Zebra balığında (Danio rerio) embriyonik kök hücrelerin üç boyutlu kültürü ve di-2-etilheksil fitalat (DEHP) etkisinin değerlendirilmesi için kullanımı
Küçük Resim Yok
Dosyalar
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Araştırmanın ilk basamağında, blastula aşamasındaki zebra balığı (Danio rerio) embriyolarından izole edilen kök hücrelerin iki boyutlu (2D) kültürleri yapıldı. Kültür kaplarının kaplanmasında ilk kez uygulanan serisin ekstraktı, daha başarılı kültivasyon için kullanışlı bir biyomateryal olarak not edildi. Ayrıca asılı damla sferoid ve biyobaskı yöntemleriyle gerçekleştirilen üç boyutlu (3D) kültürlerde fizyolojik gerçekliği daha iyi taklit edebilecek bir hücresel model elde edebilmek için yenilikçi bir çözüm olarak, kartezyen bir biyoyazıcı ile özgün bir biyomürekkep tasarlanıp geliştirildi. İkinci basamakta ise tehditkâr bir çevre kirleticisin, di-2-etilhekzil fitalat'ın (DEHP) etkilerini değerlendirmek için 2D ve 3D kültürler kullanıldı. LC50 değeri hesaplanan DEHP hücre kültürlerine subletal konsantrasyonlarda ve 24, 48 ve 72 saat süreyle uygulandı. klf4, pou5f1 ve cyp19b gen ifadeleri pluripotensi ve endokrin engelleyici biyobelirteçleri olarak istatistiksel anlamda değerlendirildi. Hücre döngüleri ile katalaz, süperoksit dizmutaz ve glutatyon peroksidaz enzimlerinin antioksidatif etkinliklerindeki sayısal değişimler ölçülüp karşılaştırıldı. Beklenildiği üzere konsantrasyon ve uygulama süresine bağlı olarak, Uygulamadan sadece gen ifadeleri değil hücre çevrimleri ve enzim etkinlikleri de önemli ölçüde etkilendi. 3D hücre kültürleri, sergiledikleri en yüksek duyarlılığa dayanılarak, kimyasalların etki mekanizmaları ile ekotoksikolojik risklerinin belirlenmesinde şiddetle önerilen materyaller olarak değerlendirildi.
Stem cells isolated from blastula-staged zebrafish (Danio reiro) embryos were two-dimensionally (2D) cultured at first step of the study. Sericin extracts that firstly used to coat culture flasks, were noted as practicable biomaterial for better cultivation. Three-dimensional (3D) cultures were also performed via hanging drop spheroid formation and bioprinting methods. In order to maintain a cellular model that better mimic the physiological reality, a novel cartesian bioprinter, and a specific bioink were designed, developed and tested as innovative solutions for 3D culture. At second step, 2D and 3D cultures were used to evaluate the effects of di-2- ethylhexyl phthalate (DEHP), a threatening environmental contaminant. LC50 value of DEHP was calculated, cell cultures were exposed to sublethal concentration of the chemical for 24, 48 and 72 hours. The expressions of the genes of klf4, pou5f1, and cyp19b, were statistically interpretated as biomarkers for pluripotency, and endocrine-disruption, respectively. Quantitative changes of cell cycles and the levels of the antioxidative activities of catalase, superoxide dismutase and glutathione peroxidase were also measured and compared. Not only gene expressions but also cell cycles and enzymatic activities were definitely affected by DEHP treatments, as expected. The effects of DEHP were noted as concentration- and time-dependent. Based on their most sensitivity to exposition, 3D cultures are evaluated as highly recommended materials to determine the mode of action and ecotoxicological risk assessment of the chemicals.
Stem cells isolated from blastula-staged zebrafish (Danio reiro) embryos were two-dimensionally (2D) cultured at first step of the study. Sericin extracts that firstly used to coat culture flasks, were noted as practicable biomaterial for better cultivation. Three-dimensional (3D) cultures were also performed via hanging drop spheroid formation and bioprinting methods. In order to maintain a cellular model that better mimic the physiological reality, a novel cartesian bioprinter, and a specific bioink were designed, developed and tested as innovative solutions for 3D culture. At second step, 2D and 3D cultures were used to evaluate the effects of di-2- ethylhexyl phthalate (DEHP), a threatening environmental contaminant. LC50 value of DEHP was calculated, cell cultures were exposed to sublethal concentration of the chemical for 24, 48 and 72 hours. The expressions of the genes of klf4, pou5f1, and cyp19b, were statistically interpretated as biomarkers for pluripotency, and endocrine-disruption, respectively. Quantitative changes of cell cycles and the levels of the antioxidative activities of catalase, superoxide dismutase and glutathione peroxidase were also measured and compared. Not only gene expressions but also cell cycles and enzymatic activities were definitely affected by DEHP treatments, as expected. The effects of DEHP were noted as concentration- and time-dependent. Based on their most sensitivity to exposition, 3D cultures are evaluated as highly recommended materials to determine the mode of action and ecotoxicological risk assessment of the chemicals.
Açıklama
Anahtar Kelimeler
Zebra Balığı, Danio Rerio, Embriyonik Kök Hücre, 3D Kültür, Biyobaskı, DEHP, Gen İfadeleri, Hücre Çevrimi, Antioksidatif Enzimler, Zebrafish, Danio Rerio, Embryonic Stem Cells, 3D Culture, Bioprinting, DEHP, Gene Expressions, Cell Cycle, Antioxidative Enzymes
