DENTAL PULPA KAYNAKLI MEZENKİMAL KÖK HÜCRELER İÇİN ELEKTROPORASYON PARAMETRELERİNİN ARAŞTIRILMASI

Amaç: Bu çalışmanın amacı dental pulpa kaynaklı mezenkimal kök hücrelere (DP-MKH) Yeşil Floresan Protein (GFP) genini transfer etmek ve elektroporasyon parametrelerini optimize etmektir. Gereç ve Yöntem: DP-MKH ler GFP geni ile Neon Transfection System kullanılarak transfekte edildi. 5 f a r k l ı e l e k t r o p o r a s y o n p a r a m e t r e s i ( 1 2 0 0 v , 2 0 m s , 1 p u l s ; 1 2 0 0 v , 2 0 m s , 2 p u l s ; 1 3 0 0 v , 4 0 m s , 1 p u l s ; 1 4 0 0 v , 2 0 m s , 1 p u l s ; 1400v,20ms,2puls) optimizasyon için karşılaştırıldı. Transfeksiyondan sonra hücrelerin canlılık ve apoptoz değerleri 0, 24 ve 48. saatlerde analiz edildi. Floresan ışık yoğunluğu floresan mikroskop altında değerlendirildi. Bulgular: DP-MKH için CD29, CD44 pozitif ve CD45 negatif bulundu. Diğer gruplara göre daha fazla canlı hücre sayısı, GFP geninin daha yüksek ekspresyonu ve daha düşük apoptoz 1200v, 20ms, 1puls elektroporasyon değerinde elde edileceği tespit edildi. Sonuç: Tavşan hayvan modeli doku mühendisliği çalışmalarında sık kullanılan bir modeldir. Çalışmamızdan elde edilen bu sonuçlar ileri tavşan DP-MKH için optimum elektroporasyon koşullarını göstermektedir

Anahtar Kelimeler:

Yeşil floresan protein (GFP), mezenkimal stromal hücre, transfeksiyon

The Evaluation of Electroporation Parameters for Dental Pulp Mesenchymal Stem Cells

Objective: The aim of this study was to perform Green Fluorescent Protein (GFP) gene delivery to dental pulp derived mesenchymal stromal cells (DP-MSC) and optimize the electroporation parameters. Materials and Methods: DP-MSCs were transfected with GFP gene (Neon Transfection System). Five different electroporation parameters (1200v,20ms,1puls; 1 2 0 0 v , 2 0 m s , 2 p u l s ; 1 3 0 0 v , 4 0 m s , 1 p u l s ; 1400v,20ms,1puls; 1400v,20ms,2puls) were compared for optimization. After transfection the viability and apoptosis of cells were analyzed in 0, 24 and 48. hours. Fluorescent light density was examined under fluorescent microscope. Results: CD 29 and CD 44 were positive and CD45 was negative for DP-MSCs. Higher number of viable cells, higher expression of GFP and less apoptosis were found in 1200v, 20ms, 1puls electroporation parameters than other groups in different parameters. Conclusion: Rabbit animal model is generally used in various tissue engineering applications. The results of our study demonstrate optimal electroporation conditions for rabbit DP-MSCs.

Keywords:

Green fluorescent protein, mesenchymal stromal cells, transfection,

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