CAJ | 학술논문

目的建立脂肪酸诱导的脂肪变肝细胞模型,探讨活化的cAMP应答元件结合蛋白H(CREBH)蛋白对该模型中C反应蛋白表达的影响.方法给予L02细胞不同比例和浓度的油酸软脂酸混合液(2∶1、1∶2和0∶3),分别干预12和24 h.CCK-8法测定细胞活力.流式测定细胞内脂肪含量.实时PCR方法检测细胞内C反应蛋白、CREBH、C/EBP同源蛋白C/EBP(CHOP)和免疫球蛋白结合蛋白(BIP) mRNA表达水平变化.Western印迹法检测细胞核内活化的CREBH蛋白含量.RNA干扰靶向沉默CREBH蛋白后再分别采用混合脂肪酸干预24h.实时PCR方法检测细胞内C反应蛋白、CREBH、CHOP和BIP mRNA表达水平变化.结果 (1)混合脂肪酸诱导肝细胞脂肪变.随着软脂酸比例的升高,细胞内C反应蛋白表达水平升高,并且具有时间依赖性.(2)混合脂肪酸可以诱导CREBH表达升高(P＜0.05),可增加核内CREBH蛋白水平.(3)混合脂肪酸中软脂酸比例越高,内质网应激标志增加越显著.(4)CREBHsiRNA可有效抑制CREBH表达.敲除CREBH基因后,C反应蛋白表达显著下降(P＜0.05).结论软脂酸可上调肝细胞C反应蛋白表达水平,但是油酸无此作用.活化的CREBH可调节软脂酸诱导的C反应蛋白表达.
목적 건립지방산유도적지방변간세포모형,탐토활화적cAMP응답원건결합단백H(CREBH)단백대해모형중C반응단백표체적영향.방법 급여L02세포불동비례화농도적유산연지산혼합액(2∶1、1∶2화0∶3),분별간예12화24 h.CCK-8법측정세포활력.류식측정세포내지방함량.실시PCR방법검측세포내C반응단백、CREBH、C/EBP동원단백C/EBP(CHOP)화면역구단백결합단백(BIP) mRNA표체수평변화.Western인적법검측세포핵내활화적CREBH단백함량.RNA간우파향침묵CREBH단백후재분별채용혼합지방산간예24h.실시PCR방법검측세포내C반응단백、CREBH、CHOP화BIP mRNA표체수평변화.결과 (1)혼합지방산유도간세포지방변.수착연지산비례적승고,세포내C반응단백표체수평승고,병차구유시간의뢰성.(2)혼합지방산가이유도CREBH표체승고(P＜0.05),가증가핵내CREBH단백수평.(3)혼합지방산중연지산비례월고,내질망응격표지증가월현저.(4)CREBHsiRNA가유효억제CREBH표체.고제CREBH기인후,C반응단백표체현저하강(P＜0.05).결론 연지산가상조간세포C반응단백표체수평,단시유산무차작용.활화적CREBH가조절연지산유도적C반응단백표체.
Objective To establish a steatosis hepatocyte model in vitro and explore the effect of activated cAMP-responsive element-binding protein H(CREBH) protein on the expression of C-reactive protein(CRP) in this model.Methods L02 cells were cultured with free fatty-acid (FFA) mixtures at different ratios of oleic acid and palmitic acid(2 ∶ 1,1 ∶ 2,and 0 ∶ 3) for 12 or 24 h to induce fat-overloading.Cell viability was investigated by CCK-8,the levels of CRP,CREBH,C/EBP homologous protein (CHOP),and immunoglobulin-binding protein (BIP) were detected by real-time PCR.The level of CREBH in nuclei was detected by western blot.Then CREBH siRNA or negative control siRNA was transfected into L02 cells,and cultured with FFA mixtures for 24 hours.The levels of CRP,CREBH,CHOP,and BIP were detected by real-time PCR.Results (1) Cells cultured with FFA mixtures showed significantly increased fat droplets compared with control group.The level of CRP was increased by FFA mixtures,depending on the proportion of palmitic acid.(2) The expression levels of CREBH were increased by FFA mixture(P＜0.05) and its expression in nuclear was also increased by FFA mixture.(3) Endoplasmic reticulum stress could be induced by FFA mixture,depending on the proportion of palmitic acid.(4)The CREBH siRNA could effectively reduce the expression level of CREBH mRNA.Compared with the negative control group,the expression levels of CRP mRNA were significantly decreased (P＜0.05).Conclusion Palmitic acid,but not oleic acid,could up-regulate the expression of CRP.Palmitic acid-induced expression of CRP was regulated by activated CREBH.