Açelya YILMAZER-AKTUNA, Hadiseh TAHERI, Alp CAN

Melanoma hücrelerinin Sendai viral vektörleri ile verimli transdüksiyonu

Amaç: Yaşayan hücrelere gen salımı yapmak üzere pek çok viral vektör geliştirilmiştir. Sendai viral (SeV) vektörleri geçici gen ifadesi, geniş konak özgüllüğü, düşük patojenite ve yüksek immünojenite gibi özellikleri sayesinde gen aktarımı için önemli vektörlerdir. SeV vektörleri gen tedavisi, aşı teknolojileri ve rejeneratif amaçlı moleküler tıpta sıklıkla kullanılır.Yöntem: Bu çalışmada, farklı melanoma hücre dizilerinde SeV vektörlerinin gen aktarım verimlilikleri floresan mikroskop ve konfokal lazer taramalı mikroskop görüntüleme teknikleri ile değerlendirilmiştir. A375, MDA- MB- 435, G361 ve WM115 hücreleri yeşil flüoresan proteini (GFP) ifade eden SeV vektörleri tarafından farklı virüs derişimlerinde (enfeksiyon çarpanı (MOI): 1, 3 ve 9) transdükte edilmiştir. GFP ifadesi virüs inkübasyonundan 24 ve 48 saat sonrasında kontrol edilmiştir. Konfokal lazer taramalı mikroskop görüntüleme ile gen salım verimliliği hesaplanmıştır. Bulgular: Floresan mikroskop görüntüleme ile düşük virüs derişimlerinde dahi (enfeksiyon çarpanı: 1), A375, MDA -MB- 435, G361 ve WM115 hücrelerinin SeV tarafından verimli şekilde transdükte edildiği gösterilmiştir. Viral transdüksiyonu takiben, GFP kontrol gen aktivitesi 24 saat içerisinde gözlemlenmeye başlanmış ve 48 saatte artış göstermiştir. Transdüksiyondan 24 saat sonrasında hücrelerde hafif toksisite gözlemlenmiş olsa da 48 saat sonrasında hücreler toksisite etkisinden kurtularak çoğalmış ve verimli şekilde gen ifadesi göstermişlerdir. Konfokal lazer taramalı mikroskop görüntüleme sonucuna göre 48 saat sonunda tüm hücre dizilerinde hücrelerin %80'inden fazlası başarılı bir şekilde GFP genini ifade etmiştir. Sonuç: Sonuç olarak, SeV vektörleri melanoma hücrelerini yüksek verimlilikle transdükte edip gen ifadesini sağlamıştır. Bu çalışma SeV vektörlerinin melanoma orijinli hücrelerdeki kullanımını açığa çıkarmış ve SeV vektörlerinin kullanımını içeren kanser tedavi ve hücre programlama alanındaki gelecek çalışmalarına destek sağlamıştır

Efficient transduction of melanoma cells with Sendai viral vectors

Objective: Various viral vectors have been developed in order to delivery genes to living cells. Sendai virus (SeV) vectors are important viral vectors due to their properties suitable for gene delivery including transient gene expression, wide host cell specificity, low pathogenicity and strong immunogenicity. SeVs vectorss are highly used in molecular medicine in gene therapy, vaccine technology and regenerative. Methods: It was evaluated the gene delivery efficiency of SeV particles in various melanoma cell lines by using fluorescence microscope and confocal laser scanning microscope imaging techniques. A375, MDAMB-435, G361 and WM115 cells have been transduced with SeV vectors expressing green fluorescent protein (GFP) at different multiplicity of infections (MOI): 1, 3, and 9. GFP expression was checked at 24 and 48 hours later following transduction. Confocal laser scanning microscopy imaging was calculated to gene delivery efficiency. Results: It was showed that A375, MDA-MB-435, G361 and WM115 cells are efficiently tranduced by seV even at low virus concentration with fluorescence microscopy imaging. GFP reporter gene activity started to be observed in 24 hours and peaked in 48 hours following viral transduction. Slight toxicity was observed following viral transduction in all cell 24 hours later; however, cells recovered and proliferated resulting in efficient gene expression 48 hours later. According to the confocal laser scanning microscopy imaging, more than 80% of all cell lines expressed GFP 48 hours after viral transduction. Conclusion: In conclusion, SeV vectors successfully transduced and expressed GFP reporter gene in various melanoma cell lines with high efficiency. This study discovered the use of SeV vectors in melanomaoriginated cells and it can open up wide range of studies involving SeV vectors in cancer therapy and cellular reprogramming fields.

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Türk Hijyen ve Deneysel Biyoloji Dergisi-Cover

ISSN: 0377-9777
Başlangıç: 1938
Yayıncı: Türkiye Halk Sağlığı Kurumu

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