Capsaici'nin in vitro ortamda erişkin sıçan spermatogenik hücre hatlarında proliferasyona ve bu süreçte gözlenen apoptozise etkileri
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Date
2004-01-20
Authors
Mızrak, Ş. Canan
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Publisher
Uludağ Üniversitesi
Abstract
Bu çalışmada, CAP'in (capsaicin) A 303 ve A 304 spermatogenik kök hücre hatlarında doza ve süreye bağlı olarak ne şekilde etki gösterdiği ve spermatogenik prosesde meydana gelen apoptozis olgusu üzerinde etkileri konularına ışık tutulması amaçlandı. A 303 ve A 304 hücre hatlarında kontrol gurubu ve deney grupları 250 uJVI, 200 uM, 150 uM, lOOuM, 50 uM ileluM olarak CAP konsantrasyonlarında oluşturuldu. Kontrol gurubunda CAP'in en yüksek konsantrasyonuna eşit DMSO (dimetilsülfoksit) ilavesi yapıldı. Oluşturulan tüm gruplarda XTT kit deneyi gerçekleştirildi. XTT deneyinin sonucunda lOOuM, 50 uM, luM grupları elenerek, 250 uM, 200 uM ve 150pM grupları ile çalışıldı. Pasajların sonlandırma zamanlan 24 ve 48 saatler olarak tespit edildi. Apoptotik hücreleri gözlemek ve görüntülemek amacıyla Nomarski mikroskobu kullanıldı. Apoptozisin immunositokimyasal anlamda belirlenmesi amacıyla anti-activated caspase 3 antikoru ile boyamalar yapılarak ışık mikroskobuyla, VR-Pin hücre hatlarında varlığını tespit etmek için ise immunofluoresan işaretleme yapılıp, sonuçlar konfokal mikroskop ile değerlendirildi. Apoptotik hücrelerin sayısal oranlarını tespit etmek amacıyla nükleer fragmantasyon belirleme protokolü uygulanmış hücreler PI (Propidium Iodide) ile boyandılar ve flow sitometri cihazında Al (apoptotik indeks) ölçümü yapıldı. Bu ölçümlerin sonucunda elde edilen apoptotik hücrelerin sayısal oranlan Kruskal Wallis NPar Tests'in Wilcoxon Signed Ranks Testi ile değerlendirildi. Bu verilerin desteklenmesi amacıyla radyoaktif timidin f3!!] bağlanması deneyi gerçekleştirildi. CAP'in 24 ve 48 saat sürelerince 3 farklı konsantrasyonla A 303 ve A 304 spermatogenik kök hücre hatlarında apoptozis olgusuna etkileri incelenip, CAP'in spermatogenik prosesi, doza ve süreye bağlı olarak etkilediği sonucuna varıldı. Bu çalışmada aynca, VR-l'in A 303 ve A 304 spermatogenik kök hücre hatlarında varlığının gösterilmesiyle hücre içi sinyal ileti mekanizmaları daha net bir şekilde ortaya konuldu.
In this study, it was aimed to determine what kind of effect does the CAP (capsaicin) with its time and dose dependent manner on A 303 and A 304 spermatogenic stem cell lines and also on apoptosis phenomenon in the spermatogenic process. In A 303 and A 304 cell lines control group, 250 uM, 200 uM, 150 uM, lOOuM, 50 uM and luM CAP concentration groups were composed. DMSO (Dimethylsulfoxide) addition was applied to the control culture, which is equivalent to the highest concentration of CAP. XTT assay was performed on all the groups. lOOuM, 50 uM, luM groups were elected depending on the results of the XTT assay and 250 uM, 200 uM and 150uM concentration groups were taken for the further assays. Ending times of the cultures were set as 24 and 48 hours. Nomarski microscope was used for observing and imaging the apoptotic cells. The stainings were performed with anti-activated caspase 3 antibody and observed under the light microscope for determination of apoptosis in terms of immunohistochemistry and immunofluoresence labelling was done to depict the existence of VR-1 on the cell lines then the results were assessed under the confocal microscope. To obtain the numerical ratio of apoptotic cells, the cells were dyed with PI (Propidium Iodide) which were applied nuclear fragmentation determination protocol, then AI (the apoptotic index) was measured with flow cytometry. After all these measurements, the apoptotic cell numbers were evaluated with Wilcoxon Signed Ranks Test of Kruskal Wallis NPar Tests. For the purpose of supporting these data, radioactive labelled thymidin I^H] incorporation assay was performed. The effects of CAP with 3 different concentrations for both 24 and 48 hours on apoptosis on A 303 and A 304 spermatogenic stem cell lines were searched, and the results were concluded saying that CAP affects the spermatogenic process with a dose and time dependent manner. Apart from that, to show the existence of VR-1 on the A 303 and A 304 spermatogenic stem cell lines put clearness on intracellular signalling machinery.
In this study, it was aimed to determine what kind of effect does the CAP (capsaicin) with its time and dose dependent manner on A 303 and A 304 spermatogenic stem cell lines and also on apoptosis phenomenon in the spermatogenic process. In A 303 and A 304 cell lines control group, 250 uM, 200 uM, 150 uM, lOOuM, 50 uM and luM CAP concentration groups were composed. DMSO (Dimethylsulfoxide) addition was applied to the control culture, which is equivalent to the highest concentration of CAP. XTT assay was performed on all the groups. lOOuM, 50 uM, luM groups were elected depending on the results of the XTT assay and 250 uM, 200 uM and 150uM concentration groups were taken for the further assays. Ending times of the cultures were set as 24 and 48 hours. Nomarski microscope was used for observing and imaging the apoptotic cells. The stainings were performed with anti-activated caspase 3 antibody and observed under the light microscope for determination of apoptosis in terms of immunohistochemistry and immunofluoresence labelling was done to depict the existence of VR-1 on the cell lines then the results were assessed under the confocal microscope. To obtain the numerical ratio of apoptotic cells, the cells were dyed with PI (Propidium Iodide) which were applied nuclear fragmentation determination protocol, then AI (the apoptotic index) was measured with flow cytometry. After all these measurements, the apoptotic cell numbers were evaluated with Wilcoxon Signed Ranks Test of Kruskal Wallis NPar Tests. For the purpose of supporting these data, radioactive labelled thymidin I^H] incorporation assay was performed. The effects of CAP with 3 different concentrations for both 24 and 48 hours on apoptosis on A 303 and A 304 spermatogenic stem cell lines were searched, and the results were concluded saying that CAP affects the spermatogenic process with a dose and time dependent manner. Apart from that, to show the existence of VR-1 on the A 303 and A 304 spermatogenic stem cell lines put clearness on intracellular signalling machinery.
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Keywords
Apoptozis, Capsaicin, Spermatogenezis, VR-1, Apoptosis, Spermatogenesis
Citation
Mızrak, Ş. C. (2004). Capsaici'nin in vitro ortamda erişkin sıçan spermatogenik hücre hatlarında proliferasyona ve bu süreçte gözlenen apoptozise etkileri. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.