SH-SY5Y Hücrelerinin Kültürlenmesinde Kollajen Kaplama, Fetal Sığır Serum Konsantrasyonu, Diferansiyasyon Ajanları ve Nörotoksin Uygulamasının Parkinson Hastalığının İn vitro Modellemesine Etkileri

Year 2024, Volume: 8 Issue: 2, 105 - 109, 30.08.2024

Fatma Gonca Koçancı

https://doi.org/10.30565/medalanya.1459470

Abstract

Amaç: Bu çalışma, Parkinson hastalığı (PH) araştırmaları için doğru in vitro hastalık modelleri geliştirmek amacıyla SH-SY5Y kültür koşullarını optimize etmeyi amaçlamaktadır. Kollajen kaplama, fetal sığır serum (FSS) konsantrasyonu, diferansiyasyon ajanları ve nörotoksin tedavileri gibi çeşitli faktörlerin hücresel davranış ve hastalık modellemesi üzerindeki etkilerini araştırmayı hedeflemektedir.

Yöntem: İnsan nöroblastoma SH-SY5Y hücre hattı, ısı ile inaktive edilmiş fetal sığır serumu, penisilin-streptomisin ve L-glutamin ile desteklenmiş DMEM/F12 içinde kültürlenmiştir. Hücre diferansiyasyonu üzerindeki etkisini değerlendirmek için kollajen kaplama uygulanmış, ideal hücre yoğunluğu ve serum oranı ise deneysel olarak belirlenmiştir. Parakuat'ın sitotoksik etkilerini değerlendirmek için MTT testi kullanılmış, dopamin seviyeleri ELISA ile ölçülmüştür. Gen ekspresyonu gerçek zamanlı qPCR ile analiz edilmiştir. Parkinson modelini doğrulamak ve hücresel morfolojiyi değerlendirmek için immünofloresan boyama ve nörit uzunluğu ölçümleri yapılmıştır.

Bulgular: 5x103 hücre/cm2 yoğunluğunda kültürlenmiş hücreler, kollajen ve %2 FSS ile retinoik asit maruziyetinde dopaminerjik nöron özellikleri sergilemiştir. Bununla birlikte, parakuat tedavisi nörotoksisiteye neden olmuş, dopamin seviyelerinde azalma ve nörit büyümesinde bozulma gözlenmiştir.

Sonuç: Bu çalışma, PH modellemesi için SH-SY5Y hücre kültürü koşullarınının optimizasyonu araştırmıştır. Temel bulgular arasında optimal hücre yoğunluğu, FSS konsantrasyonu ve kolajen kaplamanın faydalı etkileri yer almaktadır. Ek olarak, nöronal farklılaşma ve dejenerasyon konusunda gelecekteki araştırmalar için sağlam bir çerçeve sağlayan etkili bir parakuat nörotoksisite protokolü oluşturulmuştur.

Keywords

Parkinson hastalığı, SH-SY5Y hücreleri, nöronal diferansiyasyon, nörotoksisite, in vitro modelleme

Project Number

220S745

Effects of Collagen Coating, Fetal Bovine Serum Concentration, Differentiation Agents, and Neurotoxin Application on In Vitro Modeling of Parkinson's Disease Using SH-SY5Y Cell Culture

Abstract

Aim: This study aims to optimize SH-SY5Y culture conditions to develop precise in vitro disease models for Parkinson's disease (PD) research. It seeks to investigate the effects of various factors such as collagen coating, fetal bovine serum concentration, differentiation agents, and neurotoxin treatments on cellular behavior and disease modeling.

Materials and Methods: The human neuroblastoma SH-SY5Y cell line was cultured in DMEM/F12 supplemented with heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin, and L-glutamine. Collagen coating was applied to assess its impact on cell differentiation, while the ideal cell density and serum ratio for generating neurite-like cells were determined through experimentation. The MTT assay was employed to evaluate the cytotoxic effects of paraquat, while dopamine levels were quantified using ELISA. Gene expression was analyzed via real-time qPCR. Immunofluorescence staining and neurite length measurements were conducted to validate the PD model and assess cellular morphology.

Results: Cells cultured at a density of 5x103 cells/cm2 with collagen and 2% FBS exhibited characteristics of dopaminergic neurons upon exposure to retinoic acid. Conversely, paraquat treatment induced neurotoxicity, resulting in decreased dopamine levels and impaired neurite outgrowth.

Conclusion: This study investigated the optimization of SH-SY5Y cell culture conditions for PD modeling. Key findings include optimal cell density, FBS concentration, and beneficial effects of collagen coating. Additionally, an effective paraquat neurotoxicity protocol has been established, providing a solid framework for future research on neuronal differentiation and degeneration.

Keywords

Parkinson, SH-SY5Y cells, neuronal differentiation, neurotoxicity, in vitro modeling

Ethical Statement

Since our study involves in vitro cell culture techniques, ethics committee permission is not required.

Supporting Institution

TUBİTAK-220S745

Project Number

220S745

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