CD34 und Alpha-smooth-Muscle-Actin-Keratozyten-Expression nach photodynamischer Inaktivierung (PDI)

Zusammenfassung

Ziele: Die photodynamische Inaktivierung (PDI) kann als mögliche Behandlungsalternative einer therapieresistenten infektiösen Keratitis in Betracht gezogen werden. PDI kann Mikroorganismen einer infizierten Hornhaut zum einen durch freie Sauerstoffradikale schädigen, zum anderen die Aktivierung von Keratozyten und immunkompetenten Zellen unterstützen. Ziel dieser Studie war die Bestimmung der Auswirkungen von PDI auf die Aktivierung von humanen Keratozyten in der Zellkultur.

Methoden: Primäre humane Keratozyten wurden durch enzymatische Behandlung mit Kollagenase A (1 mg/ml) aus humanen Korneoskleralscheiben isoliert und in DMEM/Hamʼs Kulturmedium, versetzt mit 10 % fetalem Kälberserum kultiviert. Die Keratozyten wurden mit dem Photosensibilizator Chlorin e6 (Ce6) in den Konzentrationen von 0, 50, 150 und 250 nM für 30 Minuten inkubiert und anschließend mit einer Wellenlänge von 670 nm für 13 Minuten bestrahlt. Einen Tag nach der Behandlung wurde die Expression von CD34 und Alpha-smooth Muscle Actin mittels Durchflusszytometrie (FACS) bestimmt.

Ergebnisse: Die ausschließliche Anwendung von Ce6 oder Bestrahlung veränderte die Expression von CD34 und α-smooth-muscle-actin der Zellen nicht signifikant. Vierundzwanzig Stunden nach PDI mit 50–250 nM Ce6 blieb die CD34-Expression unverändert, während der Prozentsatz von Alpha-smooth-Muscle-Actin-positiven Keratozyten bei 250 nMol Ce6 signifikant sank (p = 0.01).

Schlussfolgerung: Die Ergebnisse unser Studie legen nahe, dass PDI in vitro als Kurzzeiteffekt die myofibroblastische Transformation der Keratozyten hemmt, jedoch keine Aktivierung der CD34-positiven kornealen Zellen bewirkt. Auf diese Weise könnte möglicherweise durch PDI die antimikrobielle Abwehr heruntergeregelt werden.

Abstract

Purpose: Photodynamic inactivation (PDI) may be a potential treatment alternative in therapy-resistant infectious keratitis. PDI may eliminate the microorganisms from the infected cornea by damage caused through free oxygen radicals, or even by supporting different stages of activation of keratocytes and inflammatory cell response. The purpose of this study was to determine the impact of PDI on activation of human keratocytes in culture.

Methods: Primary human keratocytes were isolated by digestion in collagenase A (1 mg/mL) from human corneal buttons, and cultured in DMEM/Hamʼs culture medium supplemented with 10 % foetal calf serum. Keratocytes underwent illumination (670 nm) for 13 minutes following exposure to 0, 50, 150 and 250 nMol/ml concentrations of the photosensitizer chlorin e6 (Ce6) in the culture medium. Twenty-four hours after treatment CD34 and α-smooth-muscle actin expression of the cells was analysed using flow-cytometry (FACS).

Results: Using Ce6 or illumination only, α-smooth-muscle actin expression of the cells did not change significantly. Twenty-four hours after PDI the percentage of CD34-positive keratocytes did not change significantly using 50–250 nM Ce6, however, the percentage of α-smooth-muscle actin-positive keratocytes decreased significantly at 250 nM Ce6 (p = 0.01).

Conclusions: As a short-term effect, PDI seems to inhibit myofibroblastic transformation of keratocytes, but does not have an impact on activation of CD34-positive keratocytes.

With this impact PDI possibly may reduce the antimicrobial defence of keratocytes.

Schlüsselwörter

Kornea - Pathologie - Infektiologie - photodynamische Inaktivierung (PDI) - Chlorin e6 - humane Keratozyten - alpha-SMA - CD34

Key words

cornea - infectious diseases - pathology - photodynamic inactivation (PDI) - chlorine e6 - human keratocytes - alpha-SMA - CD34

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