Intrasplenic transplantation of allogeneic hepatocytes modified by BCL-2 gene protects rats from acute liver failure

Abstract 

Background

Apoptosis of donor hepatocytes may be induced by recipient cytotoxic T lymphocytes (CTLs) during acute rejection, representing a major impediment for these cell transplants. Because the mechanisms of transplanted hepatocyte loss involve Fas-mediated pathways, BCL-2 genetic modification may protect liver cells. In the present study, we further investigated whether BCL-2 transfer into transplanted liver cells rendered them resistant to Fas ligand–induced apoptosis, and protected rats from acute liver failure.

Materials and methods

Hepatocytes isolated from Sprague-Dawley rats were infected with an adenovirus vector encoding human BCL-2 gene (AdCMVBCL-2) or a control AdCMVLacZ vector. Forty-eight hours later, cells challenged with recombinant Fas ligand (rhsFasL) were assayed for apoptosis using TUNEL staining and caspase 3 activity. Other cells were transplanted into the spleens of Wistar rats with a 90% hepatectomy 12 hours later.

Results

Western blot analysis and RT-PCR confirmed the expression of hBcl-2 in AdCMVhBcl-2–infected hepatocytes. Recombinant FasL produced a dose-dependent increase in TUNEL-positive percentage and caspase-3 activity in uninfected hepatocytes, but did not influence these features in AdCMVhBcl-2–infected cells. On challenge with 90% hepatectomy, the survival of Wistar rats receiving transplantation of AdCMVhBCL-2–infected hepatocytes was significantly prolonged compared with the controls.

Conclusion

Adenovirus-mediated BCL-2 gene transfer protects transplanted hepatocytes from Fas-mediated cytolysis, thus holding promise for a new avenue of acute liver failure treatment.