人眼角膜上皮细胞和纤维母细胞在孟加拉玫瑰红光动力疗法（RB-PDT）后的迁移和生长因子分泌，以及条件培养基的影响。
Human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium.
机构信息
Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg, Saar, Germany.
Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar, Germany.
出版信息
PLoS One. 2023 Dec 27;18(12):e0296022. doi: 10.1371/journal.pone.0296022. eCollection 2023.
PURPOSE
To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM).
METHODS
A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed.
RESULTS
RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor β (TGF-β) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-β concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-β concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT.
CONCLUSIONS
HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-β and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing.
目的
研究孟加拉玫瑰红光动力疗法（RB-PDT）后人类角膜上皮细胞和成纤维细胞的迁移以及生长因子的分泌情况，以及条件培养基（CM）的作用。
方法
使用人角膜上皮细胞系（HCE-T）、人角膜成纤维细胞（HCF）和圆锥角膜成纤维细胞（KC-HCF）。在 RB-PDT 后 24 小时，通过划痕试验确定细胞迁移率，通过 ELISA 确定细胞培养上清液中的生长因子浓度。此外，观察了 CM 的作用。
结果
与对照组相比（p≤0.02），RB-PDT 显著降低了所有细胞类型的迁移率。未经 RB-PDT 处理的 HCE-T 培养物的迁移率，在用 RB-PDT 处理后的 HCF CM 中显著高于未经 RB-PDT 处理的 HCF CM（p<0.01）。同样，未经处理的 HCF 在接受 RB-PDT 后，与未经处理的 HCE-T CM 相比，迁移率显著增加（p<0.01）。此外，单独光照和 RB-PDT 显著降低了 HCF 和 KC-HCF 上清液中角质细胞生长因子（KGF）的浓度，并且 RB-PDT 显著降低了 HCF 上清液中可溶性 N-钙黏蛋白（SN-Cad）的浓度，与对照组相比（p<0.01）。在 HCE-T CM 中，RB-PDT 增加了肝细胞生长因子（HGF）和碱性成纤维细胞生长因子（FGFb）的浓度（p≤0.02），同时降低了转化生长因子β（TGF-β）的浓度（p<0.01）。RB-PDT 使 HCF CM 中的 FGFb 浓度增加（p<0.0001），TGF-β 浓度降低（p<0.0001）。使用 RB-PDT 后，KC-HCF CM 中的表皮生长因子（EGF）、HGF 和 TGF-β 浓度降低（p≤0.03），FGFb 浓度升高（p<0.01）。
结论
RB-PDT 后 24 小时，HCE-T、HCF 和 KC-HCF 的迁移率降低。相反，在用 RB-PDT 处理后，HCE-T 细胞的迁移得到增强，并且在用 RB-PDT 处理后，HCF 细胞的迁移通过 HCE-T CM 得到增强。RB-PDT 对 EGF、KGF、HGF、FGFb、TGF-β 和 N-钙黏蛋白分泌的调节可能在角膜伤口愈合中发挥重要作用。
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