Nano alfa sepiyolitin (lületaşı) kanser ve sağlıklı insan akciğer hücreleri üzerindeki in vitro sitotoksik, genotoksik ve radyobiyolojîk etkilerinin araştırılması
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Date
2020-01-20
Authors
Akça, Çetin
Journal Title
Journal ISSN
Volume Title
Publisher
Bursa Uludağ Üniversitesi
Abstract
Bu çalışmada alfa sepiyolitten (lületaşı) mükemmel özelliklere sahip doğal nano lifler üretilmiştir. Üretilen bu doğal nano liflerin floresan özellik sergilediği, deneylerde kullanılan hücrelerin ve hücre çekirdeklerinin içerisine girebildiği, sitoplazmada veziküllerin içerisinde biriktiği çekilen TEM ve Lazer Konfakal Mikroskop görüntüleriyle gösterilmiştir. Bu çalışmanın birinci aşamasında nano alfa sepiyolitin 1605120 Ligml aralığındaki konsantrasyonları her iki hücre hattına uygulanmış olup, XTT, Trypan Blue ve Muse Count Viability Testleri ile ortalama IC (6,25;12,5;25;50;75) değerleri belirlenmiştir. Bu doz değerleri ancak yüksek dozlarda hücreler üzerinde sitotoksik etki göstermiştir. Ayrıca nano alfa sepiyolitin BEAS-2B hücrelerine göre A549 hücrelerinde seçici bir özellik sergilediği bulunmuştur. Bu çalışmada artan doz konsantrasyonuna bağlı olarak nano alfa sepiyolitin neden olduğu sitotoksik etkinin nedenleri incelenmiş olup; ROS testinde hücre içi ROS miktarı ve Komet testinde ise DNA hasarının doz artışına paralel olarak arttığı görülmüştür. Ayrıca Annexın-V testi ile nano alfa sepiyolitin BEAS-2B hücrelerini hem erken apoptoza hem de geç apoptoza sürüklediği, A549 hücrelerinde ise sadece geç apoptoza sürüklediği görülmüştür. Bu çalışmanın ikinci aşamasında nano alfa sepiyolitin radyasyonla birlikte 1Gy, 1Gy+IC6,25, 1Gy+IC12,5, 1Gy+IC25, 2Gy, 2Gy+IC6.25. 2Gy+IC12.5, 2Gy+IC25 dozlarının BEAS-2B ve A549 hücreleri üzerindeki radyobiyolojik etkileri incelenmiştir. Uygulanan 1Gy ve 2Gy dozuna göre kombine uygulanan dozların radyasyon duyarlılığını artırdığı görülmüştür. A549 hücreleri ile BEAS-2B hücrelerinin benzer dozları kıyaslandığında, A549 hücrelerinin nano alfa sepiyolit ile birlikte önemli derecede radyosensitiv etki gösterdiği bulunmuştur. Burada doz artışı ile birlikte gerçekleşen sitotoksik etkinin ROS artışı ve DNA hasarı ile ilgili olduğu yapılan testler ile gösterilmiştir. Ayrıca nano alfa sepiyolit ile birlikte radyasyonun BEAS-2B hücrelerini hem erken apoptoza hem de geç apoptoza sürüklediği, A549 hücrelerinde ise daha çok geç apoptoza sürüklediği Annexın-V testi ile bulunmuştur. Birinci ve ikinci aşamadaki apoptoz bulgmları ikili boya yöntemiyle ile de morfolojik olarak desteklenmiştir. Sonuçta nano alfa sepiyolitin hem biyouyumlu hem de radyosensitiv bir madde olduğu bulunmuştur.
