CAJ | 학술논문

目的探讨甘精胰岛素对体外培养的人脂肪细胞脂肪因子分泌及脂质合成与分解的影响.方法原代培养人皮下前脂肪细胞,采用不同浓度的甘精胰岛素(20、200、500、1 000、1 500nmol/L)干预其分化过程,酶联免疫吸附法检测分化过程中肿瘤坏死因子α(TNF-α)、瘦素、脂联素、视黄醇结合蛋白4(RBP4)的分泌情况,比色法检测细胞内甘油三酯(TG)合成及培养基中甘油的释放量,实时定量聚合酶链反应(RT-PCR)检测脂肪分化标志基因:过氧化物酶体增殖物活化受体γ(PPAR-γ)、CCAAT促进结合蛋白α(C/EBPα)以及脂联素、RBP4、脂质代谢基因脂肪细胞脂肪酸结合蛋白(aP2)、激素敏感性酯酶(LPL)转录情况.组内比较采用配对t检验,组间比较采用.结果(1)人脂肪组织可分离出前脂肪细胞,并诱导分化为成熟的脂肪细胞,随着细胞的分化成熟,TNF-α、瘦素、脂联素、RBP4的分泌逐渐增加.(2)随着细胞的分化,细胞内TG含量逐渐增加,分化第15天时达峰值[(12.16±0.19)比(0.02 ±0.00) mmol·L-1·g-1,t=11.20,P＜0.001]；培养基中甘油含量与甘精胰岛素浓度呈正相关,1 500比500nmol/L组增高[21 d:(961±15)比(611±10) μmol/L,t =3.70,P＜0.01],与分化时间无关.(3)RT-PCR结果:PPAR-γ、C/EBPα、脂联素、RBP4、aP2、LPL基因转录水平随着分化时间的延长逐渐增加,在第15 ～21天达峰值,与分化前比较,差异均有统计学意义(均P＜0.01).结论甘精胰岛素可诱导体外培养的人前脂肪细胞的分化,促进多种脂肪细胞因子的分泌,中低浓度促进脂质合成,高浓度可诱导脂质分解.
목적 탐토감정이도소대체외배양적인지방세포지방인자분비급지질합성여분해적영향.방법 원대배양인피하전지방세포,채용불동농도적감정이도소(20、200、500、1 000、1 500nmol/L)간예기분화과정,매련면역흡부법검측분화과정중종류배사인자α(TNF-α)、수소、지련소、시황순결합단백4(RBP4)적분비정황,비색법검측세포내감유삼지(TG)합성급배양기중감유적석방량,실시정량취합매련반응(RT-PCR)검측지방분화표지기인:과양화물매체증식물활화수체γ(PPAR-γ)、CCAAT촉진결합단백α(C/EBPα)이급지련소、RBP4、지질대사기인지방세포지방산결합단백(aP2)、격소민감성지매(LPL)전록정황.조내비교채용배대t검험,조간비교채용.결과(1)인지방조직가분리출전지방세포,병유도분화위성숙적지방세포,수착세포적분화성숙,TNF-α、수소、지련소、RBP4적분비축점증가.(2)수착세포적분화,세포내TG함량축점증가,분화제15천시체봉치[(12.16±0.19)비(0.02 ±0.00) mmol·L-1·g-1,t=11.20,P＜0.001]；배양기중감유함량여감정이도소농도정정상관,1 500비500nmol/L조증고[21 d:(961±15)비(611±10) μmol/L,t =3.70,P＜0.01],여분화시간무관.(3)RT-PCR결과:PPAR-γ、C/EBPα、지련소、RBP4、aP2、LPL기인전록수평수착분화시간적연장축점증가,재제15 ～21천체봉치,여분화전비교,차이균유통계학의의(균P＜0.01).결론 감정이도소가유도체외배양적인전지방세포적분화,촉진다충지방세포인자적분비,중저농도촉진지질합성,고농도가유도지질분해.
Objective To investigate the effects of insulin glargine (IG) on adipocytokines secretion,lipid synthesis and lipidolysis of human adipocytes in vitro.Methods Primary preadipocytes were isolated from human subcutaneous adipose tissue and induced to differentiation with different dose of IG (20,200,500,1 000,1 500 nmol/L).Adipocytokines such as tumor necrosis factor-α (TNF-α),leptin,adiponectin,retinol-binding protein 4 (RBP4) were detected by enzyme-linked immunosorbent assay (ELISA) during the adipogenesis process.Accumulations of intracellular triglyceride (TG) and glycerol released in the medium were used as lipid synthesis and lipolysis index by colorimetric kits.Reverse transcriptase polymerase chain reaction(RT-PCR) was used to observe the effects of IG on adipogenic genes such as PPAR-γ,C/EBPα,adipocytokines gene adiponectin,RBP4,adipocyte fatty acid binding protein (aP2) and lipoprotein lipase (LPL).t-test and AVONA were used to analysis the effects.Results (1) Human preadipocytes could be successfully isolated from adipose tissue and induced to mature adipocytes,the secretion of adipocytokines gradually increased with the differentiation.(2) With the differentiation of preadipocytes,TG content gradually increased and reached a peak at 15th days of the differentiation ((12.16 ±0.19) vs (0.02 ±0.00) mmol · L-1 · g-1,t =11.20,P ＜0.001).Moreover,glycerol content released in the medium was closely associated with IG dose but not associated with the differentiation process,after full differentiation,1 500 nmol/L group had higher glycerol content than 500 nmol/L group ((961 ± 15) vs (611 ± 10) μmol/L,t =3.70,P ＜ 0.01).(3) RT-PCR analysis showed that gene mRNA expression were increased with the differentiation process and reached the peak at 15th to 21th,days of the differentiation.Compared with undifferentiated cells,they all obviously increased (for PPAR-γ gene P ＜0.01,other genes P ＜ 0.001).Conclusion IG can induce the differentiation and secretion of human preadipocytes in vitro,it can also stimulate lipid synthesis in low to moderate concentrations,and induce lipolysis at high concentrations.