Differentiation of porcine adipose mesenchymal stem cells into adipocytes and the expression of Krüppel like factor 2

doi:10.16098/j.issn.0529-1356.2015.05.007

Abstract

Abstract:

Objective To investigate the gene expression changes of Krüppe-1 like factor (KLF)2 during the process of induced adipogenic differentiation from porcine adipose mesenchymal stem cells(AMSCs) in vitro and to reveal the foundation for regulating the differentiation of adipocyte. Methods The porcine AMSCs were isolated by typeⅠcollagen enzyme method and subcultured. F3 culture cell surface antigen was detected by flow cytometry. The differentiated adipocytes were identified by morphology and oil red O staining; The expression of KLF2 mRNA and adipogenic marker genes proliferator-activated receptor γ(PPARγ)2 were measured by Real-time PCR. Results The AMSCs expressed the MSCs cellular markers, CD29,CD44 and CD105 with a marking rate a of 91.7%,95.1% and 95.6% respectively,but did not express CD34 with a marking rate of 3.39%. Some adipocytes appeared when the AMSCs induced and differentiated for 2 days. When the induced differentiation time extended, the number of adipocytes increased. The adipogenic conversion rate reached 48.2% in the induction of 18 days. In the induction of 2,8 and 16 days, the expressions of KLF2 mRNA were 1.84±0206,1.07±0.072 and 0.83±0.095,and the expressions of PPARγ2 mRNA were 3.06±0.542,13.22±0.5736 and 15.11±1.073. Conclusion This study successfully obtained AMSCs which showed good adipogenic differentiation potential.The expressions of KLF2 was increased in early adipogenic differentiation phase, and then gradually reduced.The results suggest that the KLF2 is the negative regulation of transcription factors.

Key words: Adipose mesenchymal stem cell, Surface antigen, Induction, Differentiation, Krüppe-1 like factor 2, Real-time PCR, Porcine

LIU Jing-xia LI Fang-zheng PIAO Ying-ji JIANG Zhong-ling SONG Xue-xiong*. Differentiation of porcine adipose mesenchymal stem cells into adipocytes and the expression of Krüppel like factor 2[J]. AAS, 2015, 46(5): 616-622.

References

［1］Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses［J］. Blood, 2005, 105(4):1815-1822.
［2］Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal stem cells: nature, biology, and potential applications［J］. Stem Cells, 2001, 19(3): 180-192.
［3］Gronthos S, Franklin DM, Leddy HA, et al. Surface protein characterization of human adipose tissue-derived stromal cells［J］. J Cell Physiol, 2001, 18(9):54-63.
［4］Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement ［J］. Cytotherapy, 2006, 8(4): 315-317.
［5］Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells ［J］. Proc Natl Acad Sci USA, 2001, 98 (14):7841.
［6］Brey CW, Nelder MP, Hailemariam T, et al. Krüppel-like family of transcription factors: an emerging new frontier in fat biology［J］. Int J Biol Sci, 2009, 5(6): 622-636.
［7］Gray S, Feinberg MW, Hull S, et al. The Krüppel-like factor KLF15 regulates the insulin sensitive glucose transporter GLUT4［J］. Biol Chem, 2002, 277(9): 34322-34328.
［8］Banerjee SS, Feinberg MW, Watanabe M, et al. The Krüppel-like factor KLF2 inhibits peroxisome proliferator-activated receptor-γ expression and adipogenesis［J］. J Biol Chem, 2003, 278(4): 2581-2584.
［9］Qu ChQ. Isolation, culture and multipotential of porcine adipose mesenchymal stem cells in vitro［D］. Northwest Agriculture and Forestry University of Science and Technology,2006. (in Chinese)
屈长青. 猪脂肪间充质干细胞的分离培养及体外诱导分化研究［D］. 西北农林科技大学, 2006.
［10］Farmer SR. Transcriptional control of adipocyte formation ［J］.Cell Metab, 2006, 4(4):263-273
［11］Rosen ED, Walkey CJ, Puigserver P, et al. Transcriptionalregulation of adipogenesis［J］. Genes Dev, 2000, 14(11):1293-1307.
［12］Wu Z, Wang S. Role of kruppel-like transcription factors in adipogenesis［J］. Dev Biol, 2013, 373(2):235-243.
［13］Eisenstein A, Carroll SH, Johnston-Cox H. An adenosine receptor Krüppel-like factor 4 protein axis inhibits adipogenesis［J］. J Biol Chem, 2014, 289(30): 21071-21081.
［14］Tahmasebi S, Ghorbani M, Savage P, et al. Sumoylation of Krüppel-like factor 4 inhibits pluripotency induction but promotes adipocyte differentiation［J］. J Biol Chem, 2013, 288(3): 12791-12804.
［15］Yang X, Li QCh, Wu HH, et al. KLF2 and KLF15 genes expressed in the dynamic hBMSCs directional differentiation ［J］. Basic Medicine and Clinical, 2012, 32(8):900-905. (in Chinese)
杨珣, 李秋晨, 吴欢欢, 等. KLF2 和 KLF15基因在 hBMSCs定向分化中的动态表达［J］. 基础医学与临床, 2012, 32(8):900-905.
［16］Zhang GH, Qu ChQ, Yang GSh.Culture and adipogenesis differentiation of porcine adipose mes-enchymal stem cells［J］.Acta Zoologica Sinica, 2006, 52(5): 934-941. (in Chinese)
张国华, 屈长青, 杨公社. 猪脂肪间充质干细胞的分离培养及其成脂分化［J］. 动物学报, 2006, 52(5): 934941.

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