Brusellozlu hastalarda eksozom profilinin değerlendirilmesi
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Date
2021-06-22
Authors
Şimşek, Abdurrahman
Journal Title
Journal ISSN
Volume Title
Publisher
Bursa Uludağ Üniversitesi
Abstract
Bruselloz, etkeni Brucella bakterileri olan dünya çapında zoonotik bir enfeksiyondur. Eksozomlar, birçok hücre tipi tarafından salınan multiveziküler gövdelerden türetilen küçük lipit veziküllerdir. Eksozomal hücre kargosu, kökenlendiği hücrenin tipini yansıtır. Bu çalışmada akut, kronik bruselloz ve sağlıklı kontrollerden alınan serum örneklerinden elde edilen eksozomların Akan Hücre Ölçer (AHÖ) yardımıyla immünfenotiplemesini yapmak, farklı gruplarda eksozomların hangi hücre gruplarından köken aldığını belirlemek, brusellozun kronikleşmesinde, eksozomların etkisini ortaya koymak ve bruselloz immünpatogenezinde eksozomların potansiyel rollerine dair literatüre katkılar sunmak amaçlanmıştır. Çalışmamıza, bruselloz tanısı konmuş 10 akut ve 10 kronik hasta ile 10 sağlıklı kontrol dahil edilmiştir. Serum örneklerinden eksozomlar ticari izolasyon kiti kullanılarak elde edilmiştir. Önceden tasarlanan panelde, monoklonal antikorlarla boyandıktan sonra, AHÖ ’de eksozomların immünfenotiplemesi yapılmıştır. Eksozom varlığının doğrulanması, eksozoma spesifik monoklonal antikorlar olan CD9, CD63 ve CD81 boyamaları ve elektron mikroskobu (SEM) değerlendirmeleriyle yapılmıştır. Değerlendirme sonucuna göre granülosit kökenli eksozom değerleri akut grupta, kronik ve sağlıklı gruba göre anlamlı derecede yüksek çıkmıştır, bunun yanında kronik ve sağlıklı grup arasında anlamlı bir fark gözlenmemiştir. G-MDSC (granülositik-myeloid kökenli süpressör hücreler) kökenli eksozom değerleri, akut grupta kronik ve sağlıklı gruba göre anlamlı derecede yüksek bulunmuştur, aynı zamanda kronik grup ve sağlıklı kontrol grubu arasında anlamlı derecede farklılık gözlenmiştir. Tez çalışması sonucunda, akut dönemde hastalığın patogenezinde granülosit kökenli eksozomların rol oynadığı ve ortamda bulunan myeloid kökenli supressör hücrelerden kaynaklanan eksozomlarında granülosit kökenli eksozomlar olduğu bulunmuştur. Brusellozun farklı evrelerinde, granülosit kökenli ve G-MDSC kökenli eksozomlar arasındaki anlamlı farklar, hastalığın immünpatolojisine, yeni çalışmalarla birlikte önemli katkılar sunabilir.
Brucellosis is a worldwide zoonotic infection caused by a type of bacteria from the genus Brucella. Exosomes are small lipid vesicles derived from multi-vesicular bodies released by many cell types. The exosomal cell cargo reflects the type of cell from which it originated. In this study, we aimed to perform immunophenotyping of exosomes isolated from serum samples from acute, chronic brucellosis and healthy controls using Flow Cytometry, determine from which cell groups exosomes originate in different groups, reveal the effect of exosomes in the chronicity of brucellosis, and contribute to the literature on the potential roles of exosomes in the immunopathogenesis of brucellosis. Our study included 10 acute and 10 chronic patients diagnosed with brucellosis and 10 healthy controls. Exosomes from serum samples were extracted using a commercial isolation kit. In the pre-designated panel, after staining with monoclonal antibodies (mAbs), the exosomes were immunophenotyped using Flow Cytometry. Confirmation of the presence of exosome was performed by staining of exosome-specific mAbs CD9, CD63 and CD81 and electron microscopy (SEM) evaluations. According to the results of the evaluation, granulocyte and G-MDSC (granulocyte-like myeloid derived suppressor cells) derived exosomes (%) were significantly higher in the acute group compared to the chronic and healthy groups. On the other hand, a significant difference was observed between the chronic group and the healthy control in exosome values (%) originating from G-MDSC. In this study, we found that granulocyte and G-MDSC-derived exosomes may play a role in the acute phase responses of brucellosis. The significant differences between granulocyte and G-MDSC-derived exosomes at different phases of brucellosis may contribute to the immunopathology of the disease with new studies.
Brucellosis is a worldwide zoonotic infection caused by a type of bacteria from the genus Brucella. Exosomes are small lipid vesicles derived from multi-vesicular bodies released by many cell types. The exosomal cell cargo reflects the type of cell from which it originated. In this study, we aimed to perform immunophenotyping of exosomes isolated from serum samples from acute, chronic brucellosis and healthy controls using Flow Cytometry, determine from which cell groups exosomes originate in different groups, reveal the effect of exosomes in the chronicity of brucellosis, and contribute to the literature on the potential roles of exosomes in the immunopathogenesis of brucellosis. Our study included 10 acute and 10 chronic patients diagnosed with brucellosis and 10 healthy controls. Exosomes from serum samples were extracted using a commercial isolation kit. In the pre-designated panel, after staining with monoclonal antibodies (mAbs), the exosomes were immunophenotyped using Flow Cytometry. Confirmation of the presence of exosome was performed by staining of exosome-specific mAbs CD9, CD63 and CD81 and electron microscopy (SEM) evaluations. According to the results of the evaluation, granulocyte and G-MDSC (granulocyte-like myeloid derived suppressor cells) derived exosomes (%) were significantly higher in the acute group compared to the chronic and healthy groups. On the other hand, a significant difference was observed between the chronic group and the healthy control in exosome values (%) originating from G-MDSC. In this study, we found that granulocyte and G-MDSC-derived exosomes may play a role in the acute phase responses of brucellosis. The significant differences between granulocyte and G-MDSC-derived exosomes at different phases of brucellosis may contribute to the immunopathology of the disease with new studies.
Description
Keywords
Bruselloz, Eksozom, Kronik, Akut, Ekstrasellüler veziküller, Brucellosis, Exosome, Chronic, Acute, Extracellular vesicles
Citation
Şimşek, A. (2021). Brusellozlu hastalarda eksozom profilinin değerlendirilmesi. Yayınlanmamış yüksek lisans tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.