猪DFAT细胞成脂再分化过程中KLF2和PPARγ的表达

doi:10.11843/j.issn.0366-6964.2017.01.008

摘要/Abstract

摘要：

旨在探讨猪去分化脂肪细胞（DFAT）再分化过程中Krüppel样因子2（KLF2）和过氧化物酶体增殖物激活受体γ（PPARγ）的表达模式，为揭示猪DFAT细胞再分化机制奠定基础。采集猪成熟脂肪细胞，经“天花板”培养法获得DFAT细胞，用流式细胞仪检测表面抗原，形态学和油红O染色提取法检测成脂分化程度，Real-time PCR检测KLF2和PPARγ mRNA的表达。结果表明，猪成熟脂肪细胞接种第3天，可见明显的去分化迹象，即细胞形成犄角状突起，大脂滴分解成小脂滴并向细胞外排出脂滴，接种至第9天脂滴基本排出完毕，接种至第12天原代细胞长满底壁；间充质干细胞表面抗原CD29、CD44和CD105的阳性表达率分别达到99.0%、97.8%和99.5%，而造血干细胞表面抗原CD45和CD34的表达仅为3.55%和4.55%；将F3代细胞成脂诱导3 d，胞质中出现脂滴，并随诱导时间增加，脂滴数量增多，体积增大，诱导至12 d诱导率达80%以上；成脂诱导2、5、10和15 d时，KLF2 mRNA的相对表达量分别为0.47±0.02、0.35±0.07、0.31±0.09和0.11±0.07，PPARγ mRNA的相对表达量分别为1.62±0.01、2.03±0.04、3.22±0.04和3.49±0.06。本研究成功获得高纯度的DFAT细胞，具有间充质干细胞特性和成脂再分化能力；KLF2 mRNA的表达在成脂分化过程中随诱导时间的增加而减少，而PPARγ mRNA的表达量随诱导时间的延长逐步增加；表明KLF2在DFAT细胞的成脂再分化过程中可能发挥抑制作用，而PPARγ可能会发挥促进作用。

Abstract:

To investigate the expression patterns of Kruppel like factor 2 (KLF2) and peroxisome proliferator activated receptor gamma (PPARγ) during the adipogenic redifferentiation of porcine dedifferentiated fat (DFAT) cells and lay a foundation for revealing the mechanism of porcine DFAT cell redifferentiation, we applied the “ceiling” culture to obtain porcine DFAT cells from mature adipocytes, detected the surface antigens of DFAT cells by flow cytometry, and then, identified the adipogenic differentiation of DFAT cells by methods of morphology, oil red O staining and real-time PCR. The results showed that the dedifferentiation sign was clearly observed, showing that the horn like protrusions were formed, large lipid droplets were decomposed into small lipid droplets, and lipid droplets were extracellularly discharged after porcine mature adipocytes were seeded on the third day, the lipid droplets were almost discharged in 9 day, and the bottom wall was covered with primary cells in 12 day. The positive rates of mesenchymal stem cell surface antigen CD29, CD44 and CD105 were 99.0%, 97.8% and 99.5%, respectively, while the percentages of hematopoietic stem cell surface antigen CD45 and CD34 were only 3.55% and 4.55%. Then, after 3 days of adipogenic induction of F3 generation DFAT cells, the spindle shaped cells became wider and shorter, and lipid droplets were observed in the cytoplasm. With the increase of induction time, the number and volume of lipid droplets increased, and the induction efficiency was more than 80% after 12 day induction. During the progress of adipogenic induction, the relative expression levels of KLF2 were 0.47±0.02, 0.35±0.07, 0.31±0.09 and 0.11±0.07, and PPARγ expression levels were 1.62±0.01, 2.03±0.04, 3.22±0.04 and 3.49±0.06 on the 2, 5, 10 and 15 days, respectively. In conclusion, porcine DFAT cells with high purity were successfully obtained, owning the mesenchymal stem cell properties and the adipogenic redifferentiation competence, and, KLF2 expression levels decreased, while PPARγ expression increased with the extension of induction time during adipogenic redifferentiation of DFAT cells, indicating that KLF2 might play an inhibitory role, while PPARγ could take on a positive role during the adipogenic redifferentiation of DFAT cells.

中图分类号:

S828.2

高霞，李方正，郇延军，刘志敏，李秀，姜忠玲，牛德勋，宋学雄. 猪DFAT细胞成脂再分化过程中KLF2和PPARγ的表达[J]. 畜牧兽医学报, 2017, 48(1): 68-74.

GAO Xia, LI Fang-zheng, HUAN Yan-jun, LIU Zhi-min, LI Xiu, JIANG Zhong-ling, NIU De-xun, SONG Xue-xiong. The Expression Patterns of KLF2 and PPARγ during the Adipogenic Redifferentiation of Porcine DFAT Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(1): 68-74.

