Capsaicin'in ovaryum granuloza hücrelerinin proliferasyonu ve apoptozisi üzerine etkisi
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Date
2015-10-16
Authors
Peker, Sabire
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Publisher
Uludağ Üniversitesi
Abstract
Capsaicin, kırmızı bibere acılığı veren, alkoloid yapıda bir maddedir. Capsaicinin, sensorik sinirler üzerindeki etkilerinden dolayı, birçok çalışmada, farklı organ ve sistemler üzerindeki etkileri incelenerek geniş uygulama alanları bulmasına rağmen, ovaryum üzerindeki etkilerini araştıran çalışmalar oldukça sınırlıdır. Sunulan tez çalışmasının amacı, capsaicinin farklı doz ve zaman uygulamalarının, primer olarak elde edilen ovaryum granuloza hücreleri üzerinde meydana getirdiği proliferatif ve/veya apoptotik, direk etkilerinin incelenmesidir. Bu amaca uygun olarak, 30 günlük sıçan ovaryumlarından, belirlenen primer hücre kültürü prosedürünün takibi ile granuloza hücreleri izole edildi. Elde edilen granuloza hücreleri ile; kontrol A (hücre kontrol grubu), kontrol B (taşıt maddenin (DMSO) verildiği kontrol grubu), 10, 50, 100, 150 ve 200 µM olmak üzere capsaicinin uygulanacağı 7 farklı doz grubu ve her doz grubu için, iki farklı zaman (24 ve 48 saat) grubu oluşturuldu. Doz ve zaman uygulamalarını takiben capsaicinin, granuloza hücreleri üzerindeki olası proliferatif ekilerinin incelenmesi amacıyla PCNA primer antikoru ile immunsitokimya, immunfloresan ve western blot prosedürleri uygulandı. Ayrıca bu bulguları desteklemek amacıyla MTT hücre proliferasyon testi yapıldı. Capsaicinin apoptotik etkilerini incelemek amacıyla ise, apoptotik yolağın son elemanlarından aktif caspase-3 ve aktif PARP primer antikorları ile immunsitokimya, immunfloresan ve western blot deneyleri gerçekleştirildi. Deneylerin sonucunda, capsaicinin 50 µM'a kadar düşük uygulama dozlarıyla, granuloza hücrelerinde proliferasyonun arttığı, 100 µM dozdan sonra ise apoptozisin meydana geldiği görüldü. Bu etkilerin 24 ve 48 saatlik zaman uygulamalarında benzer olmasından dolayı, 24 saatlik capsaicin uygulamasının istenen etkilerin görülmesi için yeterli olduğu görüldü. Sonuç olarak, capsaicinin düşük dozlarının, ovaryum granuloza hücreleri üzerinde proliferatif etki gösterdiği, doz arttıkça ise apoptozisin meydana geldiği belirlendi. Bu etkilerinden dolayı, capsaicinin düşük dozlarının, reproduktif düzenin devamlılığında kullanılabileceği, yüksek dozlarının ise ovaryum kanserleri için geliştirilecek alternatif tedavi protokollerine temel oluşturabileceği söylenebilir.
Capsaicin, alkoloid substance, is pungent ingredient in red peppers. Due to the effects on sensory nerve fibers, many studies have examined the effects of capsaicin on different organs and systems. Despite the finding wide application areas on different systems, the studies of the effects on the ovary is very limited. The aim of the present thesis, the possible proliferative and/or apoptotic effects of various doses and time periods in the application of capsaicin on primarily derived granulosa cells. In accordance with this purpose, identification procedure was followed for granulosa cells isolation from 30-day rat ovaries. The obtained granulosa cells were divided seven dose groups; control A (cell control), control B ( vehicle (DMSO) control), 10, 50 , 100, 150 and 200 µM and two different time (24 and 48 hours) groups. As the results of dosage and time administration of capsaicin, the proliferative effects were examined by PCNA primary antibody and immunocytochemistry, immunofluorescence and western blot procedures were performed. In addition, the MTT cell proliferation assay was performed to support these findings. In order to examine the apoptotic effects of capsaicin, immunocytochemistry, immunofluorescence and western blot assays were performed by last elements in the apoptotic pathway; active caspase-3 and active PARP. As a result of the experiments, granulosa cell proliferation with the lower application doses of up to 50 µM, but after 100 µM and increased doses showed the apoptosis occur. Due to these effects were similar in 24 and 48 hours application time, 24 hours capsaicin application was observed to be sufficient for of the occurrence of the desired effects. In consequence of the present study, low doses of capsaicin have proliferative effects on granulosa cells and the apoptosis was occurred when the dose increase. Because of these effects, low-dose capsaicin can be used in the continuity of the reproductive pattern and higher doses in development alternative treaatment protocols on ovarian cancers.
Capsaicin, alkoloid substance, is pungent ingredient in red peppers. Due to the effects on sensory nerve fibers, many studies have examined the effects of capsaicin on different organs and systems. Despite the finding wide application areas on different systems, the studies of the effects on the ovary is very limited. The aim of the present thesis, the possible proliferative and/or apoptotic effects of various doses and time periods in the application of capsaicin on primarily derived granulosa cells. In accordance with this purpose, identification procedure was followed for granulosa cells isolation from 30-day rat ovaries. The obtained granulosa cells were divided seven dose groups; control A (cell control), control B ( vehicle (DMSO) control), 10, 50 , 100, 150 and 200 µM and two different time (24 and 48 hours) groups. As the results of dosage and time administration of capsaicin, the proliferative effects were examined by PCNA primary antibody and immunocytochemistry, immunofluorescence and western blot procedures were performed. In addition, the MTT cell proliferation assay was performed to support these findings. In order to examine the apoptotic effects of capsaicin, immunocytochemistry, immunofluorescence and western blot assays were performed by last elements in the apoptotic pathway; active caspase-3 and active PARP. As a result of the experiments, granulosa cell proliferation with the lower application doses of up to 50 µM, but after 100 µM and increased doses showed the apoptosis occur. Due to these effects were similar in 24 and 48 hours application time, 24 hours capsaicin application was observed to be sufficient for of the occurrence of the desired effects. In consequence of the present study, low doses of capsaicin have proliferative effects on granulosa cells and the apoptosis was occurred when the dose increase. Because of these effects, low-dose capsaicin can be used in the continuity of the reproductive pattern and higher doses in development alternative treaatment protocols on ovarian cancers.
Description
Keywords
Granuloza hücresi, Capsaicin, Apoptozis, Proliferasyon, Granulosa cell, Apoptosis, Proliferation
Citation
Peker, S. (2015). Capsaicin'in ovaryum granuloza hücrelerinin proliferasyonu ve apoptozisi üzerine etkisi. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.