Publication: Meme kanseri hücre hatlarında mammaglobin gen ekspresyonunun kitozan/ shrna nanopleksleri ile baskılanmasına ilişkin in vitro çalışmalar
Abstract
Meme Kanseri Hücre Hatlarında Mammaglobin Gen Ekspresyonunun Kitozan/ shRNA Nanopleksleri İle Baskılanmasına İlişkin İn Vitro ÇalışmalarÖğrencinin Adı : Aylin ÖZKANDanışmanı : Dr. Öğr. Üyesi Suna ÖZBAŞ TURANAnabilim Dalı : Farmasötik BiyoteknolojiAmaç: İnsan mammaglobin (hMAM), genellikle meme ve meme kanseri hücrelerinde eksprese edilen bir glikoproteindir. Meme spesifik hMAM’ın RNA interferans (RNAi) teknolojisi ile kısa saç tokası RNA (shRNA) kullanılarak baskılanması önemli bir gen tedavisi stratejisidir. Nükleaz degredasyonundan korunma ve hücreye giriş etkinliğini arttırmak için viral olmayan gen taşıyıcı sistem olarak kitozan; biyobozunur olması, toksik olmayışı ve katyonik yapısı sebebiyle sıklıkla tercih edilmektedir. Bu çalışmada, hazırlanan kitozan/ mammaglobin shRNA nanoplekslerin aracılığıyla hMAM geninin baskılanmasının in vitro olarak incelenmesi amaçlanmıştır.Gereç ve Yöntem: shRNA’nın spektrofotometrik ve elektroforetik kontrolleri yapılmıştır. Kitozan/ shRNA nanoplekslerinin partikül boyutu, zeta potansiyel, jel elektroforez, serum stabilitesi ve TEM görüntüleme analizleri yapılmıştır. Hücre kültüründe; tranfeksiyon sonrası mammaglobin miktarı ELISA ile ölçülmüştür ve MTT sitotoksisite, BrdU proliferasyon ve invazyon testleri yapılmıştır.Bulgular: Tam nanopleks oluşumu gözlenmiştir, nanopleks boyutlarının ve zeta potansiyel değerlerinin uygun olduğu belirlenmiştir. Nanoplekslerin shRNA’yı degredasyondan koruduğu görülmüştür. MTT sonuçlarına göre sitotoksik etki saptanmamıştır. Mammaglobinin ve invazyonun baskılandığı görülmüştür.Sonuçlar: Başarılı bir nanopleks yapısının oluştuğu, kitozanın shRNA için uygun bir taşıyıcı sistem olduğu ve kitozan/ shRNA nanoplekslerinin meme kanserinde mammaglobini baskıladığı gösterilmiştir.
In vitro Studies on Inhibition of Mammaglobin Gene Expression with Chitosan/ shRNA Nanoplexes in Breast Cancer Cell LinesStudent’s Name : Aylin ÖZKANSupervisor : Asst. Prof. Suna ÖZBAŞ TURANDepartment : Pharmaceutical BiotechnologyAim: Human mammaglobin (hMAM) is a glycoprotein and expression is limited to mammary gland and breast tumor cell lines. Suppression of breast specific hMAM using short hairpin RNA (shRNA) with RNA interference (RNAi) technology is an important gene therapy strategy. Nuclease degradation and poor cellular uptake limit the use of the molecule. Chitosan is one of most popular delivery system due to its biodegradability, non-toxicity and cationic structure. The aim of this study was to investigate the inhibition of hMAM gene in vitro conditions with chitosan/ mammaglobin shRNA nanoplexes.Material and Method: Spectrophotometric and electrophoretic controls of shRNA were performed. Particle size, zeta potential, gel electrophoresis, serum stability and TEM imaging analyzes of chitosan/ shRNA nanoplexes were carried out. In cell culture; the amount of mammaglobin after transfection was measured by ELISA and MTT cytotoxicity, BrdU proliferation and invasion tests were performed. Results: Fully nanoplexation at all ratios was observed. Appropriate particle size and zeta potential values of nanoplexes were obtained. Nanoplexes were observed to protect shRNA from degredation No cytotoxic effect was observed according to MTT results. It was observed that mammaglobin and invasion were suppressed.Conclusions: It has been shown that a successful complex structure is formed, chitosan is the safe and effective delivery system for shRNA and chitosan/ shRNA nanoplexes suppress mammaglobin in breast cancer.
In vitro Studies on Inhibition of Mammaglobin Gene Expression with Chitosan/ shRNA Nanoplexes in Breast Cancer Cell LinesStudent’s Name : Aylin ÖZKANSupervisor : Asst. Prof. Suna ÖZBAŞ TURANDepartment : Pharmaceutical BiotechnologyAim: Human mammaglobin (hMAM) is a glycoprotein and expression is limited to mammary gland and breast tumor cell lines. Suppression of breast specific hMAM using short hairpin RNA (shRNA) with RNA interference (RNAi) technology is an important gene therapy strategy. Nuclease degradation and poor cellular uptake limit the use of the molecule. Chitosan is one of most popular delivery system due to its biodegradability, non-toxicity and cationic structure. The aim of this study was to investigate the inhibition of hMAM gene in vitro conditions with chitosan/ mammaglobin shRNA nanoplexes.Material and Method: Spectrophotometric and electrophoretic controls of shRNA were performed. Particle size, zeta potential, gel electrophoresis, serum stability and TEM imaging analyzes of chitosan/ shRNA nanoplexes were carried out. In cell culture; the amount of mammaglobin after transfection was measured by ELISA and MTT cytotoxicity, BrdU proliferation and invasion tests were performed. Results: Fully nanoplexation at all ratios was observed. Appropriate particle size and zeta potential values of nanoplexes were obtained. Nanoplexes were observed to protect shRNA from degredation No cytotoxic effect was observed according to MTT results. It was observed that mammaglobin and invasion were suppressed.Conclusions: It has been shown that a successful complex structure is formed, chitosan is the safe and effective delivery system for shRNA and chitosan/ shRNA nanoplexes suppress mammaglobin in breast cancer.