OX-LDL İLE NÖTROFİL AKTİVASYONU SIRASINDA OLUŞAN DNA HASARININ KOMET ASSAY İLE DEĞERLENDİRİLMESİ

Year 2018, Volume: 4 Issue: 4, 183 - 189, 27.12.2018

Fatma Behice Serinkan-cinemre Leyla Sevinc Birsen Aydemir Hakan Cinemre

Abstract

Amaç İnsan vücudunda normal metabolizma ürünü olarak ya da ekzojen kaynaklarla oluşturulan reaktif oksijen türleri (ROT) ve diğer serbest
radikallerin, çok sayıda fi zyolojik ve patolojik olayda yer aldığı bilinmektedir. Bu reaktif türler eşleşmemiş elektrona sahip oldukları için,
hücrenin pek çok bileşeni, DNA, protein ve lipid içerikleriyle kolaylıkla reaksiyona girerek, normal hücre fonksiyonlarını bozabilme
yeteneğindedir. Bu çalışmada, oksidan stres modeli olarak seçilen nötrofi llerin okside olmuş düşük dansiteli lipoprotein (ox-LDL) ile
aktive edilmesi sonucu oluşan hasarı Komet Assay ile göstermeyi amaçladık.
Gereç ve
yöntemler
Bu çalışmada LDL, “short-run ultracentrifugation” yöntemiyle izole edildikten sonra CuCl� ile okside edildi. Ficoll-Hypaque gradientcentrifi ıgation” yöntemi ile de tam kandan izole edilen nötrofi ller ox-LDL ile sitümüle edildi. %1 LMP-agoraz içinde yatay elektroforezde
yürütüldü ve propidyum iyodidle boyanarak, fl oresan mikroskopu (Leica-DMLB) ile değerlendirildi. Sonuçlar, sayılan 100 hücreden
komet oluşturmuş hücrelerin yüzdesi olarak bildirildi.
Bulgular Komet yöntemi ile Ox-LDL ile muamele edilen hücrelerde Komet oluşumu %30,0 ± 4,6 olarak tespit edildi. Aynı oran kontrol hücre
grubunda %5,2 ± 1,9 olarak bulundu. Bu sonuçlara göre ox-LDL ile indüklenen oksidatif stres durumunda Komet oluşturan hücrelerin
yüzdesinin belirgin olarak arttığı gözlendi (p<0.001). Aynı hücre gruplarında tripan mavisi ile tespit edilen hücre canlılığı %53,4 ± 3,9
(oksidan stres durumunda); %84,7± 3,2 (kontrol grubunda) olarak belirlendi (p<0.001).
Sonuç Nötrofi llerin ox-LDL ile muameleye aktivasyonla yanıt verdiği ve bunun sonucu DNA hasarında istatistiksel olarak anlamlı bir yükselme
olduğu Komet yöntemi ile göstermek mümkün olmuştur. Bu DNA hasarının nötrofi llerin doğal immünitedeki koruyucu fonksiyonlarının
aksine kronik infl amatuar hastalıklar, kanser oluşumu gibi pek çok hastalığın etyopatogenezindeki rollerini açıklamada önemli olacaktır. 

Keywords

ox-LDL, nötrofi ller, DNA hasar, Komet Assay

Evulation of DNA Damage with Comet Assoy During Neutrophil Activation with OX-LDL

Abstract

Aim As a product of normal metabolism in the human body or produced from exogenous sources, reactive oxygen species (ROS) and other
free radicals involved in a large number of physiological and pathological events in human body. Because of their unmatched electrons,
these reactive species can easily react with many components of the cell such as DNA, proteins and lipids, and disrupt normal cell
functions. In this study, the neutrophils activated by oxidized low-density lipoprotein (ox-LDL) were selected as the model of oxidant
stress. We aimed to show the DNA damage after neutrophil activation by using Comet Assay
Material
and
Methods
In this study, LDL was oxidized with CuCl2 after isolatation by sonra short-run ultracentrifugation. Neutrophils were isolated from whole
blood by “Ficoll-Hypaque gradient-centrifugation” method and activated with ox-LDL. After activation they applied into LMP-agorase(%
1) for electrophoresis. After staining with propidium, iodide they evaluated for Comet formation under the fl uorescence microscopy
(Leica-DMLB). The results were reported as a percentage of cells in the form of comet.
Results Comet formation were %30,0 ± 4,6 and %5,2 ± 1,9 in cells treated with Ox-LDL and control, respectively. According to these results,
the percentage of Comet-forming cells in the case of oxidative stress induced by ox-LDL was signifi cantly increased (p<0.001). Cell
viability detected by trypan blue in the same cell groups were %53,4 ± 3,9 (In the case of oxidant stress); %84,7± 3,2 (in the control
group) (p<0.001).
Conclusion By using Comet Assay, it was possible to demonstrate that neutrophils responded to activation by treatment with ox-LDL, which
resulted in a statistically signifi cant increase in DNA damage. This DNA damage will be important in explaining the role of neutrophils
in the etiopathogenesis of many diseases, such as chronic infl ammatory diseases, cancer formation, as opposed to their protective
functio ns in natural immunity.

Keywords

ox-LDL, neutrophils, DNA damage, Comet Assay

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