Raw 264.7 hücre hattında CD80 ve CD86 ekspresyonun CRISPR/Cas9 gen düzenleme sistemi kullanılarak baskılanması
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Date
2019-08-29
Authors
Ardahanlı, Elif
Journal Title
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Publisher
Bursa Uludağ Üniversitesi
Abstract
İmmünolojik tolerans, bağışıklık sisteminin bir antijene yanıt vermemesi durumudur. İmmünolojik toleransın başarısızlığı otoimmünite ve otoimmün hastalıklarla sonuçlanır. Son yıllarda otoimmün hastalıkların görülme sıklığı ve prevalansında rahatsız edici bir artış vardır. Bu hastalıklar kronik yapıları, ilgili sağlık hizmetleri maliyetleri ve genç nüfuslardaki prevalansı sebebiyle önemli bir klinik problemdir. Bu çalışmada T hücrelerini uyaran profesyonel antijen sunan hücrelerden makrofajlarda, CD80 ve CD86 genlerinin susturularak, T hücrelerinde anerji oluşturulup tolerans mekanizmasının geliştirilmesi amaçlanmıştır. Çalışmada, mouse CRISPR Knockout Pooled Library kullanılarak seçilen CD80 ve CD86 genlerine özgü oligonükleotitler, LentiCRISPRv2 vektörüne klonlandı. Ligasyon yapılan CRISPR vektörlerinin virüsleri üretildi ve Raw 264.7 hücreleri bu virüslerle transdükte edildi. Bu hücreler daha sonra puromisin seleksiyonuna tabi tutuldu ve hayatta kalan hücreler, de novo sentezin baskılanıp baskılanmadığını göstermek için IFN-γ ile uyarıldı. Ardından bu hücreler CD80 ve CD86 monoklonal antikorlarıyla muamele edilerek akan hücre ölçerde okutulup analizleri yapıldı. IFN-γ ile uyarımı takiben yapılan akan hücre ölçer sonuçlarında, kontrol vektörüyle trandükte edilen hücrelerle benzer şekilde gen düzenlemesi yapılan makrofajlarda CD80 ve CD86 ekspresyonlarında artış görülmüş olup, beklenen CD80-CD86 susturulması gerçekleşmemiştir. Otoimmün hastalıklara karşı tolerans mekanizmalarının tetiklenmesine yönelik tedavi denemeleri yapılmakta ve olumlu sonuçlar alınmaktadır. Özellikle CD80 ve CD86 ekspresyonunu baskılanması üzerine yapılmış çalışmalar yeni olup bizim çalışmamız da bu denemelere örnektir. Çalışmamızda CD80-CD86 genleri susturulamasa da, sorunu çözmeye yönelik ileri çalışmalarda, CRISPR/Cas9 sistemi ile eş uyaran sinyalini baskılamaya yönelik yaklaşımlar alternatif tedavi yöntemi olarak yerini alabilir.
Immunological tolerance is a condition in which the immune system does not respond to an antigen. Failure of immunological tolerance results in autoimmunity and autoimmune diseases. There has been a disturbing increase in the incidence and prevalence of autoimmune diseases in recent years. These diseases are important clinical problems due to their chronic structure, related health care costs and prevalence in young populations. In this study, it was aimed to improve tolerance mechanism by forming anergy in T cells by silencing CD80 and CD86 genes in macrophages which are one of professional antigen presenting cells that stimulate T cells. In study, the oligonucleotides specific to CD80 and CD86 genes selected by using Mouse CRISPR Knockout Pooled Library were cloned into LentiCRISPRv2 vector. Viruses of ligated CRISPR vectors were generated and Raw 264.7 cells were transduced with these viruses. These cells were then subjected to puromycin selection and the surviving cells were stimulated with IFN-γ to show whether de nova synthesis was suppressed. These cells were then threated with CD80 and CD86 monoclonal antibodies and read and analyzed by flow cytometry. In flow cytometry results following IFN-γ stimulation showed an increase in CD80 and CD86 expression in macrophages transduced with CRISPR vector similar to cells transduced with the control vector, expected CD80-CD86 silencing did not occur. Treatment attempts are being made to trigger tolerance mechanisms against autoimmune diseases and positive results are obtained. In particular studies on the suppression of CD80 and CD86 expression are new and our study is an example of these attempts. Although CD80-CD86 genes could not be silenced in our study, in further studies aimed at solving the problem, approaches to suppress the co-stimulatory signal with CRISPR/Cas9 system may be used as an alternative treatment method.
Immunological tolerance is a condition in which the immune system does not respond to an antigen. Failure of immunological tolerance results in autoimmunity and autoimmune diseases. There has been a disturbing increase in the incidence and prevalence of autoimmune diseases in recent years. These diseases are important clinical problems due to their chronic structure, related health care costs and prevalence in young populations. In this study, it was aimed to improve tolerance mechanism by forming anergy in T cells by silencing CD80 and CD86 genes in macrophages which are one of professional antigen presenting cells that stimulate T cells. In study, the oligonucleotides specific to CD80 and CD86 genes selected by using Mouse CRISPR Knockout Pooled Library were cloned into LentiCRISPRv2 vector. Viruses of ligated CRISPR vectors were generated and Raw 264.7 cells were transduced with these viruses. These cells were then subjected to puromycin selection and the surviving cells were stimulated with IFN-γ to show whether de nova synthesis was suppressed. These cells were then threated with CD80 and CD86 monoclonal antibodies and read and analyzed by flow cytometry. In flow cytometry results following IFN-γ stimulation showed an increase in CD80 and CD86 expression in macrophages transduced with CRISPR vector similar to cells transduced with the control vector, expected CD80-CD86 silencing did not occur. Treatment attempts are being made to trigger tolerance mechanisms against autoimmune diseases and positive results are obtained. In particular studies on the suppression of CD80 and CD86 expression are new and our study is an example of these attempts. Although CD80-CD86 genes could not be silenced in our study, in further studies aimed at solving the problem, approaches to suppress the co-stimulatory signal with CRISPR/Cas9 system may be used as an alternative treatment method.
Description
Keywords
CRISPR/Cas9, Otoimmün hastalıklar, İmmün tolerans, Eş uyaran molekülleri, Autoimmun disease, Immune tolerance, Co-stimulatory molecules
Citation
Ardahanlı, E. (2019). Raw 264.7 hücre hattında CD80 ve CD86 ekspresyonun CRISPR/Cas9 gen düzenleme sistemi kullanılarak baskılanması. Yayınlanmamış yüksek lisans tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.