Leptinin Yara İyileşmesi Üzerine In Vitro Etkisinin Büyüme Faktörleri Üzerinden İncelenmesi

Yara iyileşmesi; epitelyal, endotelyal, mezenkimal ve inflamatuvar hücrelerin biraraya gelip, normal işlevlerini belirli bir sıra ve düzen içerisinde yerine getirmeleriyle karakterizedir. Leptin, ağırlıklı olarak yağ dokusundan salgılanan ve yara iyileşme süresini kısalttığı bilinen sitokin benzeri bir hormondur. Bu çalışmada; fibroblast hücrelerinde yara iyileşmesi modeli oluşturarak leptinin doz (100 ng/mL, 200 ng/mL, 400 ng/mL ve 800 ng/mL) ve süre (24 ve 48 saat) bağımlı etkilerinin in vitro yöntemlerle incelenmesi amaçlanmıştır. Yara modeli oluşturulan hücreler üzerine artan dozlarda leptin uygulaması yapıldıktan 24 ve 48 saat sonra yara alanlarının kapanma oranları hesaplandı. Hücre canlılığını belirlemek amacı ile WST-1 analizi yapıldı. Crystal Violet boyaması ile fibroblast hücreleri morfolojik olarak incelendi ve FGFR2, KGF (FGF7), TGF-α, TGF-β1 ve Ki67 ekspresyonlarını belirlemek için de immunositokimya (ICC) analizi yapılarak H-Skor değerleri hesaplandı. In vitro yara modeli analizinde fibroblast hücrelerinin en yüksek yüzde kapanma oranı ve WST-1 analizi ile en yüksek hücre canlılık yüzdesi 48 saat 800 ng/mL leptin uygulanan grupta tespit edildi. ICC sonucunda elde edilen H-Skor değerleri ise, değerlendirilen proteinlerin ekspresyonlarının fibroblast hücrelerinde leptin dozu ve süresine bağlı olarak arttığını gösterdi. Çalışma sonucunda fibroblast hücrelerinde leptinin in vitro yara iyileşmesini sağlayan en etkili dozunun 800 ng/mL olduğu belirlenmiştir. Ayrıca FGFR2, KGF (FGF7), TGF-α, TGF-β1 ve Ki67 ekspresyonlarının leptin dozuna ve uygulama süresine bağlı olarak hücrelerde arttığı ve en yüksek artışın en yüksek dozda ortaya çıktığı gösterilmiştir. Bu çalışmanın sonuçlarının, yapılacak olan in vivo yara iyileşmesi araştırmalarında leptin kullanımına öncülük edeceği düşünülmektedir.

Anahtar Kelimeler:

FGFR2, KGF, Leptin, TGF, Yara İyileşmesi

Investigation of The In Vitro Effect of Leptin on Wound Healing Through Growth Factors

Wound healing is characterized by the epithelial, endothelial, mesenchymal and inflammatory cells come together and perform their normal functions in a certain order. Leptin is a cytokine-like hormone secreted predominantly from adipose tissue and known to shorten wound healing time. In this study, it is aimed to examine the dose (100 ng/mL, 200 ng/mL, 400 ng/mL and 800 ng/mL) and time (24 and 48h) dependent effects of leptin by in vitro methods by creating wound healing model in fibroblast cells. The closure rates of the wound areas were calculated 24 and 48h after the application of increasing doses of leptin. WST-1 analysis was performed to determine cell viability. Fibroblast cells were examined morphologically by Crystal Violet staining and immunocytochemistry (ICC) analysis was performed to determine FGFR2, KGF (FGF7), TGF-α, TGF-β1 and Ki67 expressions, and H-Score values were calculated. In the in vitro wound healing model analysis, the highest percent closure rate of fibroblast cells and the highest cell viability percentage with WST-1 analysis were detected in the group that treated with 800 ng/mL leptin for 48 hours. The H-Score values obtained as a result of ICC showed that the expression of the evaluated proteins increased in fibroblast cells dose and time depended manner. As a result of the study, it was determined that the most effective dose of leptin in fibroblast cells for wound healing was 800 ng/mL in vitro. In addition, it was shown that FGFR2, KGF (FGF7), TGF-α, TGF-β1 and Ki67 expressions increased in cells depending on the leptin dose and administration time, and the highest increase occurred at the highest dose. It is thought that the results of this study will lead to the use of leptin in in vivo wound healing model studies.

Keywords:

FGFR2, KGF, Leptin, TGF, Wound Healing,

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