Hayriye SARIKAYA, Elif ÖNDER, Nazlı ÇİL, Ergun METE, Gülçin ABBAN METE

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

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

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,

PDF

___

1. Matic I, Antunovic M, Brkic S, et al. Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation. Open Access Maced J Med Sci 2016;4:9-16. https://doi.org/10.3889/oamjms.2016.008
2. Alves H, Munoz Najar U, De Wit J, et al. A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells. J Cell Mol Med 2010;14:2729-2738. https://doi.org/10.1111/j.1582-4934.2009.00931.x
3. Jang YY, Sharkis SJ. A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche. Blood 2007;110:3056-3063. https://doi.org/10.1182/blood-2007-05-087759 4. López Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell 2013;153:1194-1217. https://doi.org/10.1016/j.cell.2013.05.039
5. Lees H, Walters H, Cox LS. Animal and human models to understand ageing. Maturitas 2016;93:18-27. https://doi.org/10.1016/j.maturitas.2016.06.008
6. Durmaz C, Şen E. Kök hücrelerde DNA hasarı ve onarımı. Tıp Fakültesi Klinikleri Dergisi 2022;5:19-26. https://doi.org/10.17932/IAU.TFK.2018.008/tfk_v05i1003
7. Bengal E, Perdiguero E, Serrano AL, Muñoz Cánoves P. Rejuvenating stem cells to restore muscle regeneration in aging. F1000Res 2017;6:76. https://doi.org/10.12688/f1000research.9846.1
8. Rossi DJ, Bryder D, Zahn JM, et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci USA 2005;102:9194-9199. https://doi.org/10.1073/pnas.0503280102
9. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. The aging of hematopoietic stem cells. Nat Med 1996;2:1011-1016. https://doi.org/10.1038/nm0996-1011
10. Sudo K, Ema H, Morita Y, Nakauchi H. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med 2000;192:1273-1280. https://doi.org/10.1084/jem.192.9.1273
11. Biteau B, Hochmuth CE, Jasper H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut. Cell Stem Cell 2008;3:442-455. https://doi.org/10.1016/j.stem.2008.07.024
12. Nakada D, Oguro H, Levi BP, et al. Oestrogen increases haematopoietic stem-cell self-renewal in females and during pregnancy. Nature 2014;505:555-558. https://doi.org/10.1038/nature12932
13. Ün B, Ferah MA, Ün BÇ. Mezenkimal kök hücre ve koşullandırılmış besiyerinin ovaryum hasarı üzerindeki tedavi edici etkileri. SDÜ Tıp Fakültesi Dergisi 2021;28:179-185 https://doi.org/10.17343/sdutfd.654926
14. Igarashi M, Guarente L. mTORC1 and sırt1 cooperate to foster expansion of gut adult stem cells during calorie restriction. Cell 2016;166:436-450. https://doi.org/10.1016/j.cell.2016.05.044
15. Zhou S, Zilberman Y, Wassermann K, Bain SD, Sadovsky Y, Gazit D. Estrogen modulates estrogen receptor alpha and beta expression, osteogenic activity, and apoptosis in mesenchymal stem cells (MSCs) of osteoporotic mice. J Cell Biochem Suppl 2001;36:144-155 https://doi.org/10.1002/jcb.1096
16. Brinckmann M, Kaschina E, Altarche Xifró W, et al. Estrogen receptor alpha supports cardiomyocytes indirectly through post-infarct cardiac c-kit+ cells. J Mol Cell Cardiol 2009;47:66-75. https://doi.org/10.1016/j.yjmcc.2009.03.014
17. Oh WJ, Jacinto E. mTOR complex 2 signaling and functions. Cell Cycle 2011;10:2305-2316. https://doi.org/10.4161/cc.10.14.16586
18. Tee AR, Manning BD, Roux PP, Cantley LC, Blenis J. Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr Biol 2003;13:1259-1268. https://doi.org/10.1016/s0960-9822(03)00506-2
19. Martin SK, Fitter S, Dutta AK, et al. Brief report: the differential roles of mTORC1 and mTORC2 in mesenchymal stem cell differentiation. Stem Cells 2015;33:1359-1365. https://doi.org/10.1002/stem.1931
20. Kim E, Goraksha Hicks P, Li L, Neufeld TP, Guan KL. Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 2008;10:935-945. https://doi.org/10.1038/ncb1753
21. Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair--current views. Stem Cells 2007;25:2896-2902. https://doi.org/10.1634/stemcells.2007-0637
22. Bell A, Gagnon A, Grunder L, Parikh SJ, Smith TJ, Sorisky A. Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts. Am J Physiol Cell Physiol 2000;279:335-340. https://doi.org/10.1152/ajpcell.2000.279.2.C335
23. Cho HJ, Park J, Lee HW, Lee YS, Kim JB. Regulation of adipocyte differentiation and insulin action with rapamycin. Biochem Biophys Res Commun 2004;321:942-948. https://doi.org/10.1016/j.bbrc.2004.07.050
24. El Chaar D, A. Gagnon, and A. Sorisky. Inhibition of insulin signaling and adipogenesis by rapamycin: effect on phosphorylation of p70 S6 kinase vs eIF4E-BP1. Int J Obes Metab Disord 2004;28:191-198. https://doi.org/10.1038/sj.ijo.0802554
25. Zhang HH, Huang J, Düvel K, et al. Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway. PLoS One 2009;47:6189. https://doi.org/10.1371/journal.pone.0006189
26. Isomoto S, Hattori K, Ohgushi H, Nakajima H, Tanaka Y, Takakura Y. Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells. J Orthop Sci 2007;121:83-88. https://doi.org/10.1007/s00776-006-1079-9
27. Lee KW, Yook JY, Son MY, et al. Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway. Stem Cells Dev 2010;19:557-568. https://doi.org/10.1089/scd.2009.0147
28. Bitto A, Ito TK, Pineda VV, et al. Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. Elife 2016;5:16351. https://doi.org/10.7554/eLife.16351
29. Chen C, Liu Y, Liu Y, Zheng P. mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells. Sci Signal 2009;298:75. https://doi.org/10.1126/scisignal.2000559
30. Johnson SC, Rabinovitch PS, Kaeberlein M. mTOR is a key modulator of ageing and age-related disease. Nature 2013;493:338-345. https://doi.org/10.1038/nature11861
31. Ylmaz ÖH, Katajisto P, Lamming DW, et al. mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake. Nature. 2012;486:490-495. https://doi.org/10.1038/nature11163
32. Igarashi M, Guarente L. mTORC1 and SIRT1 cooperate to foster expansion of gut adult stem cells during calorie restriction. Cell 2016;166:436-450. https://doi.org/10.1016/j.cell.2016.05.044
33. Weichhart T, Hengstschläger M, Linke M. Regulation of innate immune cell function by mTOR. Nat Rev Immunol 2015;15:599-614. https://doi.org/10.1038/nri3901
34. Sukhbaatar N, Hengstschläger M, Weichhart T. mTOR-mediated regulation of dendritic cell differentiation and function. Trends Immunol 2016;37:778-789. https://doi.org/10.1016/j.it.2016.08.009
35. Araki K, Ellebedy AH, Ahmed R. TOR in the immune system. Curr Opin Cell Biol 2011;236:707-715. https://doi.org/10.1016/j.ceb.2011.08.006
36. Ogawa T, Tokuda M, Tomizawa K, et al. Osteoblastic differentiation is enhanced by rapamycin in rat osteoblast-like osteosarcoma (ROS 17/2.8) cells. Biochem Biophys Res Commun 1998;249:226-230. https://doi.org/10.1006/bbrc.1998.9118