米黄色脂肪细胞诱导分化方法研究
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摘要
目的建立米黄色脂肪细胞分化的方法并进行分析。方法通过诱导物刺激使脂肪前体细胞3T3-L1向米黄色脂肪细胞分化。细胞分化完成后经油红染色检测胞内脂滴的形成,经荧光定量PCR和Western blot检测米黄色脂肪细胞标志性蛋白Pparγ,Prdm16和Ucp1的表达,确定分化成熟的细胞是米黄色脂肪细胞。经转录测序分析脂肪前体细胞和米黄色脂肪细胞差异表达基因及KEGG pathway富集情况。结果采用本研究中的诱导分化方法得到的分化细胞油红染色阳性,米黄色脂肪细胞标志性蛋白Pparγ、Prdm16、Ucp1均高表达,转录测序显示分化后细胞与未分化细胞基因表达差异较大。结论本研究建立的米黄色脂肪细胞分化的方法准确可靠,可用于米黄色脂肪细胞形成调控机制等研究。
Objective To establish and analyze the method of inducing preadipocytes differentiation into beige adipocytes. Methods The preadipocytes cells 3 T3-L1 were differentiated into beige adipocytes cells by inducer. Oil red O stain was used to detect the formation of lip droplet. qPCR and western blot were performed to monitor the expression of beige adipocyes markers Peroxisome proliferator-activated receptor gamma (Pparγ),PR domain containing 16 (Prdm16) and Uncoupling protein 1 (Ucp1) for determining the differentiated mature cells to be beige fat cells. RNA-seq was implemented to analyze the different expression genes and KEGG pathway enrichment. Results The Oil red O stain positive and Pparγ,Prdm16,Ucp1 high expression beige adipocytes were obtained through the induction method in this study. RNA-seq showed that there were a huge number of different expression genes in beige adipocytes versus preadipocytes.Conclusion The method of inducing preadipocytes differentiation into beige adipocytes was established successfully. The inducing method can be used for the regulation mechanism study of beige adipocytes formation.
Objective To establish and analyze the method of inducing preadipocytes differentiation into beige adipocytes. Methods The preadipocytes cells 3 T3-L1 were differentiated into beige adipocytes cells by inducer. Oil red O stain was used to detect the formation of lip droplet. qPCR and western blot were performed to monitor the expression of beige adipocyes markers Peroxisome proliferator-activated receptor gamma (Pparγ),PR domain containing 16 (Prdm16) and Uncoupling protein 1 (Ucp1) for determining the differentiated mature cells to be beige fat cells. RNA-seq was implemented to analyze the different expression genes and KEGG pathway enrichment. Results The Oil red O stain positive and Pparγ,Prdm16,Ucp1 high expression beige adipocytes were obtained through the induction method in this study. RNA-seq showed that there were a huge number of different expression genes in beige adipocytes versus preadipocytes.Conclusion The method of inducing preadipocytes differentiation into beige adipocytes was established successfully. The inducing method can be used for the regulation mechanism study of beige adipocytes formation.
引文
[1]Wensveen FM,Valenti S,estan M,et al.The"Big Bang"in obese fat:events initiating obesity-induced adipose tissue inflammation[J].Eur J Immunol,2015,45(9):2446-2456.
[2]Fink J,Matsumoto M,Tamura Y.Potential application of testosterone replacement therapy as treatment for obesity and type 2diabetes in men[J].Steroids,2018,138:161-166.
[3]Tiller G,Fischer-Posovszky P,Laumen H,et al.Effects of TWEAK(TNF superfamily member 12)on differentiation,metabolism,and secretory function of human primary preadipocytes and adipocytes[J].Endocrinology,2009,150(12):5373-5383.
[4]Brestoff JR,Artis D.Immune regulation of metabolic homeostasis in health and disease[J].Cell,2015,161(1):146-160.
[5]Oelkrug R,Polymeropoulos ET,Jastroch M.Brown adipose tissue:physiological function and evolutionary significance[J].JComp Physiol B,2015,185(6):587-606.
[6]Sanchez-Gurmaches J,Hung CM,Sparks CA,et al.PTEN loss in the Myf5 lineage redistributes body fat and reveals subsets of white adipocytes that arise from Myf5 precursors[J].Cell Metab,2012,16(3):348-362.
[7]Lee MW,Odegaard JI,Mukundan L,et al.Activated type 2innate lymphoid cells regulate beige fat biogenesis[J].Cell,2015,160(1-2):74-87.
[8]Dempersmier J,Sambeat A,Gulyaeva O,et al.Cold-inducible Zfp516 activates UCP1 transcription to promote browning of white fat and development of brown fat[J].Mol Cell,2015,57(2):235-246.
