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dc.contributor.advisorGüç , Dicle
dc.contributor.authorGök Yavuz , Betül
dc.date.accessioned2018-07-12T13:02:28Z
dc.date.available2018-07-12T13:02:28Z
dc.date.issued2018
dc.date.submitted2018-06-25
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dc.identifier.urihttp://hdl.handle.net/11655/4658
dc.description.abstractGÖK YAVUZ B., The Effect of Cancer Associated Fibroblasts and Normal Fibroblasts on Monocyte Recruitment, Macrophage Polarization and Invasion in Breast Cancer. Hacettepe University Institute of Health Sciences Ph.D Thesis in Tumor Biology and Immunology Program, Ankara, 2018. In breast cancer, macrophages represent up to %50 of the tumor mass and there is a correlation between the number of tumor associated macrophages (TAM) and poor prognosis. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, hence favor tumor growth. Macrophages originate from blood monocytes, which differentiate into either M1 or M2 subtype macrophages depending on the environmental stimulus they receive. Fibroblasts turn into cancer associated fibroblasts (CAFs) in the tumor microenvironment. CAFs have recently been drawn attention for their function as a regulator of immune cell recruitment and function. In this study the role of normal fibroblasts, cancer associated fibroblasts and breast cancer cells on monocyte recruitment and macrophage polarization were determined in breast cancer. In this study, we found that CAFs and MDA-MB-231 cells recruited monocytes effectively. MCP-1 and SDF-1 were important chemotactic cytokines that secreted from breast cancer cells and stromal cells We showed that CAFs from invasive breast cancer differentiated monocytes to M2-like protumoral macrophages phenotypically in contrast to fibroblasts from normal breast. CAF-educated monocytes exhibited strong immune suppression unlike NF-educated monocytes and enhanced the motility/invasion of breast cancer cells. CAF-educated M1 macrophages displayed increased expression of M2 markers and production of anti-inflammatory cytokine IL-10 in contrast to decreased production of pro-inflammatory cytokine IL-12 compared with control M1 macrophages.en
dc.description.sponsorshipBu çalışma, Hacettepe Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi tarafından desteklenmiştir (Proje Kodu: THD-2016-12202).tr_TR
dc.description.tableofcontentsONAY SAYFASI iii YAYINLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI iv ETİK BEYAN v TEŞEKKÜR vi ÖZET vii ABSTRACT viii İÇİNDEKİLER ix SİMGELER VE KISALTMALAR xii ŞEKİLLER xv TABLOLAR xix 1. GİRİŞ 1 2. GENEL BİLGİLER 3 2.1. Normal Meme Dokusu 3 2.2. Meme Kanseri 4 2.2.1. Meme Kanseri Mikroçevresi 4 2.2.2. Meme Kanseri Stroma Bileşenleri 9 2.2.3. Kanserle İlişkili Fibroblastlar (KİF) 9 2.2.4. Tümörle İlişkili Makrofajlar (TİM) 17 2.2.5. Kemokinler 26 3. GEREÇ VE YÖNTEMLER 33 3.1. Çalışmada Kullanılan Maddeler 33 3.2. Hazırlanan Tamponlar ve Çözeltiler 34 3.3. İnsan Dokularıyla Deneyler 34 3.3.1. Normal ve Kanserli Doku Örnekleri Eldesi 34 3.3.2. Kan Örnekleri 37 3.4. Normal ve Kanserle ilişkili Fibroblastların Eksplant Kültür Yöntemiyle İzolasyonu 37 3.4.1. Normal Fibroblastların Eksplant Kültür Yöntemiyle İzolasyonu 37 3.4.2. Kanserle İlişkili Fibroblastların Eksplant Kültür Yöntemiyle İzolasyonu 38 3.5. İmmün Hücrelerin İzolasyonu 38 3.5.1. Periferik Kan Mononükleer Hücrelerin (PKMH) İzolasyonu 38 3.5.2. Monostlerin İzolasyonu 39 3.5.3. CD4+ Yardımcı T Hücrelerin Manyetik Aktive Hücre Ayrıştırma (MACS) Yöntemiyle İzolasyonu 40 3.6. Hücre Kültürü 41 3.6.1. Monosit Hücre Kültürü 41 3.6.2. MDA-MB-231 Hücre Kültürü 41 3.6.3. Aderan Hücre Kültürünün Tripsin-EDTA ile Pasajlanması 41 3.6.4. Kültür Sonrası Aderan Monositlerin Accutase ile Tek Hücre Süspansiyonu Getirilmesi 42 3.6.5. Hücre Sayma 42 3.6.6. Hücre Dondurma 43 3.6.7. Hücre Çözme 44 3.7. Morfolojik Analizler 44 3.7.1. İmmünsitokimya 45 3.8. In vitro M1 ve M2 Makrofaj Polarizasyonu 47 3.9. Koşullu Besiyeri (KB) Hazırlama 48 3.10. Monositlerin Koşullu Besiyeri ile Kültür Deneyleri 48 3.11. M1 Makrofajların Koşullu Besiyeri ile