Publication: Zara özgü katkı maddelerinin ve apoptoz sürecinin zarda oluşturduğu değişimlerin yeni geliştirilen floresans problarla belirlenmesi
Abstract
Bu çalışmada diğer yöntemlerle belirlenmesi zor olan, zar lipid organizasyonunda meydana gelen değişimlerin görüntülenmeleri amacıyla yeni geliştirilen floresan oransal problar karakterize edilmiş ve hücre zarları floresan yöntemler aracılığıyla incelenmiş ve görüntülenmiştir. İlk olarak sıcaklığın 3-hidroksiflavon ve türevlerinin floresans yanıtına etkileri incelenmiş ve sıcaklığa duyarlı yanıtlar verdikleri gözlenmiştir. Yeni geliştirilen 3-hidroksiflavon türevi floresan probların fotofiziksel özelliklerinin protik ve aprotik organik çözücülerde çözücü polaritesiyle korelasyonu floresans spektroskopisi aracılığıyla saptanmıştır. Biyolojik zarlarda kullanılması amaçlanan oransal floresan problarla farklı fosfolipid içeriklerine sahip tek tabakalı ve çok tabakalı lipid veziküller etiketlenerek, floresans spektroskopisi aracılığıyla probların lipid çift tabakadaki yerleşim ve davranışları incelenmiştir. Çalışmamızda, 3-HF türevi floresan boya F2N12S’ in zar lipid çift tabakasındaki farklı fazları belirleyebilirliğini göstermek amacıyla akıcı düzensiz, akıcı-düzenli (raft) ve her iki durumun birarada bulunduğu üç farklı tipte dev tek tabakalı lipid veziküller hazırlanarak floresan oransal görüntülemede akıcı düzensiz (Ld) ve akıcı düzenli (Lo) fazlara ait bölgeler net olarak görüntülenmiştir. U87MG malin astositoma hücrelerinin zar lipid organizayonlarını değiştirmek amacıyla, hücrelerin Aktiomisin D ile apoptoza indüklenmesi ve Metil- Siklodekstrin aracılığıyla kolesterol içeriğinin değiştirilmesi olmak üzere iki farklı uygulama gerçekleştirilmiştir ve bu hücreler hem akıcı-düzensiz faza hem de raftlara bağlanan F2N12S probuyla boyanarak, floresans oransal görüntüleme yöntemiyle hücre zarı lipid organizasyonundaki değişimler saptanmıştır. Anahtar Sözcükler: 3-Hidroksiflavon türevleri, floresans spektroskopi, lipid organizasyon, model zarlar, oransal floresans görüntüleme.
DETERMINATION OF CHANGES IN THE MEMBRANE GENERATED BY APOPTOSIS AND MEMBRANE-SPECIFIC ADDITIVES BY NEWLY DEVELOPED FLUORESCENT PROBES This work aimed to detect and display the changes that occur in lipid organization of the cell membrane with newly developed ratiometric florescence probes; a goal that has not yet been completely achieved. Initially, the fluorescence response of ratiometric 3-hydroxyflavones and derivatives was investigated . The response of these probes was found to depend on temperature. The photophysical properties of these novel fluorescent probes detected with fluorescence spectroscopy correlated with the solvent polarity in protic or aprotic organic solvents. As these fluorescent probes are intended to be used in biological membranes, at first unilamellar and multilamellar lipid vesicles were labelled with 3-hydroxyflavone derivatives and the location of these probes in the lipid bilayers was established. In order to demonstrate that the 3-hydroxyflavone derivative fluorescent probe F2N12S can be used to determine the different phases in the lipid bilayer, three different types of giant unilamellar vesicles were generated: one with a liquid disordered phase structure, one with liquid ordered (rafts) phase structure and one in which both of these phases were present. These different phases were clearly demonstrated by fluorescence ratiometric imaging. To alter the lipid organization of the U87MG astrocyte cell membrane, cells were either two induced to apoptosis with actinomycin D or the cholesterol content of the cell membrane was altered by Methyl- cyclodextrin. These cells were subsequently labelled with F2N12S which binds to the liquid disordered phase and also to rafts, and the changes in lipid organization of the cell membrane were determined with both fluorescence spectroscopy and fluorescence ratiometric imaging. Key words: 3-Hydroxyflavone derivatives, fluorescence spectroscopy, lipid organization, model membranes, ratiometric fluorescence imaging.
DETERMINATION OF CHANGES IN THE MEMBRANE GENERATED BY APOPTOSIS AND MEMBRANE-SPECIFIC ADDITIVES BY NEWLY DEVELOPED FLUORESCENT PROBES This work aimed to detect and display the changes that occur in lipid organization of the cell membrane with newly developed ratiometric florescence probes; a goal that has not yet been completely achieved. Initially, the fluorescence response of ratiometric 3-hydroxyflavones and derivatives was investigated . The response of these probes was found to depend on temperature. The photophysical properties of these novel fluorescent probes detected with fluorescence spectroscopy correlated with the solvent polarity in protic or aprotic organic solvents. As these fluorescent probes are intended to be used in biological membranes, at first unilamellar and multilamellar lipid vesicles were labelled with 3-hydroxyflavone derivatives and the location of these probes in the lipid bilayers was established. In order to demonstrate that the 3-hydroxyflavone derivative fluorescent probe F2N12S can be used to determine the different phases in the lipid bilayer, three different types of giant unilamellar vesicles were generated: one with a liquid disordered phase structure, one with liquid ordered (rafts) phase structure and one in which both of these phases were present. These different phases were clearly demonstrated by fluorescence ratiometric imaging. To alter the lipid organization of the U87MG astrocyte cell membrane, cells were either two induced to apoptosis with actinomycin D or the cholesterol content of the cell membrane was altered by Methyl- cyclodextrin. These cells were subsequently labelled with F2N12S which binds to the liquid disordered phase and also to rafts, and the changes in lipid organization of the cell membrane were determined with both fluorescence spectroscopy and fluorescence ratiometric imaging. Key words: 3-Hydroxyflavone derivatives, fluorescence spectroscopy, lipid organization, model membranes, ratiometric fluorescence imaging.