Effect Of COX-2on Fat Metabolism Related To Hepatocyte BRL-3A Cell And The Regulation In The Expression Of Acsl1、Acsl6Gene

【Objectives】: Research the influence of COX-2in fat metabolism in hepatocyteBRL-3A cell and the regulation in the expression of Acsl1、Acsl6gene.【Methods】:(1)Screening the best induced concentration,induced cells steatosis:Rat hepatocytesBRL-3A strains cultured in vitro,different concentration of medical intralipidinduced rat liver cells into fatty degeneration,by Oil Red O staining method toobserve the cell fatty change situation, determined by MTT test cell proliferationactivity and ELISA test before and after induction induced intracellular TG contentto determine the best concentration.(2)Experiments were divided into normal BRL-3A in group, grease BRL-3A ingroup, pYr-1.1-hU6-EGFP-COX-2shRNA group, pYr-1.1-hU6-EGFP-HK group,pcDNA3.1(+)-r-COX-2group, pcDNA3.1(+)-r-HK group, Nimesulide positivecontrol group (200ug/ml),In addition to the normal BRL-3A in group, the rest ofthe group are induced by the best concentration of6%fat emulsion after24h,transient transfection group corresponding plasmid,ELISA test groups ofintracellular TG content after transfection,RT-PCR detect the genetic expression ofCOX-2mRNA, Acsl1mRNA and Acsl6mRNA in diverse time phase(24h) in eachgroup.【Results】：(1)Different concentrations of medical fat emulsions induce BRL-3A cellsteatosis,cultured cells for24h，with the increase of concentration,Oil red stainingprompted liver cell vacuoles lipid droplets are increase,conforming to thecharacteristics of liver steatosis.ELISA detection showed that the intracellulartriglyceride levels also increased.According to the OD value determined by MTTtest decide the optimal concentration is6%medical fat emulsion group. (2)In fluorescence microscope, COX-2shRNA by transient transfection to steatosisBRL-3A cell cultured for24h, COX-2shRNA can see the green fluorescence in thecytoplasm, up to70%.(3)ELISA test the triglyceride expression content in the different cell groups beforeand after transfection,comparing with fatty change of liver cells, the TG content inpYr-1.1-hU6-EGFP-COX-2shRNA group and Nimesulide group were reduced(p<0.05),especially in pYr-1.1-hU6-EGFP-COX-2shRNA transfection group,obviou-sly lower than the remaining six group (p<0.01).Compared with fatty change ofliver cells, the TG content in pcDNA3.1(+)-r-COX-2group of transfection groupwere higher than in the remaining six group (p<0.05).The TG content in Nimesulidelgroup is lower than that of pcDNA3.1(+)-r-COX-2group but higher than that ofpYr-1.1-hU6-EGFP-COX-2shRNA group.Compared with the fatty change of livercells, the TG content in pYr-1.1-hU6-EGFP-HK group and pcDNA3.1(+)-r-HKgroup has no obvious change, no statistical significance（p>0.05）.(4)COX-2and Acsl1gene and Acsl6gene expression of seven groups:RT-PCRdetect the expression of COX-2mRNA after transfected24h. The expression inCOX-2mRNA in seven groups：comparing with the normal liver cell group，theexpression of COX-2mRNA in fat liver cell group was increased (p<0.05),comparing with the fat liver cell group，the expression of COX-2mRNA in pYr-1.1-hU6-EGFP-COX-2shRNA group and Nimesulidel group were also reduced(p<0.05),especially in pYr-1.1-hU6-EGFP-COX-2shRNA group(p<0.01),but theexpression of COX-2mRNA in pcDNA3.1(+)-r-COX-2group was increased, andthe expression of COX-2mRNA inpYr-1.1-hU6-EGFP-HK group and pcDNA3.1(+)-r-HK group has no obvious change, no statistical significance（p>0.05）.Theexpression in Acls1mRNA and Acsl6mRNA in seven groups：comparing with thenormal liver cell group，the expression of Acsl1mRNA and Acsl6mRNA in fat livercell group was increased(p<0.05),comparing with the fat liver cell group，theexpression of Acsl1mRNA and Acsl6mRNA in pYr-1.1-hU6-EGFP-COX-2shRNAgroup and Nimesulidel group were also reduced(p<0.05),especially inpYr-1.1-hU6-EGFP-COX-2shRNA group(p<0.01),but the expression of Acsl1mRNA and Acsl6mRNA in pcDNA3.1(+)-r-COX-2group was increased, andthe expression of Acsl1mRNA and Acsl6mRNA in pYr-1.1-hU6-EGFP-HK groupand pcDNA3.1(+)-r-HK group has no obvious change, has no statisticalsignificance（p>0.05）.【Conclusions】:(1)Inhibit the expression of COX-2,the fatty degeneration of liver cell werealleviated,the TG content in fatty degeneration of liver cells also reduced,At thesame time,the expression of lipid metabolism genes Acsl1、Acsl6were decreased.(2)Increase the expression of COX-2,the fatty degeneration of liver cell wereaggravated,the TG content in fatty degeneration of liver cells also increased,At thesame time,the expression of lipid metabolism genes Acsl1、Acsl6were aggrandized.