摘要
目的探索过氧化物酶体增殖物激活受体γ(PPAR-γ)激动剂吡格列酮(PGZ)对肥胖小鼠非肾上腺素能条件下原代棕色脂肪分化和功能的影响,为2型糖尿病和肥胖的治疗提供新依据。方法高脂饮食诱导的4周龄雄性C57BL/6J肥胖小鼠20只,取肩胛间区棕色脂肪组织,分离并培养C57小鼠原代棕色脂肪细胞,等量分人10个培养皿中,等分为对照组和PGZ干预组,每组5皿,使用PGZ干预细胞,建立PGZ干预的细胞模型,使用和PGZ溶液等量的生理盐水处理对照组细胞。实时定量聚合酶链反应(RT-PCR)检测细胞模型的棕色脂肪基因:解偶联蛋白1(UCP-J)、超长链脂肪酸延长酶3(ELOVL3)、PPAR-γ共激活因子α和β(PGCI-α、PGCI-β)、PR包含域16区(PRDM16)、CCAAT增强子结合蛋白β(CEBP/β)、脂联素、脂肪细胞脂质结合蛋白2(AP2)、细胞色素C1氧化酶(CYC1)、线粒体转录因子A(TFAM)等表达水平;使用油红染色定量法检测细胞模型的棕色脂肪成脂功能;Western blotting法检测PGZ干预组UCP-1蛋白表达。2组间比较采用t检验,多组间比较采用方差分析和LSD检验。结果PGZ干预组细胞的棕色脂肪特异基因(UCP-1、ELOVL3、PGCI-α、PGCI-β)、成脂基因(AP2)、线粒体功能基因(CYCI、TFAM)和脂肪分化基因(PRDM16、CESP/β)表达量均显著高于对照组(相对表达量分别为:UCP-1:1100.0±612.0、2.0±0.4;ELOVL3:1461.0±617.0、2.0±1.2;PGCI-α:8.1±2.8、2.0±1.1;PGCI-β:8.3±2.8、2.0±1.3;脂联素:2.6±0.8、1.04±0.7;AP2:5.1±2.2、1.00±0.24;CYCI:3.1±0.8、1.0±0.4;TFAM:1.2±0.4、1.00±0.25;PRDM16:4.8±2.6、2.0±0.3;CEBP/β:6×10^8±5×10^8、2.0±0.6;t=2.45~5.22,均P〈0.05);油红定量亦发现干预组成脂功能高于对照组(染色定量:1.2±0.2比1.0±0.1,t=2.45,P〈0.05)。Westernblotting检测PGZ干预组UCP-1表达高于对照组(灰度分析相对定量分别为1.24±0.25和1.00±0.14,t=2.63,P〈0.05)。结论PGZ可能通过增强棕色脂肪特异基因表达、促进细胞分化成脂、提高线粒体功能等方面增强棕色脂肪细胞功能,这可能是其改善机体代谢的原因之一。
Objective To investigate the effect of peroxisome hyperplasia activated receptor γ (PPAR-γ) agonist(PGZ) on the differentiation and function of nonadrenergic primary brown adipose tissue cells in mice with high fat diet-induced obesity, and try to provide new treatment for obesity and type 2 diabetes. Methods Primary BAT cells were separated from high fat diet-induced C57BL/6J obesity(HFD) mice and cultured in DMEM culture medium. The primary BAT cells were divided into 10 culture dishes equally, and assigned as PGZ treatment group, and control group( treated with normal saline), 5 dishes each group. The effects of PGZ on BAT cells were assessed by quantitative real-time polymerase chain reaction (RT-PCR), Gene of BAT: uncoupling protein-1 (UCP-1); elongase of very long chain fatty acids(ELOVL3) ; PPAR-γ co-activator-1α (PGCI-α) ; PPAR-γ co-activator-1β (PGCI-β) ; PRD1-BF-1-RIZ1 homologous domain containing protein-16 ( PRDM16 ) ; CCAAT/enhancer binding protein β( CEBP/β ) ; adiponectin : adipocyte fatty acid-binding protein2 ( AP2 ) ; cytochrome c oxidasel ( CYCI ) ; mitoehondrial transcription factor A (TFAM). Western blotting was used to assess the expression of UCP-1 protein; and Oil red O staining was applied to measure the adipogenesis function of BAT. The t test is used between two groups, ANOVA and LSD test is used between multiple groups. Results The expression of mRNA associated with BAT chararcteristie genes ( UCP-1, ELO VL3, PGCI-α, PGCI-β ), lipogeneic genes ( AP2 ), mitoehondrial genes( CYC1, TFAM), and differential genes (PRDM16, CEBP/β) increased in PGZ treated group when compared with those in control group, the relative expression of the genes in the two groups were listed as following : UCP-1 : 1100. 0 ± 612. 0, 2. 0 ± 0. 4 ; ELOVL3 : 1461.0 ± 617. 0,2. 0 ± 1.2 ; PGCI-α : 8.1 ±2.8,2.0±1.1;PGCI-β: 8.3 ±2.8,2.0 ± 1.3;adiponectin:2.6 ±0.8,1.0 ±0.7;AP2:5.1 ±2.2, 1.00 ±0. 24 ;CYC1:3.1 ±0. 8,1.0 ±0.4;TFAM:1. 2 ±0. 4,1.00 ±0. 25 ;PRDM16:4. 8 ±2. 6,2.0 ±0. 3 ; CEBP/β:6 ×10^8 ±5 ×10^8,2.0_±0.6; t =2.45 - 5.22,ali P 〈0.05; the expression of BAT functional protein UCP-1 assessed by Western blotting increased in PGZ treated group than that in control ( 1.24 ± 0. 25 vs 1.00 ±0. 14, t = 2.63, P 〈 0. 05 ) ; the function of BAT adipogenesis measured by oil red 0 test increased inPGZ treated group than that in control (1.2±0.2 vs 1.0 ±0.1, t =2.45, P〈0.05). Conclusion PPAR-γ agonist-pioglitazone (PGZ) may promote the differentiation and function of brown adipocytes. This effect may be one of the reasons for PGZ improving metabolism.
出处
《中华糖尿病杂志》
CAS
CSCD
2013年第3期149-154,共6页
CHINESE JOURNAL OF DIABETES MELLITUS