凋亡蛋白酶活化因子1基因在脂肪细胞诱导分化中表达及肿瘤坏死因子-α对其调节作用

目的 观察3T3-L1脂肪前体细胞诱导分化过程中（0~10 d）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 1.0 ng·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Western blot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 24 h）脂肪细胞中APAF1基因的表达水平。结果 ① 3T3-L1 前体脂肪细胞诱导分化过程中（0~10 d），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；② APAF1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。

Expression of APAF1 gene during 3T3-L1 preadipocyte differentiation and regulation of tumor necrosis factor-alpha

Objective 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 3T3-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 1-4, day 6 and day 8-10 (P＞0.05). The protein levels of APAF1 (0, 2, 4, 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 (1.0 ng·mL-1) resulted in the increase of APAF1 mRNA levels with a time dependent manner. A significant increase of APAF1 mRNA was elicited by TNF-α after as short as 6 h. Compared to the 0 h, about 2.11 folds increase of APAF1 mRNA was found at 24 h while 1.52 folds at 6 h. Except for 0-2 h, there was a significant difference (P＜0.05）between any two detected phases in the levels of APAF1 mRNA. To further confirm this, we evaluated the protein levels of APAF1 and found that there was a significant difference (P＜0.05）between any two detected phases except for 6-12 h. Conclusions ① Our results confirmed that both the mRNA and protein levels were gradually decreased during 3T3-L1 preadipocyte differentiation. Down-regulated expression of APAF1 may contribute to the differentiation, maturation and adipogenesis of adipocytes. APAF1 gene may be related to the etiology of obesity;② TNF-α up-regulates the expression of APAF1 in matured adipocytes and this up-regulation may facilitate the de-differentiated effects of TNF-α on matured adipocytes.