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参考文献

［1］HAUSMAN G,DONDSON M V,AJUWON K,et al. Board-invited review: the biology and regulation of preadipocytes and adipocytes in meat animals［J］.J Anim Sci, 2009,87（4）:1218-1246.
［2］WEI S J,ZAN L S,HAUSMAN G J,et al. Dedifferentiated adipocyte-derived progeny cells (DFAT cells) Potential stem cells of adipose tissue［J］. Adipocyte, 2013,2(3):122-127.
［3］WEI S,DUART M S,ZAN L,et al. Cellular and molecular implications of mature adipocyte dedifferentiation［J］. Genomics,2012,1:5-12.
［4］MATSUMOTO T,KANO K,KONDO D. Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential［J］. J Cell Physiol, 2008, 215（1）:210-222.
［5］KALLAPAT T,YUICHI T H, ISHIKAWA S S. Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells［J］.Human Cell,2014,27(4):151-161.
［6］NAOTAKA K,YOSHIHIRO M,YOSHIYA H,et al.Dedifferentiated fat cells differentiate into osteoblasts in titanium fiber mesh［J］.Cytotechnology，2013，65(1):15-22.
［7］CHEN J,GRIDI M,FEMYHOUGH M E. Initial differences in lipid processing leading to pig- and beef-derived mature adipocyte dedifferentiation［J］.Basic Appl Myol,2009,19: 243-246.
［8］CHEN J,DODSON M V,JIANG Z. Cellular and molecular comparison of redifferentiation of intramuscular- and viscer al-adipocyte derived progeny cells［J］.Int J Biol Sci, 2010,6(1):80-88.
［9］WEI S J,DU M,JIANG Z H,et al.Bovine dedifferentiated adipose tissue (DFAT) cells: DFAT cell isolation［J］.Adipocyte,2013,2(3)：148-159.
［10］NOBUSUE H,ENDO T,KANO K. Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue［J］.Cell Tissue Res,2008, 332（3）: 435-446.
［11］PEARSON R,FLEETWOOD J,EATON S,et al.Kruppel-like transcription factors :a functional family［J］.Int J Biochem Cell Biol, 2008,40(10):1996-2001.
［12］BREY C W, NELDER M P, 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.
［13］刘京霞,李方正,宋学雄，等. 猪脂肪源间充质干细胞诱导成脂分化及Krüppel样因子2的表达［J］.解剖学报,2015,46（5）:616-622.
LIU J X,LI F Z,SONG X X, et al. Regulation of porcine adipose mesenchymal stem cells into adipocytes and the expression of KLF2 transccription factors［J］.Acta Anatomica Sinica,2015, 46（5）:616-622.（in Chinese）
［14］VAN R L,BAYLISS C E,RONCARI D A. Cytological and enzymological characterization of adult human adipocyte precursors in culture［J］. J Clin Inves,1976,58（3）:699-704．
［15］MIYAZAKI T,KJTAGAWA Y,TORIYAM A.Isolation of two human fibroblastic cell populations multiple but distinct potential of mesenchymal differentiation by ceiling culture of mature fat cells from subcutaneous adipose tissue ［J］.Differentiation, 2005，73(2-3):69-78.
［16］YAGI K,KONDO D,OKAZAKI Y,et al. A novel preadipocyte cell line established from mouse adult mature adipocytes［J］.Biochem Biophys Res Commun,2004，321(4)：967-974.
［17］SUN W,WANG H,LI Y,et al.Acquisition of pig intramuscular preadipocytes through dedifferentiation of mature adipocytes and establishment of optimal induction conditions［J］.Genet Mol Res,2013,12（4）: 5926-5936.
［18］SUGIHARA H,YONEMITSU N,MIYABARA S.Primary cultures of unilocular fat cells: characteristics of growth in vitro and changes in differentiation properties［J］.Differentiation,1986，31（1）: 42-49.
［19］SHEN J F,SUGAWARA A,YAMASHITA J,et al.Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues ［J］.Int J Oral Sci，2011，3（3）:117-124.
［20］JUMABAY M,ZHANG R,YAO Y.Spontaneously beating cardiomyocytes derived from white mature adipocytes［J］.Cardiovasc Res,2010，185（1）: 17-27.
［21］JUMABAY M,MATSUMOTO T,YOKOYAMA S. Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats［J］.J Mol Cell Cardiol,2009, 47（5）: 565-575.
［22］SONG N, KOU L, LU X W, et al. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes［J］.Biochem Biophys Res Commun,2014, 444（4）:543-548.
［23］ATSUNORI S,SOH S.Application of dedifferentiated fat cells for periodontal tissue regeneration［J］.Human Cell, 2014,27（1）: 12-21.
［24］WANG P,RENES J.Absence of an dipogenic effect of rosiglitazone on mature 3T3-L1 adipocytes: increase of lipid catabolism and reduction of adipokine expression［J］.Diabetologia,2007, 50（3）:654-665.
［25］BANERJEE S S,FEINBERG M W,WATAANBE M,et al.The Kruppel-like factor KLF2 inhibits peroxisome proliferator-activated receptor-gamma expression and adipogenesis［J］.J Biol Chem, 2003，278(4):2581-2584.
［26］JING H W,SEETHA V S,JERRY B L.The KLF2 transcription factor does not affect the form action of preadipoeytes but inhibits their diferentiation into adipocytes［J］.Biochemistry, 2005,44（48）:98-105.
［27］PENG X,SONG T,HU X,et al.Phenotypic and functional properties of porcine dedifferentiated fat cells during the long-term culture in vitro［J］.Biomed Res Int,2015,67:36-51.
［28］张庆美，李方正，宋学雄,等.猪骨髓间充质干细胞分离培养和诱导分化脂肪细胞［J］.中国兽医学报,2015,35(1):91-96.
ZHANG Q M, LI F Z, SONG X X, et al.Isolation, culturation and differentiation of porcince bone marrow mesenchymal stem cells into adipocytes［J］.Chinese J Vet.Sci,2015,35(1):91-96. （in Chinese）
［29］ZUK P A, ZHU M, MIZUNO H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies［J］.Tissue Engineering,2001,7（2）: 211-228.
［30］JAMES E W,NIKETA A P,GAY C, et al. Comparison of markers and functional attributes of human adipose-derived stem cells and dedifferentiated adipocyte cells from subcutaneous fat of an obese diabetic donor［J］.Adv Wound Care,2014,13（3）:219-228.

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