Leyla TÜRKER ŞENER, Aycan BAŞ, İbrahim KALELİOĞLU, Suzan AYDIN ÇINAR, İşıl ALBENİZ

HEMATOPOETIK KÖK HÜCRE İZOLASYONUNDA ALDH ETKİNLİĞİNİN ÖNEMİ

Amaç: Kan hücrelerine kolaylıkla farklılaşabilen hematopoetik kök hücrelerin (HKH) saflaştırmasında tercih edilen yöntem, flow sitometrik veya magnetik ayırım sistemi ile CD34+ hücrelerin seçimidir. İnsan kök hücrelerini tamamen fenotipik hücre yüzey belirteçlerine dayanmadan etkin olarak saptayan metodlara ihtiyaç vardır. Bu metodlardan biri retinoid metabolizması ve HKH’lerin siklofosfamid gibi alkilleyici ajanlara direncinde rol oynayan sitozolik aldehid dehidrogenaz (ALDH)’dır. Farklı fonksiyonları olan kordon kanından klinik uygulamalarda kullanılabilen en primitif insan hematopoetik kök ve progenitör hücrelerin daha yüksek saflıkta eldesinin CD34+CD38- yüzey belirteçlerinin yanı sıra ALDH ile de işaretlenerek akım sitometri analizi sonrası ALDH+ hücrelerin diğer hücrelerden ayrılarak olabileceğinin belirlenmesi amaçlanmıştır. Hematopoetik hücre serilerine farklılaştırılma çalışmaları için sadece CD34+ hücrelerin seçilmesinin yeterli olmadığı ALDH+ ve CD38- negatifliğin de analiz edilmesi amaçlanmıştır.Gereç ve yöntemler: Biz çalışmamızda CD34 yüzey belirteci olan hücreleri kordon kanından magnetik nanopartiküller kullanarak ayırdıktan sonra flow sitometre cihazıyla, bu hücrelerin içerisinden ALDH etkinliği olup CD38 yüzey antijeni olmayan hücreleri saflaştırdık. Bu hücre gruplarını floresan mikroskobu ile görüntüledik.Bulgular: Yaptığımız bu çalışmada kordon kanından saflaştırdığımız CD34+ hücrelerin ALDH-FITC işaretlenmeleri sonucunda aldığımız floresan mikroskop ve flow sitometrik (facs) analizinde CD34+ olan hücrelerin ALDH+ olduğu görüldü. Sonuç: Bu çalışmamızın sonucunda kordon kanından hematopoetik kök hücre izolasyonunda ALDH+ hücre grubu CD34+CD38- hücrelerden zengin olduğu ve hücre kültüründe koloni oluşturma özelliğine sahip olduğu görülmüştür. Kordon kanından hematopoetik kök hücre izolasyonunda yalnızca CD34+ olmasının yeterli olmadığı ALDH+ ve CD38- negatifliğin de analiz edilmesi gerektiği düşünülmektedir. Anahtar kelimeler: Hematopoetik kök hücre; kordon kanı; ALDH; CD34.

Anahtar Kelimeler:

Hematopoetik kök hücre, kordon kanı, ALDH, CD34

IMPORTANCE OF ALDH ACTIVITY IN HEMATOPOIETIC STEM CELL ISOLATION

Objective: The preferred method for the purification of hematopoietic stem cells (HSCs), which can easily differentiate into blood cells, is the selection of CD34+ cells by flow cytometry or magnetic discrimination. Methods are needed that effectively determine human stem cells without entirely relying on phenotypic cell surface markers. One of these methods is cytosolic aldehyde dehydrogenase (ALDH), which plays a role in retinoid metabolism and resistance of HSCs to alkylating agents such as cyclophosphamide. The prospective isolation of human hematopoietic stem and progenitor cells with different functions, as well as surface markers, are also recommended by ALDH and flow cytometry. In the above findings, ALDH+ cells can be isolated from other cells and the most primitive hematopoietic stem cells can be isolated and used in clinical applications. Materials and methods: In our study, we separated the CD34 surface marker cells from cord blood using magnetic nanoparticles and then purified the cells with ALDH activity and non-CD38 surface antigen by flow cytometer. We visualized these cell groups with fluorescence microscopy.Results: We observed that the majority of CD34+ cells were ALDH+ in fluorescence microscopy and flow cytometry (FACS) analysis of CD34+ cells purified from cord blood as a result of ALDH-FITC markings.Conclusion: As a result of this study, it was found that the ALDH+ cells group is rich in CD34+ CD38- cells in hematopoietic stem cells isolated from cord blood, and is capable of colony formation in cell culture. It is suggested that in hematopoietic stem cell isolation from cord blood, only CD34+ cells are not sufficient and ALDH+ and CD38- negativity should also be analyzed. Keywords: Hematopoietic stem cells; cord blood; ALDH; CD34.

Keywords:

Hematopoietic stem cells, cord blood, ALDH, CD34,

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