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İnsan Lösemi Monosit Hücrelerinin Forbol-12-Miristat-13-Asetat ile Makrofaj Benzeri Hücrelere Farklılaşması

Amaç: Bu çalışmada monositlerden makrofaja farklılaşmada Phorbol-12-Myristate-13-Acetate (PMA)’nın uygulama zamanı ve dozuna yönelikuygun bir model oluşturulması amaçlanmıştır. Bu amaçla insan lösemi monosit hücre hattı (THP-1) farklı doz ve uygulama sürelerinde PMA ileuyarılarak hücrelerin makrofaja farklılaşmaları morfolojik olarak gösterilmiş ve hücre canlılığı üzerine etkileri değerlendirilmiştir.Gereç ve Yöntem: THP-1 monosit hücreleri 6 kuyucuklu plaklarda 2x105/mL hücre olacak şekilde süspanse olarak üretilmiştir. Daha sonra üzerlerine%10 FCS içeren RPMI 1640 ile 10, 20, 40 ve 60 ng/mL konsantrasyonlarda sulandırılan PMA ilave edilerek, 24, 48 ve 72 saat inkübe edilmiştir.Süspanse THP-1 monosit hücrelerin yapışan makrofaj benzeri hücrelere farklılaşması, doku kültürü mikroskobunda hücre kültürü plaklarına tutunmave fenotipik değişikliklerin gözlenmesi ile doğrulanmıştır. Canlı hücreler tripan mavisi boyama yöntemi ile belirlenmiştir.Bulgular ve Sonuç: THP-1 hücreleri için fenotipik değişiklikleri ve hücre canlılığı açısından elde edilen en iyi sonuçlar 20 ng/mL PMA konsantrasyonuve 48 saat inkübasyon süresi olarak saptanmış, bu konsantrasyon ve inkübasyon süresinde hücre canlılığı %92,2 olduğu bulunmuştur. 40 ng/mL PMAuygulaması 48. saatte 10 ve 20 ng/mL PMA uygulamasına göre hücre sayısında belirgin azalmaya neden olurken 72. saat uygulaması sonrası gruplararasında fark görülmemiştir. Bu sonuçlar in vitro makrofaj modellerinin gerçek fizyolojik şartları tam olarak yansıtması için THP-1 monositlerininmakrofaja farklılaşmasının optimize edilmesi gerekliliğini göstermektedir. Bu çalışmada en iyi hücre farklılaşmasının THP-1 hücrelerine 20 ng/mLPMA eklenmesi ve bu hücrelerin 48 saat inkübasyona bırakılması ile ortaya çıktığı sonucuna varılmıştır.

Differentiation of Human Leukemia Monocyte Cells to Macrophage-Like Cells with Phorbol- 12-Myristate-13-Acetate

Objectives: In this study, it was aimed to evaluate appropriate model of application time and dosage of Phorbol-12-Myristate-13-Acetate (PMA) to get activated macrophages from monocytic cell line THP-1 cells. The differentiation of THP-1 cells into macrophages when stimulated with different dosage and application time of phorbol myristic acid (PMA) was shown morphologically and the effects on cell viability were evaluated. Materials and Methods: THP-1 cells were produced as suspension as 2x105 cells/mL in 6-well plates. Afterwards, THP-1 monocyte cells were treated with PMA of 10, 20, 40, 60 ng/mL concentrations and incubated for 24, 48 and 72 hours. Differentiation of suspended THP-1 cells into adherent macrophage-like cells was confirmed by tissue-culture microscope by observing cell adherence to cell culture flasks and phenotypic changes. Cell viability was determined by trypan blue staining. Results and Conclusion: The best results for phenotypic changes and cell viability for THP-1 cells were obtained with 20 ng/mL PMA at 48h incubation time and the cell viability was detected as 92.2%. The administration of 40 ng/mL PMA resulted in a significant decrease in cell count compared to 10 and 20 ng/ml PMA administration at the 48h, but no difference was observed between groups after 72h application. These findings show that the differentiation of THP-1 monocytes into macrophages needs to be optimized in order to reflect the real physiologic conditions of macrophage models used in in vitro studies. These findings suggest that THP-1 cells are well differentiated by 20 ng/mL PMA at 48h incubation.

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