Effect Of COX-2 Knockout On Proliferation Of Hepatic Stellate Cells

Objective:To study the effect of COX-2 knockout on the proliferation of hepatic stellate cells,and to provide new evidence and strategies for clinical reversal of hepatic fibrosis.Methods:after cell culture and passage,lv-COX-2-sgrna lentiviral vector was transfected into hsc-t6 cells according to the most applicable viral dose MOI=10,and then hsc-t6^-/-cells were obtained.Lv-nc-sgrna was transfected into hsc-t6 cells to obtain hsc-t6-nc cells,and the cells were divided into three groups:KO group(hsc-t6-cox-2^-/-cells),NC group（hsc-t6-nc cells）,CON group（hsc-t6 cells）.after 72 hours of virus transfection,the efficiency of transfection was calculated by observing under fluorescence microscope.After successful virus transfection,cox-2 proteins in the 3 groups were extracted at 72h and 96h,respectively,and Western Blot was used for knockout verification at the gene and protein levels.Clonal formation and CCK8 assay detects cell proliferation.Results:1.lv-COX-2-sgrna lentivirus vector was transfected into hsc-t6cells for 72 h,and was observed under a fluorescence microscope.Green fluorescence was not observed in the CON group,while green fluorescence was observed in the KO and NC groups,and the transfection efficiency was above 80%,indicating successful virus transfection.2.Western Blot results showed that the cox-2 protein expression in the KO group was significantly down-regulated compared with that in the CON group and the NC group after lentivirus transfection of hsc-t6 cells at 72h and 96h,this indicated that cox-2 gene had been successfully knocked out,and there was a statistical difference between the two groups（p<0.01）.3.Clonal formation and CCK8 test results showed that the proliferation of hsc-t6 cells in KO group was significantly inhibited compared with that in NC group and CON group,This indicated that the knockout of cox-2 gene could induce the proliferation inhibition of hsc-t6 cells,and there was a statistical difference between the two groups（p<0.01）.Conclusion:Knockout of the cox-2 gene in hsc-t6 cells inhibited the proliferation of hsc-t6 cells.