Mukaddes ATMACA

Improvement of the original isolation procedure for hormone studies in short-time culture

Daha önceki çalışmalar izolasyon prosedürleri esnasında hücrelerin hormona yanıtının kaybolduğunu ve metabolik aktivitenin azaldığını göstermiştir. Bu nedenle, çalışmamda kısa süreli izole karaciğer hücre (hepatosit) kültürleri için optimal koşulların sağlanması ve hormon çalışmalarında daha uygun bir yöntem geliştirmeyi amaçladım. Hücre izolasyonu için 50 IU/ml ve 100 IU/ml kollejenaz kullanıldı. Sıçan hepatositlerin hormonlara duyarlılığının ölçümünde adrenalin kullanıldı ve glukoz çıkışı (glikojenoliz) 2 saatlik inkubasyon peryodu sonunda ölçüldü. Adrenalin'nin ($10^{-6}$M) glikojenolize etkisi yanlızca 50 IU/ml kollojenaz kullanıldığında ve viabilitenin %95'in üzerinde olduğu durumlarda anlamlı görüldü (Kontrol: 0.160±0.01 mg/2saat; Adrenalin: 0.30±0.01 mg/2saat). Düşük konsantrasyonda kollejenazla izole edilen(50IU/ml) ve canlılığı %95 üzerinde olan hepatositlerin, mikroplatlere yapıştığı süreç sonunda (lsaat) vede bunu takip eden inkubasyon sürecinin sonunda, glutatyon içeriği, metiltyotetrazolum redüksiyonu, laktatdihidrojenaz çıkışı, adenozindifosfat düzeyi ölçüldü. Bu süreçler sonunda belirtilen ölçümlerde değişiklik gözlenmedi. Sonuç olarak, bulgularımız Reese and Byard'a ait özgün izolasyon prosedürünün modifiye edilmesiyle hepatositlerin metabolik faaliyetlerinin ve membran bütünlüğünün korunabileceğini göstermektedir.

Anahtar Kelimeler:

Hücre kültür teknikleri, Hormonlar, Biyotransformasyon, Glikogenoliz, Epinefrin, L-laktat dehidrogenaz, Hücre sağkalımı, Sıçanlar, Hepatositler, Modeller, hayvan

Kısa süreli kültür ortamında hormon çalışmaları için özgün izolasyon prosedürünün iyileştirilmesi

Earlier studies indicated that hormone responsiveness of cells and metabolic activity was lost during various of experimental procedure. In the light of this observation, I aimed to investigate to obtain optimal conditions for short time cultured hepatocytes and also to determine the type of test can be used to evaluate suitablity of hepatocytes for hormones studies. During the isolation period 50 IU/ml and 100 IU/ml collagenase were used. Adrenaline ($10^{-6}$M) was used to measure sensitivity of hepatocytes to hormones and glycogenolsis was measured at the end of 2hr incubation period. Adrenaline significantly increased gylcogenolysis (Control: 0.16±0.01 mg/2hr; Adrenaline: 0.30±0.01 mg/2hr) only when the 50 IU/ml collagenase was used and the viability of the cells were over 95%. Viability tests were applied to hepatocytes that obtained by using 50 IU collagenase. Cellular glutathione, methylthiazoltetrazolium reduction, lactatedehdrogenase leakage, ATP level measured to determine viability following the attachment and incubation period. No differences were observed at the end of each period. Altogether, the present study indicated that membrane integrity and metabolic function of the hepatocytes can be improved by modifying slightly the original procedure of Reese and Byard.

Keywords:

Cell Culture Techniques, Hormones, Biotransformation, Glycogenolysis, Epinephrine, L-Lactate Dehydrogenase, Cell Survival, Rats, Hepatocytes, Models, Animal,

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