均定位，未检测到对mRNA表达的调节。单向conditions.Conclusion：我们的研究结果表明，剪切应力，因为它发生在眼表眨眼时对角膜上皮细胞产生显着的影响，如细胞形态和细胞连接的表达的变化。所述模型可用于眼表上皮细胞的体外研究，因为它比通常应用的静态培养条件代表体内情况的更多生理再现。
Purpose: The aim of this study was to establish and to evaluate an in vitro model for culturing human telomerase-immortalized corneal epithelial (hTCEpi) cells under adjustable medium flow mimicking the movements of the tear film on the ocular surface.Methods: Using an IBIDI pump system, cells were cultured under unidirectional, continuous or oscillating, discontinuous medium flow. Cell surface and cytoskeletal architecture were investigated by scanning electron microscopy and immunofluorescence. Gene expression of e-cadherin, occludin, tight junction protein (TJP), desmoplakin, desmocollin and mucins was investigated by real-time PCR. Protein expression of desmoplakin, TJP, occludin and e-cadherin was analyzed by western blot and localization was detected by immunofluorescence. Rose bengal staining was used to assess mucin (MUC) barrier integrity. MUC1, -4 and -16 proteins were localized by immunofluorescence.Results: Medium flow-induced shear stress dramatically changed cellular morphology of hTCEpi. Cells subjected to discontinuous shear stress displayed the typical flattened, polygonal cell shape of the superficial layer of stratified squamous epithelia. Cell surfaces showed less bulging under shear stress and less extracellular gaps. The mRNA expression of E-cadherin, occludin and TJP were increased under oscillatory medium flow. Desmoplakin and occludin protein were upregulated under oscillatory shear stress. Stress fiber formation was not aligned to flow direction. MUC1, -4, and -16 protein were localized under all culture conditions, a regulation on mRNA expression was not detectable. Rose Bengal uptake was diminished under unidirectional conditions.Conclusion: Our findings suggest that shear stress as it occurs at the ocular surface during blinking exerts marked effects on corneal epithelial cells, such as changes in cellular morphology and expression of cell junctions. The described model may be useful for in vitro investigations of ocular surface epithelia as it represents a much more physiologic reproduction of the in vivo situation than the commonly applied static culture conditions.