Wachstumsfaktoren und Interleukine in Amniongewebehomogenat

Zusammenfassung

Ziele: Die Anwendung von Augentropfen aus einem Amniongewebehomogenat (AMH) kann eine alternative Behandlungsmethode für therapieresistente korneale Epitheldefekte darstellen. Ziel dieser Studie ist es, die Konzentrationen an Epidermal Growth Factor (EGF), Fibroblast Growth Factor basic (bFGF), Hepatocyte Growth Factor (HGF), Keratinocyte Growth Factor (KGF), Interleukin-6 (IL-6) und Interleukin-8 (IL-8) im Amniongewebehomogenat in zu bestimmen. Methode: Amnionmembranen von 8 Plazentas wurden präpariert und bei − 80 °C nach der Standardmethode der LIONS-Hornhautbank Saar-Lor-Lux, Trier/Westpfalz tiefgefroren. Die aufgetauten Membranen wurden halbiert, in flüssigem Stickstoff homogenisiert und ein Teil in einem Zelllysepuffer, der andere Teil in phosphatgepufferter Salzlösung (PBS) aufgenommen und bei − 20 °C bis zur Analyse der Wachstumsfaktoren und Interleukine gelagert. Die Konzentration von EGF, bFGF, HGF, KGF, IL-6 und IL-8 wurde mit einem Enzyme-linked Immuno Sorbent Assay (ELISA) analysiert. Ergebnisse: Die Messwerte von KGF, IL-6 und IL-8 in den Gewebehomogenaten lagen sowohl in Zelllysepuffer als auch in PBS unterhalb der Nachweisgrenze. Die EGF-Konzentration in der mit dem Zelllysepuffer behandelten Proben war mit 2412 pg/g Gewebe nicht signifikant höher als die in PBS aufgenommen Proben (1586 pg/g Gewebe, p = 0,72). In der Konzentration von bFGF zeigten sich ebenfalls keine signifikanten Unterschiede zwischen den Homogenaten mit Zelllysepuffer (3606 pg/g Gewebe) und den in PBS aufgenommenen Proben (4649 pg/g Gewebe, p = 0,35). Allerdings war die Konzentration von HGF in den mit Zelllysepuffer behandelten Homogenaten (23 555 pg/g Gewebe) signifikant geringer als die in PBS aufgenommenen Proben (47 766 pg/g Gewebe, p = 0,007). Schlussfolgerung: Die Anwendung von Augentropfen aus Amniongewebehomogenat kann aufgrund der EGF-, bFGF- und HGF-Konzentrationen bei Mangel an IL-6 und IL-8 eine alternative Behandlungsmethode für therapieresistente korneale Epitheldefekte darstellen.

Abstract

Purpose: Application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy resistant corneal epithelial defects. The purpose of this study was to determine the concentrations of epidermal growth factor (EGF), fibroblast growth factor basic (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-6 (IL-6) and interleukin-8 (IL-8) in amniotic membrane homogenates. Methods: Amniotic membranes of 8 placentas were prepared and thereafter stored at − 80 °C using the standard methods of the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz. Following defreezing, amniotic membranes were cut in two pieces and homogenized in liquid nitrogen. One part of the homogenate was prepared in cell-lysis buffer, the other part was prepared in PBS. The tissue homogenates were stored at − 20 °C until enzyme-linked immunosorbent assay (ELISA) analysis for EGF, bFGF, HGF, KGF, IL-6 and IL-8 concentrations. Results: Concentrations of KGF, IL-6 and IL-8 were below the detection limit using both preparation techniques. The EGF concentration in tissue homogenates treated with cell-lysis buffer (2412 pg/g tissue) was not significantly different compared to that of tissue homogenates treated with PBS (1586 pg/g tissue, p = 0.72). bFGF release was also not significantly different using cell-lysis buffer (3606 pg/g tissue) or PBS treated tissue homogenates (4649 pg/g tissue, p = 0.35). HGF release was significantly lower using cell-lysis buffer (23,555 pg/g tissue), compared to PBS treated tissue (47,766 pg/g tissue, p = 0.007). Conclusion: Containing EGF, bFGF and HGF, and lacking IL-6 and IL-8, the application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy-resistant corneal epithelial defects.

Schlüsselwörter

Kornea - Amniongewebehomogenat - Wachstumsfaktoren - Interleukine - ELISA

Key words

cornea - amniotic tissue homogenate - growth factors - interleukins - ELISA

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