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. 2004 Sep 1;10(17):2567-70.
doi: 10.3748/wjg.v10.i17.2567.

Inhibition of mouse hepatocyte apoptosis via anti-Fas ribozyme

Min Zhang  1 , Wei He, Fang Liu, Ping Zou, Juan Xiao, Zhao-Dong Zhong, Zhong-Bo Hu
Affiliations

Inhibition of mouse hepatocyte apoptosis via anti-Fas ribozyme

Min Zhang et al. World J Gastroenterol. .

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.

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Figures

Figure 1
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
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
Figure 3
Detection of caspase-3 activity in all groups.
Figure 4
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
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%.
See this image and copyright information in PMC

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