Wachstumsfaktorenkonzentration im Kulturüberstand von Keratozyten mit humanem Serum in vitro

Zusammenfassung

Hintergrund Die Anwendung von Serumaugentropfen (AS) stellt eine alternative Behandlungsmethode für therapieresistente korneale Epitheldefekte dar. Bei persistierenden Epitheldefekten könnten Zytokine, die von Keratozyten im Stroma produziert werden, eine entscheidende Rolle bei der epithelialen Wundheilung spielen. Ziel dieser Studie ist es, Transforming Growth Factor β1 (TGF-β1), Keratinocyte Growth Factor (KGF), Hepatocyte Growth Factor (HGF) und Fibroblast Growth Factor basic (FGFb) im Kulturüberstand von Keratozyten mit humanem Serum (HS) in vitro zu untersuchen.

Material und Methoden Serumaugentropfen von 10 Patienten wurden nach der Standardmethode der LIONS Hornhautbank Saar-Lor-Lux präpariert und bei − 80 °C tiefgefroren. Primäre humane Keratozyten wurden durch enzymatische Behandlung mit Kollagenase A (1 mg/ml) aus humanen Korneoskleralscheiben isoliert (n = 1) und in DMEM/Hamʼs Kulturmedium, versetzt mit 5% fetalem Kälberserum (FCS), kultiviert. Für den Testansatz wurden die Keratozyten mit 15 oder 30% HS (in DMEM/F14 ohne FCS) inkubiert und nach 24 h die Konzentration von TGF-β1, KGF, HGF und FGFb mittels Enzyme-linked Immunoabsorbent Assay (ELISA) aus dem Kulturüberstand bestimmt. Als Kontrolle wurden 15 oder 30% HS ohne Keratozyten nach 24 h Inkubationszeit (unter den gleichen Bedingungen wie die Keratozyten) verwendet.

Ergebnisse Die HGF-Konzentration mit beiden HS-Konzentrationen war im Kulturüberstand von Keratozyten signifikant höher, im Vergleich zur HS-Kontrolle (ohne Keratozyten) nach 24 h Inkubationszeit (p < 0,01). Die FGFb-Konzentration war im Kulturüberstand mit 30% HS signifikant höher im Vergleich zur Kontrollgruppe ohne Keratozyten nach 24 Stunden Inkubationszeit (p < 0,01). Die TGF-β1- und KGF-Konzentrationen im Kulturüberstand blieben durch die Keratozyten unverändert.

Schlussfolgerungen Durch die Anwesenheit von Keratozyten steigt die Konzentration von HGF und FGFb im Kulturmedium mit humanem Serum innerhalb von 24 Stunden an. Diese Konzentrationsänderungen könnten die Wundheilung bei Epitheldefekten beeinflussen.

Abstract

Purpose Application of serum eye drops is an alternative treatment option for therapy-resistant corneal epithelial defects. In case of persisting epithelial defects, cytokines, which are secreted from stromal keratocytes, may play a role in epithelial wound healing. Our aim was to analyze fibroblast growth factor basic (FGFb), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and transforming growth factor β1 (TGF-β1) concentration in the supernatant of keratocytes, after incubation with human serum (HS).

Materials and Methods Serum eye drops of 10 patients were prepared using the standards of the LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz, and were stored at − 80 °C. Primary human keratocytes were isolated from human corneoscleral rings using collagenase A (1 mg/ml) (n = 1) and were cultured in DMEM/Hamʼs culture medium with 10% fetal bovine serum (FBS). Thereafter, keratocyte cultures were incubated in 15 or 30% HS (in DMEM/F14 without FBS) and FGFb, HGF, KGF and TGF-β1 concentration was determined with an enzyme-linked immunoabsorbent assay (ELISA) from the supernatant of the culture after 24 hours. We used 15 or 30% HS without keratocytes (under the same storage conditions) as controls.

Results HGF concentration was, for both HS concentrations, significantly higher in the supernatant of keratocytes, than in HS controls (without keratocytes) following 24 hours (p < 0.01). FGFb concentration was significantly increased in 30% HS with keratocytes compared to 30% HS without keratocytes after 24 hours (p < 0.01). TGF-β1 and KGF concentrations remained unchanged through keratocytes.

Conclusion HGF and FGFb concentrations increase in the supernatant of keratocytes, 24 hours after incubation with human serum. These concentration changes may play a role in wound healing of epithelial defects.

Schlüsselwörter

humanes Serum - FGFb - HGF - KGF - TGF-β1 - epitheliale Wundheilung

Key words

human serum - FGFb - HGF - KGF - TGF-β1 - epithelial wound healing

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