细胞周期蛋白依赖性激酶抑制因子3对鸡前脂肪细胞分化的抑制作用

Inhibition of Differentiation of Chicken Preadipocytes by Cyclin-dependent Kinase Inhibitor 3

【目的】解析细胞周期蛋白依赖性激酶抑制因子3(cyclin-dependent kinase inhibitor 3,CDKN3)在鸡前脂肪细胞分化中的作用,为阐明脂肪沉积的分子机制奠定理论基础。【方法】构建荧光表达载体pECFPCDKN3,转染至鸡永生化前脂肪细胞系(immortalized chicken preadipocyte cell line,ICP1),观察CDKN3蛋白的亚细胞定位;采用油酸钠诱导ICP1分化,qRT-PCR检测分化过程中CDKN3 mRNA表达水平;将pCMVHA-CDKN3和pCMV-HA分别转染到ICP1中,转染24 h后进行诱导分化,通过油红O染色检测脂肪细胞分化情况,并利用qRT-PCR检测过表达CDKN3后脂肪分化相关基因的mRNA表达水平。【结果】转染了pECFP-CDKN3的细胞主要在细胞核中呈青色荧光信号,提示鸡CDKN3定位于细胞核;在诱导ICP1成脂分化的过程中,CDKN3 mRNA表达量逐渐升高;油红O染色结果显示:过表达CDKN3细胞内脂滴聚集减少,且脂肪分化相关基因C/EBPα、PPARγ、FABP4和FAS的表达水平均呈下降趋势,其中FAS显著下降(P<0.05),PPARγ极显著下降(P<0.01)。【结论】CDKN3是核蛋白,可抑制鸡前脂肪细胞分化。

[Purpose]To elucidate the role of cyclin-dependent kinase inhibitor 3(CDKN3)in the differentiation of chicken preadipocyte,laying a theoretical foundation for elucidating the molecular mechanism of fat deposition.[Methods]The fluorescent expression vector pECFP-CDKN3 was constructed,and the subcellular localization of CDKN3 protein was observed after transfection to the immortalized chicken preadipocyte cell line(ICP1).Sodium oleate was used to induce the differentiation of ICP1,and qRT-PCR detected the expression level of CDKN3 mRNA during differentiation.pCMV-HA-CDKN3 and pCMV-HA were transfected into ICP1,and differentiation was induced after 24 hours of transfection,adipocyte differentiation was detected by oil red O staining,and the mRNA expression levels of fat differentiation-related genes after overexpression of CDKN3 were detected by qRT-PCR.[Results]The cells transfected with pECFP-CDKN3 mainly had cyan fluorescence signal in the nucleus,suggesting that chicken CDKN3 was localized in the nucleus.In the process of inducing the lipid differentiation of ICP1,the expression level of CDKN3 mRNA gradually increased.The results of oil red O staining showed that the aggregation of lipid droplets in overexpressing CDKN3 was reduced,and the expression levels of C/EBPα,PPARγ,FABP4 and FAS related to fat differentiation showed a downward trend,among which FAS decreased significantly(P<0.05)and PPARγdecreased extremely significantly(P<0.01).[Conclusion]CDKN3 is a nuclear protein that can inhibit the differentiation of chicken preadipocyte.