Effect Of CRISPR/Cas9 Knockout Ptgs2 On Apoptosis Of Hepatic Stellate Cells

Objective:Study the effect of knocking out Ptgs2 gene on apoptosis of HSC-T6 cells,and explore a new strategy for clinical prevention and treatment of liver cirrhosis.Methods:Rat HSC-T6 cells were divided into KD group(HSC-T6-Ptgs2-/-cells),negative control NC group(HSC-T6-NC cells)and blank control CON group(HSC-T6 cells).The LV-Ptgs2-sgRNA lentiviral vector was transfected into the KD group,and the negative control LV lentiviral vector was transfected into the NC group.The CON group was cultured without intervention.72 hours after transfection,observed the effect of transfection with electron fluorescence microscopy and photographed,calculated transfection rate.72 hours and 96 hours after transfection,Western blot was used to detected the expression of Ptgs2 gene protein in three groups of HSC-T6 cells.5 days after transfection,the three group were stained with Annexin V-APC single staining,and the apoptosis rates were detected by flow cytometry.Results:(1)Lentiviral transfection of HSC-T6 cells in each group:green fluorescence was observed in KD group and NC group,green fluorescence was not observed in CON group,and transfection efficiency was over 80% in KD group and NC group.The lentiviral vector was successfully transfected.(2)The expression of Ptgs2 protein in HSC-T6 cells of each group:KD group for 72 hours,the expression of Ptgs2 protein was significantly lower than that in NC group and CON group,and the difference was statistically significant(P<0.05),there was no significant difference between the NC group and the CON group.After 96 hours of transfection,the expression of Ptgs2 protein in KD group was further down-regulated.The Ptgs2 protein in NC group and CON group was significantly increased,and the KD group was significantly lower than NC group and CON group(P<0.01).(3)After 5 days of transfection,Annexin V-APC stained each group of cells,the apoptosis rate of KD group was significantly higher than that of NC group and CON group(P<0.01),NC group was higher than CON group.The difference was statistically significant(P<0.01).Conclusions:1.Target knocking out Ptgs2 gene can promote HSC-T6 cells Apoptosis.