Effect of siRNA-induced inhibition of Caspase-3 expression on apoptosis of hepatocytes induced by endotoxin

WANG Xueyin; SONG Xianmei; MA Yunyun

doi:10.3969/j.issn.1002-266X.2017.34.003

Abstract：Objective To investigate the effect of siRNA-induced inhibition of Caspase-3 expression on the apoptosis of hepatocytes induced by endotoxin.Methods Primers were designed for DNA sequence 843-1634 bp of Caspase-3.The target sequences was cloned into the lentivires expression vector pLenti-7.3 and empty lentivirus vector was set as a negative control.The virus vector and empty vector were transfected into 293T cells with Lipofectamine2000 liposomes.The virus of V-caspase-3 and V-control was harvested after 48 h.The hepatocytes HL7702 were randomly divided into the observation group and the control group, which were cultured with V-caspase-3 and V-control for 4 h, and then both groups were cultured with 20 mg/L endotoxin for 12 h.The protein expression of Caspase-3 was detected by Western blotting.The apoptosis rate of HL7702 cells was tested by flow cytometry (FACS).The apoptosis index (AI) was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL).The proliferative activity of cells was determined by MTT.Results The relative expression of Caspase-3 in HL7702 cells of the observation group was 17.32±2.16, which was lower than that in the control group (62.14±4.36) (P<0.01).The apoptotic rate of HL7702 cells in the observation group was 28.75％, which was lower than that of the control group (72.67％) (P<0.01).The AI of the observation group was 23.52±1.24, which was higher than that of the control group (54.26±1.87) (P<0.01).The proliferation rate of HL7702 cells in the observation group was 65.47％±6.24％, which was higher than that of the control group (41.39％±4.87％) (P<0.01).Conclusion Inhibition of caspase-3 by siRNA can effectively decrease the level of apoptosis of endotoxin-induced hepatocytes HL7702, thereby enhance the cell proliferation activity.