Serap ERDEM KURUCA, Dolay Damla ÇELİK, Gülderen DEMİREL, Dilşad ÖZERKAN

İNSAN PERİFERİK KANINDAN ÇOK KÜÇÜK EMBRİYONİK (VSEL) KÖK HÜCRELERİN ELDE EDİLMESİ VE TANIMLANMASI

Amaç: Kök hücre (KH) nakli malin kan hastalıkları, kemik iliği yetmezlikleri ve doğumsal kan hastalıklarının tedavisinde kullanılan yöntemdir. Bu amaçla, periferik kan projenitör hücreleri (PKPH) oldukça fazla kullanılmaktadır. Son yıllarda insan embriyonik kök hücrelerinin sahip olduğu pluripotent özelliği bulunan yeni KH türü tanımlanmıştır. Çok küçük embriyonik benzeri (VSEL) kök hücreler olarak adlandırılan bu hücrelerin, yetişkin bireyde periferik kanda (PK) bulunduğu düşünülmektedir. Bu çalışmada VSEL kök hücrelerinin periferik kan kaynağı kullanarak elde edilmesi ve tanımlanması amaçlanmıştır. Gereç ve Yöntem: Donörlerden alınan materyallerden lizis ve fikol gradient yöntemleri kullanılarak elde edilen mononükleer ve eritrosit katmanınlarından, VSEL hücreleri izole edilmiştir. Flov sitometri ve immünfloresan boyama ile NANOG, OCT4, SSEA-4 ve CXCR-4 embriyonal kök hücre belirteçlerinin varlığı incelenmiştir. Western blot yöntemiyle ise, NANOG ve OCT4 proteinlerinin varlığı araştırılmıştır. Bulgular: Flov sitometri sonuçlarına göre debris katmanında, VSEL belirteci taşıyan hücre sayısı CD45- popülasyonuna göre daha yüksek bulunmuştur. Elde edilen western ve immünfloresan sonuçlarına göre yüksek miktarda OCT4 ve NANOG ekspresyonu görülmüştür. Aynı zamanda bu proteinlerin hücre içinde hem sitoplazmada hem de çekirdekte bulunduğu gösterilmiştir. Sonuç: Pluripotent kök hücre belirteci olarak bilinen bu proteinlerin yetişkin periferik kanında yüksek miktarda ekspresyonunun, farklılaşmış dokularda nasıl görev yaptığı sorusunu akıllara getirmektedir. Bu bulgular; periferik kanda pluripotent belirteçler taşıyan yeni bir kök hücre populasyonu bulunduğu tezini güçlendirmekte ve bu konuda yapılacak ileriye dönük klinik çalışmalar içinde temel oluşturmaktadır.

Anahtar Kelimeler:

Çok küçük embriyonik benzeri (VSEL) kök hücreler, Periferik kan, Pluripotent kök hücre markırları

CHARACTERIZATION AND ISOLATION OF VERY SMALL EMBRYONIC-LIKE (VSEL) STEM CELLS OBTAINED FROM HUMAN PERIPHERAL BLOOD

Objective: Stem cell transplantation is considered to be one of the available treatments for malign or hereditary blood diseases and bone marrow failure. Peripheral blood progenitor cells (PBPC) are widely used for this technique. Recently a new type of stem cell with a pluripotent potential has been identified. These cells, called very small embryonic-like (VSEL) stem cells, are thought to be found in peripheral blood (PB) in adult individuals. The aim of this study was to obtain and identify VSEL stem cells using a peripheral blood source. Material and Method: VSEL cells were isolated from mononuclear and erythrocyte layers obtained by using lysis and ficoll gradient methods from the materials taken from donors. The presence of NANOG, OCT4, SSEA-4 and CXCR-4 embryonal stem cell markers by flow cytometry and immunofluorescence staining was investigated. The presence of NANOG and OCT4 proteins was investigated using the Western blot method. Results: According to the flow cytometry results, the number of cells carrying the VSEL marker was higher in the debris layer than in the CD45-population. Western and immunofluorescence results showed high levels of OCT4 and NANOG expression. It was also determined that these proteins are present in both the cytoplasm and the nucleus. Conclusions: High expression of these proteins, known as pluripotent stem cell markers, in adult peripheral blood brings to mind the question of how they work in differentiated tissues. These findings strengthen the thesis of a new stem cell population that have pluripotent markers in peripheral blood and provide the basis for future clinical studies about this point.

Keywords:

Very Small Embryonic-like (VSEL) stem cells, Peripheral blood, Pluripotent stem cell markers,

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