PPAR-gamma信号通路对Hem-MSCs体外成脂分化的影响
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摘要
目的研究PPAR-gamma信号通路对血管瘤来源的间充质干细胞(Hemangioma derived mesenchymal stem cells,Hem-MSCs)成脂分化的影响。方法本研究以不同的条件培养基培养Hem-MSCs,实验分5组:普通培养基组(A组)、普通培养基+罗格列酮组(B组)、成脂诱导分化培养基组(C组)、成脂诱导分化培养基+罗格列酮组(D组)及成脂诱导分化培养基+罗格列酮+GW9662拮抗剂组(E组)。光镜下观察各组Hem-MSCs的成脂分化过程,以油红O染色观察被诱导细胞中脂滴的面积和数量;采用Western-blot分析各组Hem-MSCs培养2周后的perilipin A表达量,以量化各组细胞脂肪分化程度;以实时定量PCR分析各组成脂分化相关基因(如CEBPA、LPL、PLIN1、PPARG等)在HemMSCs成脂诱导分化过程中不同时间点的表达情况。结果油红O染色显示,成脂诱导分化培养基+罗格列酮组可促进Hem-MSCs的成脂分化过程;Western-blot证实罗格列酮能明显促进成脂诱导分化时perilipin A蛋白的表达;实时定量PCR进一步证实,在诱导分化的第1周及第2周时,罗格列酮对Hem-MSCs的成脂分化相关基因(CEBPA、LPL、PLIN1、PPARG等)的表达均有促进作用;而PPAR-gamma信号通路的拮抗剂GW9662能够阻断上述作用。结论激活PPARgamma信号通路可促进Hem-MSCs在体外的成脂分化能力。
Objective To investigate the effect of PPAR-gamma signaling pathway in the adipogenic differentiation of hemangioma derived mesenchymal stem cells(Hem-MSCs) in vitro. Methods Hem-MSCs were cultured in 5 groups of different media, including group A(ordinary culture medium), group B(ordinary culture medium +rosiglitazone), group C(adipogenic medium), group D(adipogenic medium+rosiglitazone), group E(adipogenic medium+rosiglitazone+GW9662). The adipogenic differentiation of hem-MSCs was observed by light microscope, and the range and number of lipid droplets were observed by oil red O staining. The expression of perilipin A in each group was detected by Western-blot after two weeks of culture to quantify the degree of adipogenic differentiation. The expressions of adipogenesis-related genes(CEBPA, LPL,PLIN1, PPARG) of each group at different time points were detected by real-time quantitative PCR. Results Oil red O staining showed that rosiglitazone could promote the adipogenic differentiation of hem-MSCs in adipogenic medium.Western-blot confirmed that rosiglitazone could significantly promote the expression of perilipin A protein in the induced differentiation group. Real-time quantitative PCR further confirmed that rosiglitazone could promote the expression of adipogenesis-related genes(CEBPA, LPL, PLIN1, PPARG) in hem-MSCs at the first and second weeks. GW9662, an antagonist of PPAR-gamma signaling pathway, could block the above effects. Conclusion Activated PPAR-gamma signaling pathway promotes the adipogenic differentiation of Hem-MSCs in vitro.
Objective To investigate the effect of PPAR-gamma signaling pathway in the adipogenic differentiation of hemangioma derived mesenchymal stem cells(Hem-MSCs) in vitro. Methods Hem-MSCs were cultured in 5 groups of different media, including group A(ordinary culture medium), group B(ordinary culture medium +rosiglitazone), group C(adipogenic medium), group D(adipogenic medium+rosiglitazone), group E(adipogenic medium+rosiglitazone+GW9662). The adipogenic differentiation of hem-MSCs was observed by light microscope, and the range and number of lipid droplets were observed by oil red O staining. The expression of perilipin A in each group was detected by Western-blot after two weeks of culture to quantify the degree of adipogenic differentiation. The expressions of adipogenesis-related genes(CEBPA, LPL,PLIN1, PPARG) of each group at different time points were detected by real-time quantitative PCR. Results Oil red O staining showed that rosiglitazone could promote the adipogenic differentiation of hem-MSCs in adipogenic medium.Western-blot confirmed that rosiglitazone could significantly promote the expression of perilipin A protein in the induced differentiation group. Real-time quantitative PCR further confirmed that rosiglitazone could promote the expression of adipogenesis-related genes(CEBPA, LPL, PLIN1, PPARG) in hem-MSCs at the first and second weeks. GW9662, an antagonist of PPAR-gamma signaling pathway, could block the above effects. Conclusion Activated PPAR-gamma signaling pathway promotes the adipogenic differentiation of Hem-MSCs in vitro.
引文
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[11]赵哲媛,郭亮.周细胞参与婴幼儿血管瘤消退机制的研究进展[J].组织工程与重建外科杂志,2014,10(4):222-224.
[12]Siersbaek R,Nielsen R,Mandrup S.Transcriptional networks and chromatin remodeling controlling adipogenesis[J].Trends Endocrinol Metab,2012,23(2):56-64.
[2]袁斯明,姜会庆,欧阳天祥,等.婴幼儿血管瘤病理结构变化与临床演变过程的联系[J].中国美容整形外科杂志,2006,17(5):388-391.
[3]Khan ZA,Boscolo E,Picard A,et al.Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice[J].J Clin Invest,2008,118(7):2592-2599.
[4]Yuan SM,Chen RL,Shen WM,et al.Mesenchymal stem cells in infantile hemangioma reside in the perivascular region[J].Pediatr Dev Pathol,2012,15(1):5-12.
[5]Gerhold DL,Liu F,Jiang G,et al.Gene expression profile of adipocyte differentiation and its regulation by peroxisome proliferator-activated receptor-gamma agonists[J].Endocrinology,2002,143(6):2106-2118.
[6]Yanik SC,Baker AH,Mann KK,et al.Organotins are potent activators of PPARγand adipocyte differentiation in bone marrow multipotent mesenchymal stromal cells[J].Toxicol Sci,2011,122(2):476-488.
[7]袁斯明,陈荣亮,王修坤,等.婴幼儿血管瘤间充质干细胞的分离鉴定及其在血管瘤组织中的定位观察[J].中国美容整形外科杂志,2011,22(3):178-181.
[8]鞠大鹏,詹丽杏.脂肪细胞分化及其调控的研究进展[J].中国细胞生物学学报,2010,32(5):690-695.
[9]Keshet R,Bryansker Kraitshtein Z,Shanzer M,et al.c-Abl tyrosine kinase promotes adipocyte differentiation by targeting PPAR-gamma2[J].Proc Natl Acad Sci U S A,2014,111(46):16365-16370.
[10]Yu Y,Fuhr J,Boye E,et al.Mesenchymal stem cells and adipogenesis in hemangioma involution[J].Stem Cells,2006,24(6):1605-1612.
[11]赵哲媛,郭亮.周细胞参与婴幼儿血管瘤消退机制的研究进展[J].组织工程与重建外科杂志,2014,10(4):222-224.
[12]Siersbaek R,Nielsen R,Mandrup S.Transcriptional networks and chromatin remodeling controlling adipogenesis[J].Trends Endocrinol Metab,2012,23(2):56-64.