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Atopik dermatit hücre kültür modelinde sert suyun kallikrein-ilişkili peptidazlar ve oksidatif stres üzerine etkisinin incelenmesi Skip to content Tez Künye Durumu Atopik dermatit hücre kültür modelinde sert suyun kallikrein-ilişkili peptidazlar ve oksidatif stres üzerine etkisinin incelenmesi / Investigation of the effect of hard water on kallikrein-related peptidases and oxidative stress in atopic dermatitis cell culture model Yazar:ALİ DORUK UZUNKAYA Danışman: PROF. DR. İBRAHİM ÇAĞATAY KARAASLAN Yer Bilgisi: Hacettepe Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı / Moleküler Biyoloji Bilim Dalı Konu:Biyoloji = Biology Dizin:Atopik egzama = ; Eozinofiller = Eosinophils ; Keratinositler = Keratinocytes ; Proteaz = ; Reseptörler-toll like = Receptors-toll like ; Su sertliği = Water hardness Onaylandı Yüksek Lisans Türkçe 2023 131 s. Atopik dermatit (AD), çocuklarda daha sık görülmekle birlikte her yaştan insanı etkileyen en yaygın inflamatuvar deri hastalığıdır. Cilt kuruluğu, kaşıntı ve kızarıklıkla karakterize olan AD, deri bariyer fonksiyon bozukluğu ve immün sistemin aşırı aktivasyonu ile tanımlanmaktadır. AD patogenezinde sıklıkla tip-2 immün cevap mekanizması gözlenmektedir. Hastalıkla ilişkili sigara ve hava kirliliği gibi etkenlerin yanı sıra, kireçli su ile karşılaşmanın da deri bariyer bütünlüğünü bozarak, AD şiddetinde etkili olduğu bulunmuştur. İçeriğindeki yüksek kalsiyum ve magnezyum iyonlarıyla, normal sulara göre daha alkalidir (pH>8.5) ve derinin fizyolojik pH seviyesinin (pH:4.5-5.3) arttırmaktadır. Derideki proteaz gruplarından olan kallikrein-ilişkili peptidazlar (KLK) sağlıklı bir deride yaşlanan derinin dökülmesinde rol oynarken yüksek pH seviyelerinde ise artan proteolitik aktiviteleri sonucunda derinin geçirgenliğinin artmasına ve alerjenlere karşı savunmasız kalmasına neden olmaktadırlar. pH değişikliğine bağlı aktivitesi artan KLK-5 ve -7’nin, deride epitel hücre yüzeyinde bulunan reseptörlerden TLR-2 veya PAR-2 aracılığıyla inflamasyona neden oldukları bulunmuştur. Hücre dışına salınan KLK’ların, anti-mikrobiyal peptit olan LL-37’yi aktifleştirirken, inflamatuvar hücrelerde ise reaktif oksijen türlerinin (ROS) artışına neden olduğu bilinmektedir. Tez çalışması kapsamında kireçli su ile gerçekleşen pH ve iyon değişiminin KLK-5 ve -7 ile LL-37 üzerindeki etkisi hem AD durumunda (AD(+)) hem de AD olmayan (AD(-)) durumdaki farklılaştırılmış keratinositlerde gen (gerçek zamanlı PZR) ve protein (ELISA) düzeyinde araştırılmıştır. Keratinositlerde, TLR-2 ve PAR-2 blokanları kullanılarak KLK’ların epitel hücrede hangi reseptör/reseptörler üzerinden etkili olduğu incelenmiştir. Yapılan keratinosit-eozinofil ko-kültür modeli ile kireçli su ile uyarılan epitel hücreden salınan KLK’ların ve LL-37’nin eozinofillerde ROT üretimindeki rolü gerçek-zamanlı PZR deneyi aracılığıyla araştırılmıştır. HaCaT hücrelerinin hem tek başına olduğu hem de Eol-1 hücre hattı ile ko-kültüre alındıktan sonra gerçekleştirilen uyarımlar sonucu HaCaT hücrelerinden üretilen KLK-5 seviyesi ELISA yöntemi ile KLK-7 ve LL-37 gen ifadeleri ise gerçek-zamanlı PZR deneyi ile tayin edilmiştir. Gerçekleştirilen ELISA ve gerçek-zamanlı PZR deneyleri sonucunda KLK-5 seviyesi 12 saat ve 24 saatlik uyarımlarda 180 ve 200 mg/L CaCO3 ile özellikle AD(-) koşullarında artış gösterirken, bu artış C29 ve SsnB blokanlarının varlığıyla tersine çevrilmiş veya engellenmiştir. Bu sonuçların yanında, KLK-5 seviyesi AD(+) koşullarda değişkenlik göstermiş, bazı koşullarda CaCO3 uyarımıyla azalırken C29 ve SsnB blokanlarının kullanıldığı her iki CaCO3 konsantrasyonunda da artmış tespit edilmiştir. KLK-7 ve LL-37 gen ifadeleri ise 12 saatlik AD(-) uyarım koşulunda CaCO3 ile azalmış, ancak C29 blokanı bu etkiyi tersine çevirerek gen ifadelerini önemli ölçüde artırmıştır. Buna karşılık, AD(+) koşulda, KLK-7 ifadesi 200 mg/L CaCO3 ile yükselmiş, C29 ile düşmüş, ancak SsnB ile tekrar yükselmiştir. LL-37 ifadesi ise tüm koşullarda artış göstermiştir. 