DENTAL PULPA KAYNAKLI MEZENKİMAL KÖK HÜCRELER İÇİN ELEKTROPORASYON PARAMETRELERİNİN ARAŞTIRILMASI

Yıl 2016, Cilt: 25 Sayı: 3, 144 - 147, 01.12.2016

Zeynep Burçin Gönen

Öz

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

Öz

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.

Anahtar Kelimeler

Green fluorescent protein, mesenchymal stromal cells, transfection

Kaynakça

• 1. Moraleda JM, Blanquer M, Bleda P, et al. Adult stem cell therapy: Dream or reality. Transpl Immunol 2006; 17:74–77.
• 2. Caplan AL, Bruder SP. Mesenchymal Stem cells: Building blocks for molecular medicine in the 21st century. Trends Mol Med 2001; 7:259–264.
• 3. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sc 2000; 97:13625–13630.
• 4. Miura M, Gronthos S, Zhao M, et al. SHED: Stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci 2003; 100:5807–5812.
• 5. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004; 364:149–155.
• 6. Morsczeck C, Gotz W, Schierholz J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol 2005; 24:155–165.
• 7. Sonoyama W, Liu Y, Fang D, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One 2006; 79:1–8.
• 8. 8. Sharpe PT, Young CS. Tube-tube teeth. Sci Am 2005; 293:34–41.
• 9. Loomba K, Bains R, Bains VK, et al. Tissue engineering and its application in endodontics: An overview. ENDO (Lond Engl) 2012; 6:105–112.
• 10. Nakashima M. Tissue engineering in endodontics. Aust Endod J 2005; 31:111–113.
• 11. Iohara K, Nakashima M, Ito M, et al. Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenic protein 2. J Dent Res 2004; 83:590–595.
• 12. Karaoz E, Dogan BN, Aksoy A, et al. Isolation and in vitro characterization of dental pulp stem cells from natal teeth. Histochemistry and Cell Biology 2010; 133:95–112.
• 13. Suchánek J, Soukup T, Ivancaková R, et al. Human dental pulp stem cells-isolation and long term cultivation. Acta Medica (Hradec Kralove) 2007; 50: 195-201.
• 14. Lee UL , Jeon SH, Park JY, Choung PH. Effect of platelet-rich plasma on dental stem cells derived from human impacted third molars. Regen Med 2011; 6: 67-79.
• 15. Rizk A, Rabie BM. Electroporation for transfection and differentiation of dental pulp stem cells. BioResearch 2013; 2:155-162.
• 16. Rizk A, Rabie AB. Human dental pulp stem cells expressing transforming growth factor β3 transgene for cartilage-like tissue engineering. Cytotherapy 2013; 15:712-725.
• 17. Rane MJ, Arthur JM, Prossnitz ER, McLeish KR. Activation of mitogen-activated protein kinases by formyl peptide receptors is regulated by the cytoplasmictail.JBiolChem1998; 273:20916-20923.
• 18. Meier J, Vannier C, Sergé A, et al. Fast and reversible trapping of surface glycine receptors by gephyrin. Nat Neurosci 2001; 4:253-260.
• 19. Cao X, Deng W, Wei Y. Incorporating ptgf-β1/calcium phosphate nanoparticles with fibronectin into 3- dimensional collagen/chitosan Scaffolds: Effıcient, sustained gene delivery to stem cells for Chondrogenic differentiation. Eur Cell Mater 2012;23:81-93.
• 20. Ferreira E, Potier E, Logeart-Avramoglou D, et al. Optimization of a gene electrotransfer method for mesenchymal stem cell transfection. Gene Ther 2008; 15:537–544.
• 21. Yalvac ME, Ramazanoglu M, Gumru OZ, et al. Comparison and optimisation of transfection of human dental follicle cells, a novel source of stem cells, with different chemical methods and electroporation. Neurochem Res 2009; 34: 1272- 1277.
• 22. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1982;1: 841-845.
• 23. Rols MP. Electropermeabilization, a physical method for the delivery of therapeutic molecules into cells. Biochim Biophys Acta 2006; 1758:423-428.