Kültür Deneyleri 49 3.12. Akım Sitometri ile Hücrelerin Fenotipik Analizi 50 3.13. Karbosiflöresein Süksinimidil Ester (CFSE) Proliferasyon Deneyi 51 3.14. Enzyme-Linked İmmunosorbent Assay (ELISA) 53 3.14.1. İnsan IL-10 ELİSA 53 3.14.2. İnsan IL-12 ELİSA 54 3.15. İn Vitro Migrasyon Deneyleri 56 3.16. İn Vitro İnvazyon Deneyleri 57 3.17. İstatistiksel Analizler 59 4. BULGULAR 60 4.1. Fibroblastların İzolasyonu 60 4.1.1. Normal Fibroblastların İzolasyonu 61 4.1.2. Kanserle İlişkili Fibroblastların İzolasyonu 62 4.2. Fibroblast Karakterizasyonları-İmmünsitokimya 63 4.3. Monosit İzolasyonu 66 4.3.1. Yapıştırma Yöntemiyle izolasyon 66 4.3.2. Manyetik Aktive Edilmiş Hücre Ayrıştırma (MACS) ile Monosit Eldesi 67 4.4. In Vitro M1 ve M2 Makrofaj Polarizasyonları 67 4.4.1. Morfolojik Analizler 68 4.4.2. Akım Sitometri ile Fenotipik Analizi 68 4.5. Monositlerin Koşullu Besiyeri ile Yapılan Kültür Deneyleri 70 4.6. M1 Makrofajların Koşullu Besiyeri ile Kültür Deneyleri 73 4.7. Karboksiflöresein Süksinimidil Ester (CFSE) Proliferasyon Deneyi 74 4.8. İn Vitro Migrasyon Deneyleri 79 4.9. İn Vitro İnvazyon Deneyleri 82 4.10. ELİSA 85 4.10.1. İnsan IL-10 ELİSA 85 4.10.2. İnsan IL-12 ELİSA 87 5.TARTIŞMA 89 6. SONUÇ VE ÖNERİLER 98 7. KAYNAKLAR 101 8. EKLER EK-1. Tez Çalışması ile İlgili Etik Kurul İzni EK-2. Tez Çalışması ile İlgili Bildiriler EK-3. Tez Çalışması ile İlgili Yayınlar 9. ÖZGEÇMİŞtr_TR
dc.language.isoturtr_TR
dc.publisherKanser Enstitüsütr_TR
dc.rightsinfo:eu-repo/semantics/openAccesstr_TR
dc.subjectMeme kanseritr_TR
dc.subjectTümör mikroçevresi
dc.subjectKanserle ilişkili fibroblast
dc.subjectMonosit
dc.subjectMakrofaj
dc.titleMeme Kanserinde, Normal Fibroblastlar ve Kanserle İlişkili Fibroblastların Monosit Çağrılması, Makrofaj Polarizasyonu ve İnvazyona Etkisitr_TR
dc.typeinfo:eu-repo/semantics/doctoralThesistr_TR
dc.description.ozetGÖK YAVUZ B., Meme Kanserinde, Normal Fibroblastlar ve Kanserle İlişkili Fibroblastların Monosit Çağrılması, Makrofaj Polarizasyonu ve İnvazyona Etkisi. Hacettepe Üniversitesi Sağlık Bilimleri Enstitüsü Tümör Biyolojisi ve İmmunolojisi Programı Doktora Tezi, Ankara, 2018. Meme kanserinde, makrofajlar tümör kütlesinin %50’sini temsil eder ve tümörle ilişkili makrofajların (TİM) sayısı ile kötü prognoz arasında bir bağlantı vardır. TİM’ler genellikle M2 makrofajlara benzerler. Pro-inflamatuvar ve anti-kanser fonksiyonları olan M1 makrofajların aksine, M2 makrofajlar tümör büyümesini desteklerler. Makrofajlar monositlerden köken alır. Monositler çevresel sinyallere göre M1 ya da M2 tip makrofaja farklılaşırlar. Fibroblastlar tümör mikroçevresinde kanserle ilişkili fibroblastlara (KİF) dönüşürler. KİF’ler, immün hücrelerin tümör alanına çağrılması ve fonksiyonları üzerindeki düzenleyici rolleri açısından dikkat çekmektedir. Meme kanserinde, stromal fibroblastların monosit/makrofaj üzerindeki rolünü araştıran çalışmalar oldukça sınırlıdır. Bu çalışmada, normal fibroblastların (NF), kanserle ilişkili fibroblastların ve meme kanser hücre hatlarının monositlerin kemotaksisi ve polarizasyonundaki rolleri araştırılmıştır. Çalışmamızda, KİF’lerin ve MDA-MB-231 hücrelerinin monosit migrasyonunu etkili bir şekilde sağladığı ve bu süreçte MCP-1 ve SDF-1 sitokinlerinin rolü olduğu gösterilmiştir. KİF’ler monositleri, normal fibroblastların aksine fenotipik olarak M2 benzeri tümörü destekleyici makrofajlara farklılaştırmıştır. KİF-koşullu besiyeri ile kültür edilmiş monositlerin meme kanseri hücre invazyonunu arttırdığı ve bu monositlerin, NF-koşullu besiyeri ile kültür edilmiş monositlere göre T hücre proliferasyonunu daha fazla baskıladığı gösterilmiştir. MDA-MB-231 ve KİF koşullu besiyeri ile kültür edilmiş M1 makrofajlarda kontrol M1 makrofajlara göre, M2 makrofaj belirteçlerinin ekspresyonu ve anti-inflamatuvar IL-10 sitokin sekresyonu artarken pro-inflamatuvar IL-12 sitokin düzeyi azalmıştır. Bu sonuçlar meme KİF’lerinin tümörü destekleyici TİM populasyonunu arttırabildiğini göstermektedir.tr_TR
dc.contributor.departmentTemel Onkolojitr_TR
dc.contributor.authorID10199870tr_TR


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