干扰FABP4基因对绵羊子宫内膜上皮细胞脂质积累及迁移的影响

Effects of interfering FABP4 gene on lipid accumulation and migration of endometrial epithelial cells in sheep

研究旨在探究脂肪酸结合蛋白4(FABP4)基因对绵羊子宫内膜上皮细胞脂质积累及迁移的影响。设计3条FABP4干扰片段,将片段与pGBplv-U6-mcherry-puro连接构建干扰载体,以空载质粒为对照(NC组),3条干扰重组质粒分别为FABP4-shRNA1、FABP4-shRNA2、FABP4-shRNA3,对重组质粒进行酶切鉴定。质粒转染后RT-qPCR鉴定干扰效率,Western Blot对载体干扰效果进行进一步验证。将载体转染至体外培养的子宫内膜上皮细胞中,BODIPY法检测脂滴聚集情况,使用试剂盒检测细胞中甘油三酯含量,RT-qPCR检测脂代谢及增殖迁移相关基因,细胞划痕试验检测细胞迁移情况。结果显示:所有载体均可有效干扰FABP4基因的表达,其中FABP4-shRNA2载体干扰效果最好。在干扰子宫内膜上皮细胞FABP4基因表达后,与NC组相比,细胞中脂滴聚集能力下降,甘油三酯含量降低,细胞脂质代谢和增殖迁移相关基因mRNA表达下降,划痕愈合速度降低;降低子宫内膜上皮细胞FABP4基因表达后,细胞脂质积累水平及细胞迁移能力下降。研究表明,FABP4基因可能对绵羊妊娠和胚胎正常发育具有重要作用,值得在育种实践中进行更深入研究。

The purpose of this study was to investigate the effect of FABP4 gene on lipid accumulation and migration of endometrial epithelial cells in sheep.Three FABP4 interference fragments were designed and ligated with pGBplv-U6-mcherry-puro to construct interference vectors.The empty plasmid was used as the control(NC group),and the three interference recombinant plasmids were FABP4-shRNA1,FABP4-shRNA2,and FABP4-shRNA3,respectively.The recombinant plasmids were identified by enzyme digestion.After plasmid transfection,RT-qPCR was used to identify the interference efficiency.Western Blot was used to further verify the interference effect of the vector.The vector was transfected into endometrial epithelial cells cultured in vitro.The accumulation of lipid droplets was detected by BODIPY method.The triglyceride content in the cells was detected by detection kit.RT-qPCR was used to detect genes related to lipid metabolism,proliferation and migration.Cell scratch test was used to detect cell migration.The results showed that all vectors can effectively interfere with the expression of the FABP4 gene,with the FABP4-shRNA2 vector showing the best interference effect.After interfering with the expression of the FABP4 gene in endometrial epithelial cells,compared with the NC group,the ability of cells to aggregate lipid droplets decreased,the content of triglycerides was reduced,the expression of mRNA,related to cellular lipid metabolism and proliferation and migration decreased,and the speed of scratch healing was reduced.After reducing the expression of the FABP4 gene in endometrial epithelial cells,the level of cellular lipid accumulation and the ability of cells to migrate decreased.The study indicates that the FABP4 gene may play an important role in the pregnancy of sheep and the normal development of embryos,and it is worth further in-depth study in breeding practice.