丝裂霉素 C 对白细胞介素 1 受体表达、白细胞介素 1 相关肝细胞生长因子分泌和角膜上皮细胞迁移的影响。

Effect of mitomycin C on IL-1R expression, IL-1-related hepatocyte growth factor secretion and corneal epithelial cell migration.

机构信息

Departments of Ophthalmology, Far Eastern Memorial Hospital, Taipei, Taiwan.

出版信息

Invest Ophthalmol Vis Sci. 2010 Mar;51(3):1389-96. doi: 10.1167/iovs.09-3494. Epub 2009 Dec 3.

DOI:10.1167/iovs.09-3494

PMID:19959635

Abstract

PURPOSE

To investigate how mitomycin C (MMC) modulates hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) secretions in human corneal fibroblasts and regulates human corneal epithelial (HCE) cell migration.

METHODS

Primary human corneal fibroblasts were treated with MMC (0.05, 0.1, or 0.2 mg/mL for 5 minutes) and were cultivated with or without interleukin (IL)-1beta. Transcript and secretion of HGF and KGF were determined by quantitative real-time RT-PCR and Western blot analysis, respectively. The effect of MMC-treated fibroblasts on HCE cell migration was evaluated using a transwell migration assay. The influence of MMC on HGF expression/secretion and HCE cell migration was further confirmed by RNA interference. The number of IL-1 receptors (IL-1R) on the fibroblast surface was analyzed by flow cytometry.

RESULTS

MMC alone did not affect endogenous HGF expression, whereas IL-1beta alone significantly upregulated HGF transcripts and secretion. By modifying IL-1R numbers, MMC further upregulated IL-1beta-related HGF expression at a concentration of 0.05 mg/mL but to a lesser extent at 0.1 and 0.2 mg/mL. KGF transcripts and intracellular expression were suppressed by MMC dose dependently in the presence or absence of IL-1beta, whereas KGF secretion was not affected. Conditioned medium from MMC-treated fibroblasts exerted a similar concentration-dependent effect on HCE cell migration, enhancing migration most significantly at 0.05 mg/mL MMC in the presence of IL-1beta. The MMC dose-dependent modulation of HCE cell migration was abolished in HGF-silenced fibroblasts.

CONCLUSIONS

MMC differentially modulated IL-1R expression at various concentrations and regulated HGF and KGF differently. MMC alone did not alter HGF expression. In the presence of IL-1beta, MMC-treated corneal fibroblasts modified HCE cell migration through IL-1beta-induced HGF secretion.

摘要

目的

研究丝裂霉素 C（MMC）如何调节人角膜成纤维细胞中肝细胞生长因子（HGF）和角质细胞生长因子（KGF）的分泌，并调节人角膜上皮（HCE）细胞的迁移。

方法

用 MMC（0.05、0.1 或 0.2 mg/mL 处理 5 分钟）处理原代人角膜成纤维细胞，并在有无白细胞介素（IL）-1β的情况下进行培养。通过定量实时 RT-PCR 和 Western blot 分析分别测定 HGF 和 KGF 的转录和分泌。通过 Transwell 迁移实验评估 MMC 处理的成纤维细胞对 HCE 细胞迁移的影响。通过 RNA 干扰进一步证实 MMC 对 HGF 表达/分泌和 HCE 细胞迁移的影响。通过流式细胞术分析成纤维细胞表面的 IL-1 受体（IL-1R）数量。

结果

MMC 本身不影响内源性 HGF 的表达，而 IL-1β 本身则显著上调 HGF 转录本和分泌。通过修饰 IL-1R 数量，MMC 在浓度为 0.05 mg/mL 时进一步上调了与 IL-1β 相关的 HGF 表达，但在 0.1 和 0.2 mg/mL 时则较少。无论是否存在 IL-1β，MMC 均呈剂量依赖性地下调 KGF 转录本和细胞内表达，但不影响 KGF 分泌。来自 MMC 处理的成纤维细胞的条件培养基对 HCE 细胞迁移具有相似的浓度依赖性效应，在存在 IL-1β 的情况下，在 0.05 mg/mL MMC 时增强迁移的效果最为显著。在 HGF 沉默的成纤维细胞中，MMC 剂量依赖性地调节 HCE 细胞迁移的作用被消除。