Inhibition of mouse hepatocyte apoptosis via anti-Fas ribozyme

Abstract

Aim: To investigate the effects of anti-Fas ribozyme on Fas expression and apoptosis in primary cultured mouse hepatocytes.

Methods: Mouse hepatocytes were isolated by using collagenase irrigation. A hammerhead ribozyme targeting the Fas mRNA was constructed, and transfected into mouse hepatocytes via Effectene. Then Fas expression in mouse hepatocytes was detected by RT-PCR and western blotting. After being treated with anti-Fas antibody (JO2), hepatocytes viability was measured with MTT assay. Caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to Annexin V-FITC apoptosis detection kit.

Results: Fas expressed in primary mouse hepatocytes. Fas expression in hepatocytes transfected with anti-Fas ribozyme was decreased remarkably and correlated with the resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity.

Conclusion: Anti-Fas ribozyme can remarkably decrease the Fas expression in mouse hepatocytes, thus inhibit Fas-mediated apoptosis in hepatocytes. It is suggested that anti-Fas ribozyme could significantly increase the resistance of transplanted hepatocytes to apoptosis and improve the survival of transplanted hepatocytes.

Figure 1

Fas gene transcription in all groups. M: DL-2000 DNA marker, Lanes 1-3: Empty control, mock-transfected group and pU6-RZ596-transfected group.

Figure 2

Fas protein expression in all groups. M: Marker; Lanes 1-3: Empty control, mock-transfected group and pU6-RZ596-transfected group.

Figure 3

Detection of caspase-3 activity in all groups.

Figure 4

Stimulated with JO₂ for 24 h, cells viabilities of control, mock-transfected and pU6-RZ596-transfected groups were detected by MTT assay.

Figure 5

Detection of apoptosis of hepatocytes through cytometry. A: Apoptosis rate of control group was 86%; B: Apoptosis rate of mock-transfected cells was 87%; C: Apoptosis rate of pU6-RZ596-transfected cells was 35%.