İn this study, natural nano fibers with excellent properties has been produced from alpha sepiolite (meerschaum). It has been demonstrated with the images of TEM and Laser Confocal Microscopes that these natural nanofibres produced have fluorescent properties, could enter into the cells and cell nucleus used in the experiments, and accumulate in the vesicles in the cytoplasm. In the first stage of this study, concentrations of nano-alpha sepiolite in the range of 160-5120pg/ml were applied to both cell lines, and the mean IC (6.25; 12.5; 25.50; 75) values were determined by XTT, Trypan Blue and Muse Count Viability Tests. These dose values were found to show cytotoxicity only on cells at high doses. In addition, it has been observed to show a selective feature in A549 cells compared to BEAS-2B cells. In this study, the causes of inhibition caused by nano alpha sepiolite due to increasing dose concentration were investigated. It was observed that the amount of intracellular ROS in the ROS test and DNA damage in the Comet test increased in parallel with the dose increase. In addition, it’s been observed by Annexin-V test that while nano-alpha sepiolite drains BEAS-2B cells into both early apoptosis and late apoptosis, A549 cells are only dragged into late apoptosis. In the second part of this study, radiobiological effects of 1Gy, 1Gy+IC6,25, 1Gy+IC12,5, 1Gy+IC25, 2Gy, 2Gy+IC6,25, 2Gy+IC12,5, 2Gy+IC25 doses of nano alpha sepiolite with radiation on the cells were investigated. It was seen that combined doses increased radiation sensitivity compared to 1Gy and 2Gy doses. When similar doses of A549 cells and BEAS-2B cells were compared, it was found that A549 cells showed significant radiosensitive effect with nano alpha sepiolite. It has been shown here that the cytotoxicity associated with the dose increase is related to ROS increase and DNA damage. In addition, thanks to Annexin-V test, it’s been observed by that radiation together with nano alpha sepiolite drains BEAS-2B cells into both early apoptosis and late apoptosis, A549 cells are only dragged into late apoptosis. Apoptosis findings in the first and second stages were also supported morphologically by dual dye method. As a result, nano alpha sepiolite was found to be both a biocompatible and a radiosensitive substance.
İn this study, natural nano fibers with excellent properties has been produced from alpha sepiolite (meerschaum). It has been demonstrated with the images of TEM and Laser Confocal Microscopes that these natural nanofibres produced have fluorescent properties, could enter into the cells and cell nucleus used in the experiments, and accumulate in the vesicles in the cytoplasm. In the first stage of this study, concentrations of nano-alpha sepiolite in the range of 160-5120pg/ml were applied to both cell lines, and the mean IC (6.25; 12.5; 25.50; 75) values were determined by XTT, Trypan Blue and Muse Count Viability Tests. These dose values were found to show cytotoxicity only on cells at high doses. In addition, it has been observed to show a selective feature in A549 cells compared to BEAS-2B cells. In this study, the causes of inhibition caused by nano alpha sepiolite due to increasing dose concentration were investigated. It was observed that the amount of intracellular ROS in the ROS test and DNA damage in the Comet test increased in parallel with the dose increase. In addition, it’s been observed by Annexin-V test that while nano-alpha sepiolite drains BEAS-2B cells into both early apoptosis and late apoptosis, A549 cells are only dragged into late apoptosis. In the second part of this study, radiobiological effects of 1Gy, 1Gy+IC6,25, 1Gy+IC12,5, 1Gy+IC25, 2Gy, 2Gy+IC6,25, 2Gy+IC12,5, 2Gy+IC25 doses of nano alpha sepiolite with radiation on the cells were investigated. It was seen that combined doses increased radiation sensitivity compared to 1Gy and 2Gy doses. When similar doses of A549 cells and BEAS-2B cells were compared, it was found that A549 cells showed significant radiosensitive effect with nano alpha sepiolite. It has been shown here that the cytotoxicity associated with the dose increase is related to ROS increase and DNA damage. In addition, thanks to Annexin-V test, it’s been observed by that radiation together with nano alpha sepiolite drains BEAS-2B cells into both early apoptosis and late apoptosis, A549 cells are only dragged into late apoptosis. Apoptosis findings in the first and second stages were also supported morphologically by dual dye method. As a result, nano alpha sepiolite was found to be both a biocompatible and a radiosensitive substance.
Description
Keywords
Lületaşı, Meerschaum, Nano alfa sepiyolit, Sitotoksisite, Genotoksisite, Radyobiyolojik etki, In vitro, Nano alpha sepiolite, Cytotoxicity, Genotoxicity, Radiobiological
Citation
Akça, Ç. (2020). Nano alfa sepiyolitin (lületaşı) kanser ve sağlıklı insan akciğer hücreleri üzerindeki in vitro sitotoksik, genotoksik ve radyobiyolojik etkilerinin araştırılması. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Fen Bilimleri Enstitüsü.