NR4A1对小鼠卵巢颗粒细胞脂肪代谢相关调控因子的调节作用

NR4A1 orphan nuclear receptors regulated the expression of lipid metabolism-related factors in mouse granulosa cells

目的观察NR4A1对小鼠卵巢颗粒细胞中脂肪代谢相关调控因子脂联素受体2(Adipo-R2)、解偶联蛋白2(UCP2)、B类清道夫受体(CD36)的调节作用。方法分别构建NR4A1超表达腺病毒载体(AdCMV-NR4A1)及干涉腺病毒载体(AdH1-siRNA/NR4A1)。体外培养小鼠卵巢颗粒细胞,贴壁生长至90%时,分为病毒感染组、胰岛素(100nmol/L)与胰岛素增敏剂(10μmol/L)组和病毒十胰岛素组。Trizol裂解细胞提取总RNA,实时荧光定量PCR法检测颗粒细胞中Adipo-R2,UCP2,CD36的mRNA表达水平。结果在小鼠卵巢颗粒细胞中超表达NR4A1能够促进Adipo-R2,UCP2,CD36基因的转录(P<0.05),RNA干扰NR4A1后则可以抑制以上基因的表达(P<0.05);胰岛素可以促进UCP2,CD36基因的表达(P<0.05),而对Adipo-R2的作用不明显;另外胰岛素增敏剂曲格列酮也能够促进Adipo-R2,UCP2,CD36的表达(P<0.05);RNA干扰NR4A1 24h后,再向细胞中加入生理剂量(100nmol/L)胰岛素30min后Adipo-R2,UCP2,CD36的表达水平与单独干涉组比均可逆转升高(P<0.05),其中UCP2的表达水平可升高到胰岛素组同样的水平,但Adipo-R2,CD36的逆转升高水平并没有达到仅加入胰岛素时的升高水平(P<0.05)。结论 NR4A1对Adipo-R2,UCP2,CD36具有正向调节作用,同时与胰岛素或胰岛素增敏剂可能有协同调节作用,进而发挥对脂肪代谢的调控。

Objective. To examine the effect of NR4A1 on the regulation of three lipid metabolism-related factors, adiponeetin receptor 2 （Adipo-R2）, uncoupling protein 2 （UCP2） and scavenger receptor B （CD36） in mouse granulosa cells. Methods： Recombinant adenovirus AdCMV-NR4A1 and AdHI-siRNA/NR4A1 were constructed to enhance and knock down the expression of NR4A1, respectively. Mouse granulosa cells were isolated and cultured in vitro to reach a confluency of 90% or more. Cultured cells were infected with recombinant adenovirus, or treated with insulin （100 nmol/L） or troglitazone （10 μmol/L）, or infected with recombinant adenovirus plus insulin treatment. Total RNA of the cultured cells were extracted by Trizol after the treatment and the mRNA expressions of Adipo-R2, UCP2, CD36 were detected by Real-time polymerase chain reaction （PCR）.Results： The mRNA expressions of Adipo-R2, UCP2, CD36 were significantly increased in mouse granulosa cells infected with AdCMV-NR4A1 （P〈0.05）, while significantly inhibited in those infected with AdHI-siRNA/NR4A1 （P〈0.05） ; Troglitazone increased the mRNA expressions of all three studied factors in the cultured cells （P〈0.05） ; Expressions of UCP2 and CD36 were up-regulated by insulin, but Adipo-R2 expression was unchanged; The expressions of Adipo-R2, UCP2, CD36 in the cells infected with AdHI-siRNA/NR4A1 were significantly increased by insulin stimulation, when compared with those in the cells infected with AdHI-siRNA/NR4A1 alone （P〈0.05）, and Adipo-R2 expression could reach the same levels as in the cells treated with insulin alone. Conclusions： NR4A1 could up-regulate the expressions of Adipo-R2, UCP2 and CD36 in cultured mouse granulose cells, and may have a synergistic effect with insulin or troglitazone.