RESUMEN
We have used high-titer (10(8) ffu/ml) recombinant retroviral vectors to transfer the beta-galactosidase (beta-Gal) gene to rat hepatocytes in vivo. In animals injected twice in the portal blood stream the next day after partial hepatectomy, half of the hepatocytes (46 +/- 17%) expressed the marker at the end of liver regeneration. The number of positive cells closely correlated with the viral titer as well as with beta-Gal enzymatic activity present in the whole liver. Because genes transferred via retroviral vectors in the liver are known to be expressed permanently, our present results open new possibilities for the development of gene therapy protocols for hereditary liver diseases using recombinant retroviral vectors.
Asunto(s)
Técnicas de Transferencia de Gen , Hígado/virología , Retroviridae/genética , Animales , Vectores Genéticos/farmacología , Hepatectomía , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
AIMS/BACKGROUND: We studied the fate of hepatocytes in the rat liver after D-galactosamine injury by genetic labeling using recombinant retroviruses carrying the Escherichia coli lacZ gene coupled to a nuclear localization signal. METHODS: Hepatocytes were either labeled by direct injection of 2.5 ml high-titer retrovirus-containing medium in the regenerating liver parenchyma after administration of a single dose of D-galactosamine. Alternatively hepatocytes were pre-labeled, 24 h after a two-thirds hepatectomy, by injecting the same volume of retroviral solution in the portal vein and D-galactosamine was administered 15 days later. Gamma-glutamyl transpeptidase and beta-galactosidase activities were assessed on cryostat sections, along with localization of the hepatocyte-specific HES6 antigen. RESULTS: Morphological observations, as well as beta-galactosidase activity detection, showed that hepatocytes actively divide as early as 1 day after D-galactosamine injection. Gamma-glutamyl transpeptidase activity was detected in biliary cells, but also in mature hepatocytes, pre-labeled with beta-galactosidase before D-galactosamine administration. CONCLUSIONS: These experiments demonstrate that hepatocytes can divide to restore the liver mass after D-galactosamine liver injury. Furthermore, we also show that gamma-glutamyl transpeptidase, which has been reported to be expressed only by fetal or preneoplastic hepatocytes, can be re-expressed by mature hepatocytes during the recovery process.