Fas siRNA reduces apoptotic cell death of allogeneic-transplanted hepatocytes in mouse spleen.
BACKGROUND
Synthetic siRNAs 21 to 23 nucleotides in length silence gene expression posttranscriptionally, and RNA interference targeting Fas protects mice from fulminant hepatitis. Fas-mediated apoptosis has also been implied in the mechanism of hepatocyte apoptosis upon allogenic hepatocyte transplantation (HTx), and blockade of Fas and Fas ligand interaction successfully promotes the repopulation of allogenic liver cells in recipient spleens. In the present study, we further investigated the protective effects of Fas silencing on allogeneic hepatocytes transplanted into mouse spleens.


METHODS
Hepatocytes were isolated from BALB/c mice and mock transfected or transfected with Fas siRNA or GFP siRNA (n = 8/group). The expression of Fas was examined by RT-PCR and flow cytometric analysis. Forty-eight hours later, the cells were transplanted into spleens of allogenic B6 mice. Spleens were harvested on day 21 after transplantation. Apoptosis was assessed by TUNEL assay, survival of hepatocytes by alanine transaminase (ALT) assay.


RESULTS
Fas siRNA transfection reduced Fas expression on hepatocytes at both mRNA and protein levels (P <.05). Upon transplanting into recipient spleens, hepatocytes transfected with Fas siRNA demonstrated a lower percentage of apoptosis detected by TUNEL (6 +/- 3% in Fas siRNA group vs 12 +/- 5% in GFP siRNA group and 10 +/- 3% in mock transfected group; P <.05), and increased survival as determined by ALT assay (38.2 +/- 10.6 IU/g in Fas siRNA group vs 21.3 +/- 8.4 IU/g in GFP siRNA group and 18.5 +/- 5.9 IU/g in mock-transfected animals).


CONCLUSIONS
Fas silencing by RNA interference reduces apoptosis and increases survival of allogenic transplanted hepatocytes, and thus holds promise to inhibit acute rejection after hepatocyte transplantation.
Wang
J
J
Department of Surgery, Sun-Yat-Sen Memorial Hospital of Sun-Yat-Sen University of Medical Science, Guangzhou, China. gzsumsjw@yahoo.com.cn
Li
W
W
Min
J
J
Ou
Q
Q
Chen
J
J
D000818:Animals; D017209:Apoptosis; D016923:Cell Death; D017690:Cell Transplantation / Q000379:methods*; D006085:Graft Survival / Q000502:physiology*; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology* / Q000637:transplantation; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D023421:Models, Animal; D034741:RNA, Small Interfering / Q000235:genetics*; D013154:Spleen; D014162:Transfection; D014184:Transplantation, Homologous / Q000502:physiology*; D019014:fas Receptor / Q000235:genetics*
D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D019014:fas Receptor; D049452:Green Fluorescent Proteins