TSG-6基因在3T3-L1脂肪细胞诱导分化中表达水平的变化及TNF-α对其调节作用的研究

目的观察3T3-L1脂肪细胞诱导分化过程中TSG-6基因mRNA表达水平的变化,探讨TNF-α对成熟脂肪细胞中TSG-6基因表达水平的调节作用.方法体外培养3T3-L1脂肪细胞,在诱导3T3-L1脂肪细胞分化成熟的基础上,应用重组TNF-α干预分化成熟的脂肪细胞,采用RT-PCR技术检测诱导分化不同时段及TNF-α干预后不同时间脂肪细胞中TSG-6基因的表达水平.结果 (1)随脂肪细胞逐渐分化成熟,TSG-6 基因mRNA表达水平逐渐升高.TSG-6 基因表达水平除在细胞分化第0～2天、第3～5天、第4～6天和第7～10天各时段内差异无显著意义(P>0.05)外,其余各时段之间表达水平,差异均有显著意义(P＜0.05);(2)不同浓度重组TNF-α(0.1～10.0 ng/ml)均能显著抑制成熟脂肪细胞中TSG-6基因mRNA的表达,除外TNF-α浓度1.0 ng/ml时6～24 h时段,其抑制作用呈现随TNF-α浓度增加和刺激时间延长而明显增强的总体趋势.TNF-α浓度为0.1 ng/ml时,TSG-6基因表达水平6 h内下降33.73%,12 h内下降97.39%;TNF-α浓度为1.0 ng/ml时,TSG-6基因表达水平6 h内下降78.68%,并持续至24 h;TNF-α浓度达10.0 ng/ml时,TSG-6基因表达水平2 h内即下降96.27%,TSG-6基因的表达几乎被完全抑制.结论 (1)TSG-6基因可能参与脂肪细胞分化及脂质形成过程;(2)TNF-α对脂肪细胞中TSG-6基因表达具有抑制作用,其抑制效应总体趋势上呈现剂量反应性特征.

Expression of TSG-6 gene during 3T3-L1 preadipocyte differentiation and regulative role of tumor necrosis factor-alpha

OBJECTIVE: Tumor necrosis factor alpha-stimulated gene-6 (TSG-6 gene) differentially expressed in adipose tissue of obese and normal human subjects or rats. To explore the relationship between the differential expression of TSG-6 and adipocyte differentiation, adipogenesis and obesity, the present study aimed to investigate the changes of TSG-6 gene expression during 3T3-L1 preadipocyte differentiation and to analyze the regulative role of TNF-alpha on TSG-6 gene expression in matured 3T3-L1 adipocytes. METHODS: 3T3-L1 preadipocytes were cultured in vitro and differentiated into the matured adipocytes. TNF-alpha in different concentrations (0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml) was added into the culture medium of fully differentiated adipocytes (day 10) for various times (0.5 h, 2 h, 6 h, 12 h, 24 h). Total RNA from these adipocytes was extracted and the levels of TSG-6 gene mRNA expression were evaluated by RT-PCR. RESULTS: (1) In preadipocytes, the level of TSG-6 gene mRNA expression remained low. In the presence of dexamethasone (Dex), MIX and insulin, with the 3T3-L1 preadipocytes being differentiated into the matured adipocytes, the level of TSG-6 gene mRNA expression was upregulated and reached the higher level in fully differentiated adipocytes. There is a significant difference between any two detected phases in the levels of TSG-6 gene mRNA expression (P < 0.05), except that the levels of TSG-6 gene mRNA expression did not increase obviously on day 0 to day 2, day 3 to day 5, day 4 to day 6 and day 7 to day 10 (P > 0.05). (2) Treatment of day 10 3T3-L1 adipocytes with TNF-alpha of different concentrations (0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml) resulted in a significant decrease in the level of TSG-6 gene mRNA expression. The inhibition effect of TNF-alpha on TSG-6 gene mRNA expression generally tended to be reinforced with the increasing concentrations of TNF-alpha and the elongation of time course, except for the period of 6 - 24 h after the stimulation of 10.0 ng/ml TNF-alpha. When 0.1 ng/ml TNF-alpha was applied, the level of TSG-6 gene expression decreased by 33.73% at 6 h, 97.39% at 12 h. While 1.0 ng/ml TNF-alpha was used, the level of TSG-6 gene expression decreased by 78.68% at 6 h, which remained until 24 h. At a concentration of TNF-alpha up to 10.0 ng/ml, the level of TSG-6 gene expression decreased by 96.27% at 2 h. TSG-6 gene expression was almost fully inhibited. CONCLUSION: (1) TSG-6 gene may be involved in adipocyte differentiation and adipogenesis. (2) TNF-alpha can downregulate the mRNA expression of TSG-6 gene in matured adipocytes. The inhibitory effect of TNF-alpha on TSG-6 gene expression is generally dose-correlated.