| 摘要: |
| 【目的】研究PLIN2基因对秦川牛脂肪细胞分化过程中脂滴形成的功能。【方法】以秦川牛原代脂肪细胞为试验材料，采用高干扰效率的si-PLIN2及对照组si-NC，超表达PLIN2基因真核表达载体pcDNA3.1(+)-PLIN2及对照pcDNA3.1(+)-empty vector（pcDNA3.1(+)-EV），转染秦川牛脂肪细胞并进行相关基因mRNA及蛋白水平表达量验证，并对转录因子C/EBPα是否能结合在PLIN2基因启动子区域进行验证。【结果】si-PLIN2处理组中CREB基因表达量无明显变化，C/EBPβ和C/EBPα基因mRNA水平表达量与si-NC对照组相比分别出现显著或极显著下调。与对照组pcDNA3.1(+)-EV相比，过表达组pcDNA3.1(+)-PLIN2CREB相对表达量极显著上调，C/EBPα和C/EBPβ基因相对表达量上调，但差异不显著。Western blot蛋白表达水平检测发现，si-PLIN2中磷酸化水平CREB(phospho-CREB,p-CREB)、C/EBPβ、C/EBPα的表达量显著低于对照组si-NC，过表达组上述转录因子表达量未出现明显上调。ChIP试验结果表明，转录因子C/EBPα可以结合在PLIN2基因启动子区域，并调控秦川牛PLIN2基因的表达。【结论】下调秦川牛PLIN2基因可反馈抑制上游cAMP-PKA信号通路p-CREB、C/EBPβ、C/EBPα 3个转录因子的表达，从而抑制秦川牛脂肪细胞分化过程中脂滴生成。 |
| 关键词: 秦川牛 PLIN2基因 脂滴形成 调控机制 |
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| 基金项目:国家自然科学基金项目（31972994）；国家肉牛牦牛产业技术体系建设项目（CARS-37）；国家重点研发计划项目（2018YFD0501700）；陕西省农业科技创新转化项目（NYKJ-2018-YL09）；宁夏回族自治区重点研发计划项目（2019BEF02004） |
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| The function of PLIN2 in regulating lipid droplets formation of Qinchuan cattle |
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LI Peiwei,ZAN Linsen
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| Abstract: |
| 【Objective】This study aimed to study the functions of PLIN2 gene in lipid droplets formation during adipocytes differentiation of Qinchuan cattle.【Method】The primary adipocytes of Qinchuan cattle were selected for induction differentiation test and verification.Using high interference efficiency of si-PLIN2,the control group si-NC,over-expression pcDNA3.1(+)-PLIN2 and pcDNA3.1(+)-empty vector (pcDNA3.1(+)-EV) were transfected in Qinchuan cattle adipocytes,and the related PLIN2 mRNA and protein expression level were verified.The combination of transcription factor C/EBPα in the promoter region of PLIN2 gene was also detected.【Result】The expression of CREB gene changed insignificantly in si-PLIN2,while mRNA levels of C/EBPβ and C/EBPα were significantly or extremely significantly down regulated compared to si-NC.The relative expression levels of CREB and C/EBPα and C/EBPβ genes in pcDNA3.1(+)-PLIN2 were up-regulated compared to pcDNA3.1(+)-EV,but the differences were not significant.Western blot analysis revealed that expression levels of phospho-CREB,C/EBPβ and C/EBPα in si-PLIN2 were significantly lower than si-NC.However,there was no significantly up-regulation trend in overexpression group.ChIP assay indicated that the transcription factor C/EBPα could bind to the promoter region of PLIN2 gene and regulate the expression of PLIN2.【Conclusion】Inhibiting PLIN2 expression could decrease the expression of p-CREB,C/EBPβ and C/EBPα in upstream cAMP-PKA signaling pathway and restrain lipid droplets formation during adipocytes differentiation of Qinchuan cattle. |
| Key words: Qinchuan cattle PLIN2 gene lipid droplets formation regulation mechanism |
