Adipoz dokudan izole edilen mezenkimal kök hücrelerin donör yaşına bağlı olarak mTOR aktivitesinin belirlenmesi

Öz

Amaç: Canlı organizmalarda yaşlanma kaçınılmaz bir fizyolojik sonuçtur. Hücresel yaşlanma sadece farklılaşmasını tamamlamış hücrelerde değil, kök hücrelerde de meydana gelir. Rapamisin protein kompleksinin memeli hedefi (mTOR), hücre büyümesinde ve metabolizmasında önemli bir role sahiptir. Hücre çoğalmasında önemli rol oynayan mTOR, aynı zamanda hücresel yaşlanmayı düzenler ve biyoenerjetik altyapıyı yönlendirir. Çalışmanın amacı, donör yaşına bağlı olarak yağ dokusundan elde edilen mezenkimal kök hücrelerin (MSC) mTOR ekspresyonunu belirlemektir. Gereç ve yöntem: Altı haftalık pubertal sıçanlar Grup 1 (n=6), 10-12 haftalık reprodüktif dönem sıçanları Grup 2 (n=6) ve 20 aylık sıçanlar Grup 3 (n=6) olarak isimlendirildi. Gruplardan alınan yağ dokusundan primer eksplant kültür metodu ile MSC izolasyonu yapıldı. Elde edilen MSC'de karakterizasyon ve farklılaşma deneyleri yapıldı. Kök hücrelerdeki mTOR aktivitesi (mTORC1 ve mTORC2) qRT-PCR yöntemi ile belirlendi. Caspase 3, 8, 9, Bax ve Bcl-2 ekspresyonları Real-time polymerase chain reaction (qRT-PCR) yöntemi ile değerlendirildi. Bulgular: Çalışmamızda apoptotik belirteçlerin en yüksek ekspresyonunun Grup 1'de, en düşük ekspresyonun Grup 2'de olduğu belirlendi. mTOR ekspresyonu değerlendirildiğinde, mTORC1 en yüksek Grup 2'de, en düşük Grup 1'de bulundu. Grup 1'deki mTORC2 ifadesi diğer gruplara göre daha düşüktü. Grup 3'teki mTORC1 ve mTORC2 ekspresyonu Grup 2'deki kadar yüksek olmasa da istatistiksel olarak anlamlıydı (p<0.05). Sonuç: Bu çalışmada hem mTORC1 hem de mTORC2'nin donör yaşına bağlı olarak kök hücrelerde farklı şekilde eksprese edildiğini bulduk. Bu farkın fonksiyonel sonuçlarını daha iyi anlamak için daha fazla çalışmaya ihtiyaç vardır.

Anahtar Kelimeler

• Yaşlanma
• yağ doku
• mTOR
• mezenkimal kök hücreler

Determination of mTOR activity depending on donor age of mesenchymal stem cells isolated from adipose tissue

Abstract

Purpose: Aging in living organisms is an inevitable physiological consequence. Cellular senescence occurs not only in cells that have completed their differentiation, but also in stem cells. Mammalian target of Rapamycin protein complex (mTOR) has an important role in cell growth and metabolism. The mTOR, which plays an important role in cell proliferation, also regulates cellular aging and directs the bioenergetic infrastructure. The aim of the study is to determine the mTOR expression of mesenchymal stem cell (MSC) obtained from adipose tissue depending on the donor age. Materials and methods: Six-week-old pubertal rats were named Group 1 (n=6), 10-12-week-old reproductive period rats were named Group 2 (n=6), and 20-month-old rats were named Group 3 (n=6). The isolation of MSC was performed by primary explant culture method from adipose tissue taken from groups. Characterization and differentiation experiments were performed in MSC obtained. The activity of mTOR (mTORC1 and mTORC2) in MSC was determined by qRT-PCR method. Caspase 3, 8, 9, Bax and Bcl-2 expressions were evaluated by Real-time polymerase chain reaction (qRT-PCR) method. Results: In our study, it was determined that the highest expression of apoptotic markers was in Group 1 and the lowest expression was in Group 2. When mTOR expression was evaluated, mTORC1 was found to be highest in Group 2 and lowest in Group 1. mTORC2 expression in Group 1 was lower than in other groups. Although the expression of mTORC1 and mTORC2 in Group 3 was not as high as in Group 2, it was statistically significant (p<0.05). Conclusion: In this study, we found that both mTORC1 and mTORC2 are differentially expressed in stem cells depending on donor age. Further studies are needed to better understand the functional consequences of this difference.

Keywords

• Aging
• adipose tissue
• mTOR
• mesenchymal stem cells

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