24 saatlik AD(-) deney düzeneğinde, KLK-7 ifadesi tüm koşullarda artış göstermiştir. LL-37 ifadesi de CaCO3 ve C29 ile artmıştır. Bununla birlikte, AD(+) koşullarda KLK-7 ifadesi 200 mg/L CaCO3 ile 32 katlık bir artış gösterirken bu artış ortama blokanların eklenmesiyle hafiflemiştir. Eol-1 hücre hattından izole edilen RNA’lar ile yapılan ROS gen profillerinin incelendiği deneyde SOD1 ve ApoE gibi AD hastalığında biyobelirteç olarak da kullanılan genlerin ifadelerinde 180 mg/L CaCO3 ile karşılaşılması sonucu artış tespit edilirken, kalsiyum karbonatın etkisi TLR-2 reseptör blokanlarının varlığıyla tersine çevrilmiştir. Elde edilen bu sonuca rağmen SsnB blokanının varlığıyla geriye kalan oksidatif stres genlerinin ifadesi ile AD-modelden bağımsız olarak hem 12 hem de 24 saatlik uyarımlar sonucu yükselmiştir. Gerçekleştirilen tüm deneyler sonucunda 2 farklı konsantrasyonda CaCO3 ile uyarılan HaCaT hücre hattında AD durumundan bağımsız bir şekilde KLK-5, KLK-7 ve LL-37 hem gen (gerçek-zamanlı PZR) hem de protein (ELISA) seviyelerinde 12 ve 24 saatte belirli koşullarda artış gözlenmiştir. CaCO3 uyarımlarının yanı sıra TLR-2 reseptör blokanı olarak kullanılan inhibitörlerin ortamda bulunmasıyla ilgili genlerin hem ifadesinde hem de protein düzeylerinde artış tespit edilmiştir. TLR-2 reseptör blokanlarıyla elde edilen bu sonuçlar CaCO3’ün HaCaT hücreleri üzerindeki etkisinin TLR-2 reseptörü yerine başka reseptör üzerinden gidebileceği ve kullanılan reseptör blokanlarının da HaCaT hücrelerinde farklı hücresel yolakları etkileyerek KLK-5, KLK-7 ve LL-37’nin gen ve protein düzeyinde daha yüksek seyretmesine neden olabileceği gözlemlenmiştir. Atopic dermatitis (AD) is the most common inflammatory skin disease affecting people of all ages, although it is more common in children. AD, which is characterized by skin dryness, itching and redness, is defined by skin barrier dysfunction and overactivation of the immune system. Type-2 immune response mechanism is frequently observed in the pathogenesis of AD. In addition to factors such as smoking and air pollution associated with the disease, exposure to chalky water has been found to affect the severity of AD by disrupting the integrity of the skin barrier. With its high calcium and magnesium ions, it is more alkaline (pH>8.5) than normal water and increases the physiological pH level of the skin (pH:4.5-5.3). While kallikrein-related peptidases (KLK), one of the protease groups in the skin, play a role in the shedding of aging skin in a healthy skin, at high pH levels, as a result of their increased proteolytic activity, they increase the permeability of the skin and make it vulnerable to allergens. It has been found that KLK-5 and -7, whose activity increases due to pH change, cause inflammation through the receptors TLR-2 or PAR-2 located on the epithelial cell surface of the skin. It is known that KLKs released out of the cell activate the anti-microbial peptide LL-37 and cause an increase in reactive oxygen species (ROS) in inflammatory cells. Within the scope of the thesis, the effects of pH and ion exchange with calcareous water on KLK-5, KLK-7 and LL-37 were investigated at gene (real-time PCR) and protein (ELISA) levels in differentiated keratinocytes in both AD and non-AD states. In