İnsan indolamin -2,3-dioksijenaz (İDO) geninin ELAM-1 promotoru kontrolünde klonlanması ve ekspresyonu
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Date
2017-04-28
Authors
Karaçay, Mehmet
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Publisher
Uludağ Üniversitesi
Abstract
İmmünolojik tolerans, antijen ile karşılaşan lenfositlerin yanıtsız kalması olarak tanımlanmıştır. İmmün sistem bazı patolojik durumlarda ve immün toleransın kırılması sonucunda, öz antijenlerine karşı duyarlı hale gelerek, kendi doku antijenlerine karşı reaksiyona geçmektedir. Böylece immün tolerans bozularak otoimmün hastalıklar ortaya çıkmaktadır. Otoimmün hastalıkların görülme sıklığı toplum genelinde %1-2 oranındadır. Birçok otoimmün hastalıkta triptofan aminoasit katabolizmasıyla ilgili teoriler ortaya atılmıştır. Triptofan aminoasit katabolizmasının başlangıç ve hız sınırlayıcı adımını gerçekleştiren İndolamin 2, 3-dioksijenaz (IDO), hücre içi bir enzimdir. IDO kullanımı sonucunda T hücre aktivasyonu baskılanırken, triptofanın katabolik ürünü olan kinürinin ve türevleri, serbest oksijen radikalleri gibi moleküller T hücre proliferasyonunu ve yaşamlarını düzenlerler. Endotel Lökosit Adezyon Molekülü- 1 (ELAM-1) lökositlerin enflamasyonlu dokulara göçünde yer alan bir molekülüdür. Normal koşullar altında endotel hücrelerde düşük düzeylerde eksprese edilir. Ayrıca endotel hücre kültürlerinde IL-1 (Interlökin-1) ve TNF (Tümör Nekroz Faktör) gibi sitokinler tarafından 4-6 saat gibi kısa bir sürede uyarılabilir. Bu tez çalışmasının amacı ELAM-1 promotor kontrolünde klonlanan IDO geninin sadece enflamasyon koşulları altında ekspresyonunu sağlamaktır. Bu amaçla hazırlanan plazmit memeli hücre hattı olan HeLa hücrelerine aktarılmış ve sitokinler aracılığı ile uyarılarak IDO aktivasyonu gözlemlenmiştir. IDO ekspresyonu ELISA, qRT-PZR ve Western blot analizleriyle gösterilmiştir. Bu çalışmanın devamında yapılması planlanan hayvan modelleme çalışmalarıyla birçok otoimmün hastalığa yeni bir gen tedavi yaklaşımı olarak kullanılıp kullanılmayacağı belirlenecektir.
Immunological tolerance is defined as the unresponsiveness of lymphocytes facing with antigen. In some pathological conditions and as a result of breaking of the immunological tolerance, the immune system becomes susceptible to its own antigens and undergoes a reaction against its own tissue antigens. Thus, immunological tolerance is impaired and autoimmune diseases arise. The prevalence of autoimmune diseases is 1-2% in the general population. In many autoimmune diseases there are theories about the tryptophan amino acid catabolism. Indolamine 2, 3-dioxygenase (IDO) is an intracellular enzyme that initiates and rate-limits the catabolism of tryptophan amino acid. As a result of IDO usage T cell activation is repressed while catabolic products of tryptophan such as kynurenine, kynurenine derivatives and free oxygen radicals, regulate T cell proliferation and survival. Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1) is a molecule which is involved in the migration of leukocytes to inflammatory tissues. Under normal conditions it is expressed at low levels in endothelial cells. It can also be stimulated with cytokines such as IL-1 (Interleukin-I beta) and TNF (Tumor Necrosis Factor) in endothelial cell cultures in a short time as 4-6 hours. The aim of this thesis is to provide expression of the IDO gene, which is cloned under ELAM-1 promoter control, during only inflammatory conditions. The plasmid that is prepared for this purpose was transferred to mammalian cell line HeLa and IDO activation was observed by stimulation with cytokines. Expression of IDO was shown by ELISA, qRT-PCR and Western blot analysis. The animal modeling studies that will be carried out in the future, will determine if we can use it as a new gene therapy approach to many autoimmune diseases or not.
Immunological tolerance is defined as the unresponsiveness of lymphocytes facing with antigen. In some pathological conditions and as a result of breaking of the immunological tolerance, the immune system becomes susceptible to its own antigens and undergoes a reaction against its own tissue antigens. Thus, immunological tolerance is impaired and autoimmune diseases arise. The prevalence of autoimmune diseases is 1-2% in the general population. In many autoimmune diseases there are theories about the tryptophan amino acid catabolism. Indolamine 2, 3-dioxygenase (IDO) is an intracellular enzyme that initiates and rate-limits the catabolism of tryptophan amino acid. As a result of IDO usage T cell activation is repressed while catabolic products of tryptophan such as kynurenine, kynurenine derivatives and free oxygen radicals, regulate T cell proliferation and survival. Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1) is a molecule which is involved in the migration of leukocytes to inflammatory tissues. Under normal conditions it is expressed at low levels in endothelial cells. It can also be stimulated with cytokines such as IL-1 (Interleukin-I beta) and TNF (Tumor Necrosis Factor) in endothelial cell cultures in a short time as 4-6 hours. The aim of this thesis is to provide expression of the IDO gene, which is cloned under ELAM-1 promoter control, during only inflammatory conditions. The plasmid that is prepared for this purpose was transferred to mammalian cell line HeLa and IDO activation was observed by stimulation with cytokines. Expression of IDO was shown by ELISA, qRT-PCR and Western blot analysis. The animal modeling studies that will be carried out in the future, will determine if we can use it as a new gene therapy approach to many autoimmune diseases or not.
Description
Keywords
Indolamin 2, 3-dioksijenaz (IDO), ELAM-1 promotor, Gen tedavisi, Klonlama, ELISA, qRT-PZR, Western blot analizi, Indolamine 2, 3-dioxygenase (IDO), Gene therapy, Clonning, qRT-PCR, Western blotting
Citation
Karaçay, M. (2017). İnsan indolamin -2,3-dioksijenaz (İDO) geninin ELAM-1 promotoru kontrolünde klonlanması ve ekspresyonu. Yayınlanmamış yüksek lisans tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.