摘要
目的观察3T3-L1脂肪前体细胞诱导分化过程中（0～10d）凋亡蛋白酶活化因子1（APAF1）基因表达水平的变化趋势，探讨肿瘤坏死因子-α（TNF-α）对成熟脂肪细胞中APAF1基因表达水平的调节作用。方法体外培养3T3-L1脂肪前体细胞，通过油红O染色鉴定其分化成熟的基础上，应用人重组TNF-α．1．0ng·mL^-1干预分化成熟的脂肪细胞，抽提脂肪细胞总RNA和总蛋白后，采用RT-PCR及Westernblot技术检测诱导分化不同时段及TNF-α干预后不同时间（2、6、12和24h）脂肪细胞中APAF1基因的表达水平。结果①3T3-L1前体脂肪细胞诱导分化过程中（0～10d），APAF1基因表达水平呈现逐渐减低的趋势；②人重组TNF-α对成熟脂肪细胞中APAF1基因的表达具有显著的增强作用，且TNF-α对APAF1基因表达的上调作用呈现随刺激时间延长而明显增强的总体趋势。结论①在3T3-L1前体脂肪细胞分化过程中，APAF1基因的表达逐渐下调可能有利于脂肪细胞的分化成熟和脂质积聚；②APAF1基因在人重组TNF-α刺激成熟脂肪细胞过程中呈明显上调趋势，这种上调可能具有协同TNF-α促进成熟脂肪细胞去分化的作用。
Objectlve This study was to investigate the changes of APAF1 （ apoptotic protease activating factor 1 ） gene expression during 3T3-L1 preadipocyte differentiation （ 0 - 10 d ）, and to analyze the regulation of TNF-α on APAF1 gene expression in matured 3T3-L1 adipocytes. Methods 3T3-L1 preadipocytes were cultured in vitro and differentiated into matured adipocytes by adding of insulin, 3-isobutyl-1-methyxanthine （ MIX）, and dexamethasone （DEX）. Oil Red O staining was used to identify the matured adipocytes. To further evaluate the linkage between APAF1 gene and differentiation or de-differentiation, TNF- α which can promote the matured adipocytes to de-differentiation was used to interfere 3T3-L1 matured adipocytes. TNF-alpha （ 1.0 ng-mL^-1 ） was added to the culture medium of differentiated 333-L1 cells （day 10） for various periods （2, 6, 12, 24 h）. Total RNA and protein of these cells were then extracted. The levels of APAF1 mRNA was evaluated by RT-PCR. Western-Blot was used to detect the protein levels of APAF1. Results ① In preadipocytes, the levels of APAF1 gene mRNA remained higher. In the presence of DEX, MIX and insulin, 3T3-L1 preadipocytes were gradually differentiated into matured adipocytes. In preadipocytes （day 0）, lipid droplets were rarely visible, while many more lipid droplets were visible in matured adipocytes （day 10 ）.② The levels of APAF1 mRNA was down-regulated and reached the lower level in fully differentiated adipocytes. Compared to the day 0, approximately 55% decrease of APAF1 mRNA expression was found in day 10. There was a significant difference between any two detected phases in the levels of APAF1 mRNA （ P 〈 0. 05 ）, except for at the stage of day 1 - g, day 6 and day 8 - 10 （ P 〉 0. 05 ）. The protein levels of APAF1 （0, 2, g, 6, 8,10 d） were detected and found that, with the differentiation, there was a similar decreasing trend in protein levels except for day 2 - 6. ③Treatment of day 10 3T3-L1 adipocytes with TNF-α （
出处
《中国循证儿科杂志》
CSCD
2007年第1期38-42,共5页
Chinese Journal of Evidence Based Pediatrics
基金
国家自然科学基金(30371502)
江苏省自然科学基金(BK2001120)
关键词
凋亡蛋白酶活化因子1基因
脂肪细胞分化
肿瘤坏死因子-Α
Apoptotic protease activating factor 1 gene
Adipocyte differentiation
Tumor necrosis factor-alpha
