Adhesion, Migration, and Proliferation of Cultured Human Corneal Endothelial Cells by Laminin-5
Adhesion, Migration, and Proliferation of Cultured Human Corneal Endothelial Cells by Laminin-5
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DOI：
10.1167/iovs.10-5555
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发表时间：
2011-02-01
影响因子：
4.4
通讯作者：
Yamagami, Satoru
中科院分区：
文献类型：
--
作者：
Yamaguchi, Masahiro;Ebihara, Nobuyuki;Yamagami, Satoru
目的.探讨层粘连蛋白-5（LM 5）及其受体在人角膜内皮细胞（HCEC）中的表达，以及重组人LM 5是否影响培养的HCEC的粘附、增殖和迁移。方法。通过免疫组织化学、逆转录-聚合酶链反应和流式细胞术检测人供体角膜中LM 5及其受体的表达。在无血清条件下培养的HCEC用于分析LM 5的生物学效应。通过计数细胞数量来评估由于LM 5引起的HCEC粘附和增殖的变化。通过使用图像处理和分析软件程序在伤口愈合测定中定量伤口闭合的百分比来评估HCEC迁移。成人HCEC表达LM 5受体α 3 β 1整合素，但不表达LM 5本身。在细胞粘附测定中，粘附到重组LM 5（1.0 μ g/mL）包被的培养皿上的细胞明显多于未包被的培养皿。在细胞增殖测定中，LM 5（1.0 μ g/mL）和可溶性LM 5（20 ng/mL和50 ng/mL）适度促进培养的HCEC的增殖。在伤口愈合试验中，使用含有可溶性LM 5的培养基比使用对照培养基获得了显著更高的伤口闭合百分比。HCEC表达LM 5受体α 3 β 1整联蛋白。重组LM 5促进培养的HCEC的粘附、迁移和适度增殖。这可能是促进HCEC培养的关键因素，并可能有助于组织工程HCEC的实际应用。（Invest Ophthalmol维斯科学。2011;52：679-684）DOI：10.1167/iovs.10-5555
PURPOSE. To investigate the expression of laminin-5 (LM5) and its receptors by human corneal endothelial cells (HCECs) and whether recombinant human LM5 influences adhesion, proliferation, and migration of cultured HCECs.METHODS. The expression of LM5 and its receptors was examined in human donor corneas by immunohistochemistry, reverse transcription-polymerase chain reaction, and flow cytometry. HCECs cultured under serum-free conditions were used for analysis of the biological effects of LM5. Changes in HCEC adhesion and proliferation due to LM5 were evaluated by counting the number of cells. HCEC migration was assessed by quantifying the percentage of wound closure in the wound-healing assay with an image-processing and -analysis software program.RESULTS. Adult HCECs expressed the LM5 receptor alpha 3 beta 1 integrin, but not LM5 itself. Significantly more cells became adherent to recombinant LM5 (1.0 mu g/mL)-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by LM5 (1.0 mu g/mL) and soluble LM5 (20 ng/mL and 50 ng/mL) in the cell proliferation assay. A significantly higher percentage of wound closure was obtained with medium containing soluble LM5 than with control medium in the wound-healing assay.CONCLUSIONS. HCECs express the LM5 receptor alpha 3 beta 1 integrin. Recombinant LM5 promotes adhesion, migration, and moderate proliferation of cultured HCECs. It may be a critical factor in promoting HCEC culture and may contribute to the practical use of tissue-engineered HCECs. (Invest Ophthalmol Vis Sci. 2011;52:679-684) DOI:10.1167/iovs.10-5555