干扰PPARγ基因对绵羊滋养层细胞增殖、凋亡、迁移和脂质积累的影响  

Effects of Interference with PPARγGene on Proliferation,Apoptosis,Migration and Lipid Accumulation of Trophoblast Cells in Sheep

作　　者：刘畅 郝科兴 陈岩 曾维斌[1] 喻恒彬 陈磊[1] 王静[1] 胡广东 LIU Chang;HAO Kexing;CHEN Yan;ZENG Weibin;YU Hengbin;CHEN Lei;WANG Jing;HU Guangdong(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China)

机构地区：[1]石河子大学动物科技学院,石河子832003

出　　处：《畜牧兽医学报》2024年第6期2421-2430,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(32060751);兵团重点领域科技攻关计划项目(2021AB014);石河子大学高层次人才科研启动资金专项项目(RCZK201941)。

摘　　要：旨在通过干扰PPARγ基因的表达,探究PPARγ对绵羊滋养层细胞增殖活性、细胞凋亡、细胞迁移率以及脂质累积的影响,为深入研究PPARγ在绵羊滋养层细胞中的分子机制提供基础依据。本研究设计并合成干扰PPARγ基因的siRNA干扰片段,通过Western Blot和RT-qPCR筛选干扰效率最高的siRNA,并转染至分离培养的绵羊滋养层细胞中。通过BODIPY染色检测干扰PPARγ基因后滋养层细胞中脂滴的聚积,比色法检测细胞中甘油三酯的含量,并利用RT-qPCR检测脂代谢相关基因的转录水平。采用CCK-8法、划痕试验和流式细胞法,分别探究干扰PPARγ基因对滋养层细胞增殖活性、细胞迁移率及细胞凋亡的影响。筛选得到了干扰效率最高的siPPARγ-1片段用于转染滋养层细胞,在干扰滋养层细胞PPARγ基因表达后,细胞中脂滴聚积能力下降,甘油三酯含量降低(P<0.01),脂代谢相关基因CD36、FABP4和PLIN2的转录水平也受到抑制(P<0.01)。同时,滋养层细胞的增殖活力也显著降低(P<0.05),细胞迁移率减少(P<0.01),而细胞凋亡率显著上升(P<0.01)。本研究表明PPARγ基因在调控滋养层细胞功能方面起重要作用,可对其具体分子机制进行深入研究,以期用于繁殖育种实践中。The aim of this study was to explore the effects of PPARγon the cell viability,apoptosis,cell migration rate and lipid accumulation of sheep trophoblast cells by interfering with the expression of PPARγgene,so as to provide a basis for further study of the molecular mechanism of PPARγin trophoblast cells.To design and synthesize siRNA interference fragment of PPARγgene,the siRNA with the highest interference efficiency was screened by Western blot and RT-qPCR.Then,the siRNA was transfected into the sheep trophoblast cells.After interfering with the PPARγgene,the accumulation of lipid droplets in trophoblast cells was measured by BODIPY staining,the amount of triglycerides by colorimetric method,and the transcript levels of genes involved in lipid metabolism was detected using RT-qPCR(real-time quantitative PCR).CCK-8 assay,wound healing test and flow cytometry were used to detect the effects of PPARγinterference on cell viability,migration rate and apoptosis of trophoblast cells.siPPARγ-1 fragment with the highest interference efficiency was screened and used to transfect trophoblast cells to interfere with the expression of PPARγgene.Interference with PPARγgene expression in trophoblast cells reduced the accumulation capacity of lipid droplets and cellular triglyceride content(P<0.01),and the transcription levels of related genes CD36,FABP 4 and PLIN 2 were also inhibited(P<0.01).At the same time,interference with PPARγgene expression significantly reduced cell viability(P<0.01)and migration rate(P<0.05),while the cell apoptosis rate was increased significantly(P<0.01).This study showed that PPARγgene plays an important role in regulating the function of trophoblast cells and its specific molecular mechanism can be studied in depth with a view to using it in reproductive breeding practices.

关 键 词：绵羊 滋养层细胞 PPARΓ 干扰 

分 类 号：S826.3[农业科学—畜牧学]