Anti-CD2 antikor kaplanmış Wharton Jeli kaynaklı mezenkimal kök hücrelerinin uyarılmış CD3+ T lenfositlerine etkisinin incelenmesi
Investigation the effect of Anti-CD2 antibody coated Wharton's Jelly derived mesencyhmal stem cells on stimulated CD3+ T lymphocytes
- Tez No: 847535
- Danışmanlar: PROF. DR. YUSUFHAN YAZIR
- Tez Türü: Yüksek Lisans
- Konular: Allerji ve İmmünoloji, Biyomühendislik, Moleküler Tıp, Allergy and Immunology, Bioengineering, Molecular Medicine
- Anahtar Kelimeler: Human Wharton's Jelly Mesenchymal Stem Cell, Paracrine, Immunoregulatory, CD2, CD3+ T lymphocytes
- Yıl: 2023
- Dil: Türkçe
- Üniversite: Kocaeli Üniversitesi
- Enstitü: Sağlık Bilimleri Enstitüsü
- Ana Bilim Dalı: Kök Hücre Ana Bilim Dalı
- Bilim Dalı: Kök Hücre ve Doku Yenilenmesi Bilim Dalı
- Sayfa Sayısı: 72
Özet
Amaç: Bu çalışmada, anti-CD2 antikorunun hücre membran elemanı olacak şekilde yağ asitleriyle birlikte aktarılmasıyla geliştirilecek insan Wharton jeli mezenkimal kök hücrelerinin (iWJ-MKH), fitohemaglutinle (PHA) uyarılmış CD2+/CD3+ T lenfositlerine bağlanma oranının artırılması amaçlanmaktadır. Anti-CD2, CD3+ T hücre yüzey belirteçlerinden biri olan CD2'yi (LFA2) hedefleyen bir antikordur. MKH'lerde olmayan anti-CD2 yüzey belirtecini hücrelere aktarılmasıyla MKH/T-lenfosit hücre etkileşimini artırılması sağlanacaktır. Böylece MKH'lerinin immün modülatör özelliklerinin geliştirilmesi ulaşılmak istenen bir çıktı olacaktır. Yöntem: In vitro kültür ortamında yapılan tez çalışmasında öncelikli olarak kaplamanın hücreler üzerindeki toksik etkisi WST-8 testi ile değerlendirilmiş ve iWJ-MKH'leri palmitlenmiş protein G (PPG) ve anti-CD2 ile kaplanmıştır. Kaplama ve T hücresi izolasyonları sonrasında 48 saatlik ko-kültür gerçekleştirilmiştir. Bu ikili hücre kültüründe kaplamanın hücreler üzerindeki etkinin incelenmesi için WST-8, canlı-ölü, CFSE, DCFDA, CD3 akış sitometrisi analizleri ve Real Time PCR tekniği ile gen ifade analizleri yapılmıştır. Bu analizler ile ikili kültürde bulunan T lenfositlerinin canlılık, proliferasyon, ROS oluşumuna katkısı ve periferik kan mononükleer hücrelerde (PBMC) bulunma miktarları incelenmiştir. Bulgular: MKH'lerin karakterizasyonu yapılmış ve CD73, CD105, CD90 (+) belirteçleri pozitiflik gösterirken, negatif belirteçler olan CD3, CD45, HLA-DR, CD34 ve CD2'de okuma alınamamıştır. Kaplama için PPG'nin %80 canlılığı koruyan kültürdeki dozu 20 µg/ml olarak belirlenmiştir (P
Özet (Çeviri)
Objective: In this study, it is aimed to increase the binding rate of human Wharton's Jelly mesenchymal stem cells (iWJ-MSC), which will be developed by transferring anti-CD2 antibody together with fatty acids as a cell membrane element, to phytohemagglutin (PHA)-stimulated CD2+/CD3+ T lymphocytes. Thus, it is desired to improve the effectiveness of these developed MSCs against CD3+ T lymphocytes stimulated by paracrine effect. Anti-CD2 is an antibody that targets one of the CD3+ T cell surface markers, CD2 (LFA2). By transferring the anti-CD2 surface marker, which is not involve in MSCs, to the cells, the MSC/T-lymphocyte cell interaction will be increased. Thus, improving the immune modulatory properties of MSCs will be a desired outcome. Method: In the thesis study carried out in in vitro culture environment, primarily the toxic effect of the coating on cells was evaluated with the WST-8 test and iWJ-MSCs were coated with PPG and anti-CD2. After coating and T cell isolations, 48 hours of co-culture was performed. WST-8, live-dead, CFSE, DCFDA, CD3 flow cytometry analyzes and gene expression analyzes with Real Time PCR were performed for the interactions of the coating between cells in cell culture. With these analyses, the viability, proliferation, contribution of T lymphocytes in culture to ROS formation and their amount in peripheral blood mononuclear cells (PBMC) were examined. Results: MSCs have been characterized and CD73, CD105, CD90 (+) markers were proven to be positive, while the negative markers CD3, CD45, HLA-DR, CD34 and CD2 were negative. The dose of PPG in the culture, which maintains 80% viability for coating, was determined as 20 µg/ml. Purifications of commercially available anti-CD2 and PPG (IgG1) antibodies were performed by FPLC method. CD25, CD2 markers of the isolated T cells were determined before and after PHA stimulation. There increase in CD25 and CD2 markers after stimulation with PHA. It was determined that there were CD3+T cells in the range of 70-90% in the groups formed by CD3 flow cytometry antibody staining. In the CFSE staining performed within the scope of the thesis study, the intensity increased in the group with the coated MSCs compared to the group without coating. In DCFDA staining irradiation, the irradiation intensity was decreased in the coated group compared to the other groups. In the WST-8 viability test, it was found that the % viability decreased in the groups with the coated MSCs compared to the uncoated group. With live-dead staining, dead and live cells in groups were detected under fluorescent microscopy. As a result of gene expression analysis; differentiation markers was found that the cell did not undergo any differentiation after plating and immune suppression properties increased and proliferation did not change. Antioxidant enzyme gene expressions were determined that the antioxidant defense system to reduce reactive oxygen species was activated and the stress began to decrease in the co-culture with coated MSCs. Conclusions: iWJ-MSCs were coated with anti-CD2 antibody conjugated with palmitated protein G (PPG), and viability, proliferation and metabolic activities of T lymphocytes after coating were examined. The coated group decrease in the amount of ROS compared with the uncoated group. In the group with only CD2, the amount of ROS is higher than the group in which the coating is applied. Besides these findings, MSC coating decreased T cell proliferation. The group in which the coating was made contains 88% CD3+ T cells. In the two data sets examined, the group with the coated MSCs decreased in % viability. Coated MSCs activated antioxidant defense system, which is responsible for reducing reactive oxygen, and the coating doesn't affect MSC differentiation, its potency is preserved.
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