Obezite Araştırmaları İçin İn Vitro Adiposit Hücre Kültürü İle Etkili Model Oluşturma: Ön Çalışma

Abstract

Amaç: 3T3-L1 preadipositlerin farklılaşma etkinliği obezite, diyabet ve ilgili bozukluklarla ilişkili hücresel mekanizmalar üzerine yapılan çalışmaları etkileyen önemli bir faktördür. 3T3-L1 hücrelerinin farklılaşması genellikle insülin, deksametazon (DEX) ve 3-izobütil- 1-metilksantin (IBMX) içeren bir adipojenik kokteyl ile indüklenmektedir. 3T3-L1 hücreleri çoğaltılırken dikkat edilmesi gereken önemli noktalar içeren bir sürece sahip hassas bir hücre grubu olup ilerleyen pasajlarda farklılaşma yeteneğini kaybedebilmektedir. Bu sorunu çözmek için, çalışmamızın amacı doğrultusunda, yaygın olarak kullanılan üç farklı adipojenik kokteylin farklılaşma verimliliği karşılaştırılmıştır. Bununla birlikte, birtakım parametreler değiştirilerek 3T3-L1 hücrelerinin farklılaşma yetenekleri test edilmiştir. Gereç ve Yöntemler: Çalışmamızda 3T3-L1 hücre kültürü, fibroblast hücrelerin olgun adiposit hücrelere farklılaştırılması, Oil Red O boyama ile lipitlerin mikroskop altında gösterilmesi ve kantitatif analiz için boyanın spektrofotometrik ölçümü yöntemleri kullanılmıştır. Bulgular: Deney sonuçları, 0,5 mM IBMX, 0,25 μM DEX ve 1 μg/mL insülin içeren adipogenik kokteylin 3T3-L1 hücre farklılaşması için en etkili kokteyl olduğunu (p<0,0001) ve farklılaşmanın etkinliğinin adipojenik ajanların konsantrasyonları ile yakından ilişkili olduğunu göstermiştir. Sonuç: Çalışmamızda ATCC’nin 3T3-L1 hücreleri için vermiş olduğu protokolün alternatifi olarak in vitro preklinik obezite modeli için kullanılabilecek önemli modifikasyonlar bildirilmiştir.

Keywords

• Obezite
• hücre kültürü
• 3T3-L1
• adipogenez
• adiposit

Supporting Institution

Tübitak, Dokuz Eylül Üniversitesi

Project Number

TÜBİTAK 222S104, TDK-2022-2810 DEU BAP

Creating An Effective Model with In Vitro Adipocyte Cell Culture for Obesity Research: Preliminary Study

Abstract

Aim: The differentiation efficiency of 3T3-L1 preadipocytes is a crucial factor affecting studies on cellular mechanisms associated with obesity, diabetes and related disorders. Differentiation of 3T3-L1 cells is usually induced by an Adipogenic cocktail containing insulin, dexamethasone (DEX), and 3-isobutyl-1-methylxanthine (IBMX). There is a process that includes important points to consider when growing 3T3-L1 cells. It is a sensitive cell group and may löse its ability to differentiate in later passages. To solve this problem, for the purpose of our study, the differentiation efficiency of three different commonly used Adipogenic cocktails was compared. Additionally, the differentiation abilities of 3T3-L1 cells were tested by changing some parameters. Material and Methods: In our study, 3T3-L1 cell culture, differentiation of fibroblast cells into mature adipocyte cells, demonstration of lipids under the microscope with Oil Red O staining, and spectrophotometric measurement of the dye for quantitative analysis were used. Results: Experimental results Showed that the Adipogenic cocktail containing 0.5 mM IBMX, 0.25 μM DEX and 1 μg/mL insülin was the most effective cocktail for 3T3-L1 cell differentiation (p<0.0001) and the effectiveness of differentiation was closely related to the concentrations of adipogenic agents. Conclusion: In our study, important modifications that can be used for the in vitro preclinical obesity model have been reported as an alternative to the protocol given by ATCC for 3T3-L1 cells.

Keywords

• Obesity
• cell culture
• 3T3-L1
• adipogenesis
• adipocyte

Project Number

TÜBİTAK 222S104, TDK-2022-2810 DEU BAP

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