CAJ | 학술논문

目的探讨人皮下、网膜脂肪组织来源的前脂肪细胞形态、功能的差异,观察不同浓度甘精胰岛素对其增殖、分化的影响.方法胶原酶分离法原代培养人皮下、网膜前脂肪细胞,倒置显微镜下观察不同来源的人前脂肪细胞在形态方面的差异,不同浓度甘精胰岛素(20、200、500、1000、1500 nmol/L)干预其增殖及分化过程,四甲基偶氮唑盐(MTT)法检测二者增殖的差异,实时定量PCR检测脂肪分化相关基因表达的差异.结果 (1)可从人皮下、网膜脂肪组织分离出前脂肪细胞,并在体外扩增,皮下前脂肪细胞更细长,容易增殖,而网膜前脂肪细胞呈多角形,易于老化.(2) MTT法结果显示甘精胰岛素抑制网膜前脂肪细胞的体外增殖,且其抑制作用具有剂量依赖性,1000 nmol/L干预72 h后,与未加胰岛素组相比,网膜前脂肪细胞的增殖明显低[吸光度(A)值:0.144±0.021比0.267±0.040,P＜0.01]；皮下前脂肪细胞作用不明显(A值:0.305±0.045比0.350±0.037,P＞0.05).(3)500 nmol/L的胰岛素浓度是人前脂肪细胞分化的适宜浓度,PCR结果显示该浓度时,对于皮下前脂肪细胞,脂肪分化的标志基因过氧化物酶增殖物激活受体(PPAR)γ(F=31.31,P＜0.01)和CCAAT/增强子结合蛋白α(C/EBPα)mRNA表达最高(F =9.86,P＜0.05),前脂肪细胞的标志基因Pref-1表达最低(皮下F=68.95,P＜0.01;网膜F=30.38,P ＜0.01),但是胰岛素浓度对网膜脂肪细胞PPARγ、C/EBPα mRNA影响不大(均P＞0.05).结论甘精胰岛素可抑制人网膜前脂肪细胞的增殖,促进皮下、网膜前脂肪细胞的分化.
목적 탐토인피하、망막지방조직래원적전지방세포형태、공능적차이,관찰불동농도감정이도소대기증식、분화적영향.방법 효원매분리법원대배양인피하、망막전지방세포,도치현미경하관찰불동래원적인전지방세포재형태방면적차이,불동농도감정이도소(20、200、500、1000、1500 nmol/L)간예기증식급분화과정,사갑기우담서염(MTT)법검측이자증식적차이,실시정량PCR검측지방분화상관기인표체적차이.결과 (1)가종인피하、망막지방조직분리출전지방세포,병재체외확증,피하전지방세포경세장,용역증식,이망막전지방세포정다각형,역우노화.(2) MTT법결과현시감정이도소억제망막전지방세포적체외증식,차기억제작용구유제량의뢰성,1000 nmol/L간예72 h후,여미가이도소조상비,망막전지방세포적증식명현저[흡광도(A)치:0.144±0.021비0.267±0.040,P＜0.01]；피하전지방세포작용불명현(A치:0.305±0.045비0.350±0.037,P＞0.05).(3)500 nmol/L적이도소농도시인전지방세포분화적괄의농도,PCR결과현시해농도시,대우피하전지방세포,지방분화적표지기인과양화물매증식물격활수체(PPAR)γ(F=31.31,P＜0.01)화CCAAT/증강자결합단백α(C/EBPα)mRNA표체최고(F =9.86,P＜0.05),전지방세포적표지기인Pref-1표체최저(피하F=68.95,P＜0.01;망막F=30.38,P ＜0.01),단시이도소농도대망막지방세포PPARγ、C/EBPα mRNA영향불대(균P＞0.05).결론 감정이도소가억제인망막전지방세포적증식,촉진피하、망막전지방세포적분화.
Objective To compare the morphological and functional differences of human primary preadipocytes from different fat depots and explore the effects of insulin glargine on their proliferation and differentiation.Methods Primary preadipocytes isolated from human subcutaneous and omental adipose tissue by collagenase I were passaged in vitro.Inverted phase contrast microscope was used to observe the morphological differences of two kinds of preadipocytes.Then two kinds of preadipocytes were cultured or induced to differentiation with different doses of insulin glargine.The methyl thiazolyl tetrazolium (MTT) assay was used to detect their proliferative differences.Reverse transcription-polymerase chain reaction (RT-PCR) was used to observe the effects of insulin on adipogenic gene expression.Results (1) Both preadipocytes could be successfully cultured from adipose tissue and amplified in vitro.Subcutaneous preadipocytes were more slender and proliferated more quickly while omental preadipocytes were polygonal and aged easily.(2) MTT results showed that insulin glargine could inhibit the proliferation of omental preadipocytes in a dose-dependent fashion.After 72 h incubation,compared with negative control,the absorbance (A) value of 1000 nmol/L insulin glargine group decreased greatly (0.144 ±0.021 vs 0.267 ±0.040,P ＜ 0.01).But it had no effect on subcutaneous preadipocytes (0.305 ± 0.045 vs 0.350 ± 0.037,P ＞ 0.05).(3) Insulin at 500 nmol/L was a suitable concentration for inducing differentiation.RT-PCR analysis showed that,for subcutaneous adipocytes,adipogenic genes such as peroxisome proliferator-activated receptor gamma (PPARγ)(F=31.31,P ＜ 0.01)and CCAAT enhancer binding protein α (C/EBPα) (F =9.86,P ＜ 0.05) had the highest mRNA expression while preadipocytic gene Pref-1 had the lowest expression at this concentration.But insulin dose had no obvious effect on PPARγor C/EBPα mRNA (P ＞ 0.05) for omental adipocytes.Conclusion Insulin glargine could inhibit the proliferation of omental preadipocytes,and enhance the differentiation of subcutaneous and omental preadipocytes.