Okumura Naoki, Kakutani Kazuya, Numata Ryohei, Nakahara Makiko, Schlötzer-Schrehardt Ursula, Kruse Friedrich, Kinoshita Shigeru, Koizumi Noriko
Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan 2Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan.
Invest Ophthalmol Vis Sci. 2015 May;56(5):2933-42. doi: 10.1167/iovs.14-15163.
The purpose of this study was to investigate the usefulness of laminin isoforms as substrates for culturing human corneal endothelial cells (HCECs) for clinical application of tissue engineering therapy.
Expression of specific laminin chains in human corneal endothelium and Descemet's membrane was analyzed at the mRNA and protein levels. The effect of laminin-511 and -521 on cell adhesion and proliferation was evaluated. Recombinant laminin E8 fragments (E8s), which represent functionally minimal forms of laminins, were also evaluated for their effects on cell density and cellular phenotype. The potential involvement of α3β1 and α6β1 integrins in laminin signal transduction was also investigated using neutralizing antibodies.
Laminin-511 and -521 were expressed in Descemet's membrane and corneal endothelium. These laminin isoforms significantly enhanced the in vitro adhesion and proliferation, and differentiation of HCECs. A cell density of 2200 to 2400 cells/mm2 was achieved when HCECs were cultured on laminin-511 or -521, whereas the density was only 1100 cells/mm2 on an uncoated control. E8s also supported HCEC cultivation with a similar efficacy to that obtained with full-length laminin. Functional blocking of α3β1 and α6β1 integrins suppressed the adhesion of HCECs even in the presence of laminin-511.
Laminin-511 and -521 were the laminin isoforms present in Descemet's membrane, and these laminins modulate the adhesion and proliferation of CECs. Laminin E8s represent an ideal xeno-free defined substrate for the culture of CECs for clinical applications.
本研究旨在探讨层粘连蛋白异构体作为培养人角膜内皮细胞（HCECs）的基质在组织工程治疗临床应用中的效用。
在mRNA和蛋白质水平分析人角膜内皮和Descemet膜中特定层粘连蛋白链的表达。评估层粘连蛋白-511和-521对细胞黏附和增殖的影响。还评估了重组层粘连蛋白E8片段（E8s），其代表层粘连蛋白的功能最小形式，对细胞密度和细胞表型的影响。使用中和抗体研究α3β1和α6β1整合素在层粘连蛋白信号转导中的潜在作用。
层粘连蛋白-511和-521在Descemet膜和角膜内皮中表达。这些层粘连蛋白异构体显著增强了HCECs的体外黏附、增殖和分化。当HCECs在层粘连蛋白-511或-521上培养时，细胞密度达到2200至2400个细胞/mm2，而在未包被的对照上密度仅为1100个细胞/mm2。E8s也支持HCEC培养，其功效与全长层粘连蛋白相似。α3β1和α6β1整合素的功能阻断即使在存在层粘连蛋白-511的情况下也会抑制HCECs的黏附。
层粘连蛋白-511和-521是Descemet膜中存在的层粘连蛋白异构体，这些层粘连蛋白调节角膜内皮细胞的黏附和增殖。层粘连蛋白E8s是用于临床应用的角膜内皮细胞培养的理想无动物源限定基质。
