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目的观察3T3-L1脂肪细胞诱导分化过程中生长抑制和DNA损伤诱导家族45β(GADD45β)基因mRNA表达水平的变化,探讨TNF-α对成熟脂肪细胞中GADD45β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,采用逆转录-聚合酶链反应(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GADD45β基因表达水平。结果随脂肪细胞逐渐分化成熟,GADD45β基因mRNA表达水平渐降低。GADD45β基因表达水平除在细胞分化d1~7、d8~10各时段内差异无显著意义(P>0.05)外,余各时段之间表达水平差异均具有显著意义(P<0.05)。不同质量浓度重组TNF-α(0.1~10.0μg/L)均能显著促进成熟脂肪细胞中GADD45β基因mRNA的表达。TNF-α质量浓度为0.1μg/L时,GADD45β基因表达水平6 h内上升51.39%,24 h内上升100.15%;TNF-α质量浓度为1.0μg/L时,GADD45β基因表达水平6 h内上升44.15%,24 h内上升100.63%;TNF-α质量浓度达10.0μg/L,GADD45β基因表达水平6 h内即上升104.26%,24 h内上升122.17%。结论GADD45β基因可能参与脂肪细胞分化及脂质形成过程;TNF-α对成熟脂肪细胞中GADD45β基因表达具有促进作用,其促进效应总体趋势呈现时间依赖性特征。
Objective To observe the changes of GADD45β mRNA expression induced by DNA damage and growth inhibition induced by 3T3-L1 adipocytes, and to investigate the regulatory effect of TNF-α on GADD45β gene expression in mature adipocytes. Methods 3T3-L1 adipocytes were cultured in vitro and induced to differentiate into 3T3-L1 adipocytes. Recombinant TNF-α was used to differentiate mature adipocytes, and RT-PCR was used to detect the induction Differentiation at different time points and intervention of TNF-α at different time adipocytes GADD45β gene expression levels. Results With the gradual differentiation and maturation of adipocytes, the mRNA expression level of GADD45β gradually decreased. The expression level of GADD45β was significantly different (P <0.05) except for the differences in cell differentiation between d1 ~ 7 and d8 ~ 10 (P> 0.05). Different concentrations of recombinant TNF-α (0.1-10.0 μg / L) could significantly promote the expression of GADD45β mRNA in mature adipocytes. When the concentration of TNF-α was 0.1 μg / L, the expression level of GADD45β increased by 51.39% within 6 h and increased by 100.15% within 24 h. When the concentration of TNF-α was 1.0 μg / L, the expression level of GADD45β increased within 6 h 44.15%, increased by 100.63% within 24 h; the concentration of TNF-α reached 10.0 μg / L, the expression level of GADD45β increased by 104.26% within 6 h and increased by 122.17% within 24 h. Conclusion GADD45β may play an important role in adipocyte differentiation and lipid formation. TNF-α may promote the expression of GADD45β in mature adipocytes, and the general trend of its promotion effect is time-dependent.