keratinocytes, TLR-2 and PAR-2 blockers were used to investigate which receptor KLKs are more effective on epithelial cells. With the keratinocyte-eosinophil co-culture model, the role of KLKs and LL-37 released from epithelial cells stimulated with lime water in ROS production in eosinophils was investigated. Within the scope of the thesis, the effects of pH and ion exchange with lime water on KLK-5 and -7 and LL-37 were investigated at gene (real-time PCR) and protein (ELISA) levels in differentiated keratinocytes in both AD (AD(+)) and non-AD (AD(-)) states. In keratinocytes, TLR-2 and PAR-2 blockers were used to examine the receptor(s) through which KLKs act on epithelial cells. With the keratinocyte-eosinophil co-culture model, the role of KLKs and LL-37 released from the epithelial cell stimulated with lime water and the role of LL-37 in ROS production in eosinophils was investigated by real-time PCR assay. The level of KLK-5 produced from HaCaT cells as a result of stimulation of HaCaT cells both alone and after co-culturing with Eol-1 cell line was determined by ELISA method and KLK-7 and LL-37 gene expressions were determined by real-time PCR assay. As a result of ELISA and real-time PCR experiments, KLK-5 level increased in 12 h and 24 h stimulation with 180 and 200 mg/L CaCO3, especially in AD(-) conditions, and this increase was reversed or prevented by the presence of C29 and SsnB blockers. In addition to these results, KLK-5 levels were variable in AD(+) conditions, decreasing in some conditions with CaCO3 stimulation, but increasing in both CaCO3 concentrations with C29 and SsnB blockers. KLK-7 and LL-37 gene expressions were decreased by CaCO3 in the 12 h AD(-) stimulation condition, but C29 blocker reversed this effect and significantly increased gene expressions. In contrast, in AD(+) condition, KLK-7 expression increased with 200 mg/L CaCO3, decreased with C29, but increased again with SsnB. LL-37 expression increased in all conditions. In the 24 h AD(-) experimental setup, KLK-7 expression increased in all conditions. LL-37 expression also increased with CaCO3 and C29. However, in AD(+) conditions, KLK-7 expression increased 32-fold with 200 mg/L CaCO3 and this increase was attenuated by the addition of blockers to the medium. In the experiment in which ROS gene profiles were examined with RNAs isolated from Eol-1 cell line, the expression of genes such as SOD1 and ApoE, which are also used as biomarkers in AD disease, increased as a result of exposure to 180 mg/L CaCO3, while the effect of calcium carbonate was reversed by the presence of TLR-2 receptor blockers. Despite this result, the expression of the remaining oxidative stress genes was increased by the presence of SsnB blocker in both 12 and 24 hours of stimulation, independent of the AD-model. As a result of all the experiments performed, an increase in both gene (real-time PCR) and protein (ELISA) levels of KLK-5, KLK-7 and LL-37 was observed in the HaCaT cell line stimulated with CaCO3 at 2 different concentrations at 12 and 24 hours under certain conditions, independent of AD status. In addition to CaCO3 stimulation, the presence of inhibitors used as TLR-2 receptor blockers in the environment increased both the expression and protein levels of the related genes. These results obtained with TLR-2 receptor blockers suggest that the effect of CaCO3 on HaCaT cells may go through another receptor instead of the TLR-2 receptor, and the receptor blockers used may affect different cellular pathways in HaCaT cells and cause KLK-5, KLK-7 and LL-37 to be higher in gene and protein levels.
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