Silencing Of The COX-2 Gene And Its Effect And Preliminary Discussion Of The Mechanism On The Senescence Of Hepatic Stellate Cells

Objective: Construct the short hairpin RNA eukaryotic expression vector for COX-2 gene,observe its effect on the senescence of hepatic stellate cells,and explore its regulatory mechanism and influence on the hepatic stellate cells from the cellular and molecular level ?Methods1.Take the hepatic stellate cells(HSC-T6)as the object of the experimental,this experiment is divided into four groups:empty Gsensor group;COX-2sh RNA group,empty Gsensor + Fumonisin B1 group;COX-2sh RNA+ Fumonisin B1 group(40 mu mol/L)?2.Transfer p Yr-1.1COXsh RNA into HCS by Lipofectamine2000,observe the transfection efficiency by fluorescence microscope.3.Detection the senescence.of hepatic stellate cells by ?-galactosidase staining?4.Use PCR method to detect the expression of COX-2 ?LASS2?p21?Rb?Results:1.Fluorescence microscopy show that the transfection efficiency of COX-2 sh RNA is above 70%;2.?-galactosidase staining showed,compared with empty Gsensor group,cellular senescence of the HSC were increased in COX-2sh RNA group(p<0.01),cellular senescence decreased significantly in COX-2sh RNA+FB1 group compared to the COX-2sh RNA group(p<0.01).3.Quantitative PCR showed,compared to the empty gsensor group,the COX-2sh RNA group can dectease the expression of COX-2 ?upregulation the expression of the Rb(p<0.01),downregulation the expression of the p21 and LASS2(p<0.05);Compared to the COX-2sh RNA group,the COX-2sh RNA+FB1 group can downregulation the expression of Rb?upregulation the expression of the p21(p<0.01),downregulation the expression of LASS2 ?upregulation of the expression of the COX-2(p<0.05)?Conclusions1.Silencing of the COX-2 gene promotes senescence of the hepatic stellate cells.2.Inhibition the activity of the Longevity assurance2 can reverse COX-2sh RNA's effect on the senescence of the hepatic stellate cells.,indicated that the silencing of the COX-2 gene may play the role of promoting the senescence of the hepatic stellate cells through ceramide pathway.