[9]Ouellet V,Routhier-Labadie A,Bellemare W,et al.Outdoor temperature,age,sex,body mass index,and diabetic status determine the prevalence,mass,and glucose-uptake activity of18F-FDG-detected BAT in humans[J].J Clin Endocrinol Metab,2011,96(1):192-199.
[10]Jing S,Wu W,Huang S,et al.PKA-RIIB deficiency induces brown fat-like adipocytes in inguinal WAT and promotes energy expenditure in male FVB/NJ mice[J].Endocrinology,2017,158(3):578-591.
[11]Rosen ED,Sarraf P,Troy AE,et al.PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro[J].Mol Cell,1999,4(4):611-617.
[12]Afshin A,Forouzanfar MH,Reitsma MB,et al.Health effects of overweight and obesity in 195 countries over 25 years[J].NEngl J Med,2017,377(1):13-27.
[13]Collins KH,Herzog W,MacDonald GZ.,et al.Obesity,metabolic syndrome,and musculoskeletal disease:common inflammatory pathways suggest a central role for loss of muscle integrity[J].Front Physiol,2018,9:112-137.
[14]Stone TW,McPherson M,Gail Darlington L.Obesity and cancer:existing and new hypotheses for a causal connection[J].Ebio Med,2018,30:14-28.
[15]Brestoff JR,Artis D.Immune regulation of metabolic homeostasis in health and disease[J].Cell,2015,161(1):146-160.
[16]Shi J,Yu M,Sheng M.Angiogenesis and inflammation in peritoneal dialysis:the role of adipocytes[J].Kidney Blood Press Res,2017,42(2):209-219.
[2]Fink J,Matsumoto M,Tamura Y.Potential application of testosterone replacement therapy as treatment for obesity and type 2diabetes in men[J].Steroids,2018,138:161-166.
[3]Tiller G,Fischer-Posovszky P,Laumen H,et al.Effects of TWEAK(TNF superfamily member 12)on differentiation,metabolism,and secretory function of human primary preadipocytes and adipocytes[J].Endocrinology,2009,150(12):5373-5383.
[4]Brestoff JR,Artis D.Immune regulation of metabolic homeostasis in health and disease[J].Cell,2015,161(1):146-160.
[5]Oelkrug R,Polymeropoulos ET,Jastroch M.Brown adipose tissue:physiological function and evolutionary significance[J].JComp Physiol B,2015,185(6):587-606.
[6]Sanchez-Gurmaches J,Hung CM,Sparks CA,et al.PTEN loss in the Myf5 lineage redistributes body fat and reveals subsets of white adipocytes that arise from Myf5 precursors[J].Cell Metab,2012,16(3):348-362.
[7]Lee MW,Odegaard JI,Mukundan L,et al.Activated type 2innate lymphoid cells regulate beige fat biogenesis[J].Cell,2015,160(1-2):74-87.
[8]Dempersmier J,Sambeat A,Gulyaeva O,et al.Cold-inducible Zfp516 activates UCP1 transcription to promote browning of white fat and development of brown fat[J].Mol Cell,2015,57(2):235-246.
[9]Ouellet V,Routhier-Labadie A,Bellemare W,et al.Outdoor temperature,age,sex,body mass index,and diabetic status determine the prevalence,mass,and glucose-uptake activity of18F-FDG-detected BAT in humans[J].J Clin Endocrinol Metab,2011,96(1):192-199.
[10]Jing S,Wu W,Huang S,et al.PKA-RIIB deficiency induces brown fat-like adipocytes in inguinal WAT and promotes energy expenditure in male FVB/NJ mice[J].Endocrinology,2017,158(3):578-591.
[11]Rosen ED,Sarraf P,Troy AE,et al.PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro[J].Mol Cell,1999,4(4):611-617.
[12]Afshin A,Forouzanfar MH,Reitsma MB,et al.Health effects of overweight and obesity in 195 countries over 25 years[J].NEngl J Med,2017,377(1):13-27.
[13]Collins KH,Herzog W,MacDonald GZ.,et al.Obesity,metabolic syndrome,and musculoskeletal disease:common inflammatory pathways suggest a central role for loss of muscle integrity[J].Front Physiol,2018,9:112-137.
[14]Stone TW,McPherson M,Gail Darlington L.Obesity and cancer:existing and new hypotheses for a causal connection[J].Ebio Med,2018,30:14-28.
[15]Brestoff JR,Artis D.Immune regulation of metabolic homeostasis in health and disease[J].Cell,2015,161(1):146-160.
[16]Shi J,Yu M,Sheng M.Angiogenesis and inflammation in peritoneal dialysis:the role of adipocytes[J].Kidney Blood Press Res,2017,42(2):209-219.