Transfection of the Albumin Promoter Driven-Oncogenes to Isolated Rodent Hepatocytes, and Transplantation of the Hepatocytes into Livers and Spleens of Syngenic Animals.

本研究的目的是建立一个肝癌发生模型，在体外将激活的癌基因转染到离体的啮齿类动物肝细胞中，随后将其移植到同基因动物的肝脏或身体的其他部位。将癌基因插入白蛋白基因启动子下游，这可能有助于肝细胞内转基因的表达。1)通过PCR扩增包括白蛋白基因的核心启动子的片段并克隆到质粒载体中。使用该DNA，我们构建了pAlbElA载体，其中ElA插入到白蛋白启动子的下游。当将该载体导入分离的肝细胞中时，该基因在细胞中瞬时表达。2）为了将该基因有效地导入分离的肝细胞中，使用β-gal基因作为报告基因测试了逆转录病毒方法。我们可以成功地将β-gal基因导入培养的新生大鼠肝细胞中。3)使用F344大鼠和F344同源性白蛋白血症大鼠测试分离的肝细胞的可移植性。我们通过肝内白蛋白mRNA和白蛋白阳性细胞的增加以及细胞输注到门静脉后30-90天血清白蛋白的升高证实了F344大鼠肝细胞在无白蛋白血症大鼠肝脏内的存在。4)然后测试培养的肝细胞向肝脏和脾脏中的可移植性。将F344大鼠原代分离的肝细胞进行球形培养，并移植到无白蛋白血症大鼠的肝脏和脾脏中。我们证实，移植的肝细胞在受体器官内保持正常的结构和功能。转染到分离的F344大鼠肝细胞中的β-gal基因在移植后在脾脏内暂时表达。

The purpose of this study is to establish a hepatic carcinogenesis model in which the activated oncogenes were transfected into isolated rodent hepatocytes in vitro, and they were subsequently transplanted into the liver or other parts of the body of the syngenic animals. The oncogenes were inserted downstream to the albumin gene promoter, which may facilitate the expression of the transgenes within the hepatocytes. 1)The fragments including core promoters of the albumin gene were amplified by PCR and cloned into a plasmid vector. Using this DNA, we constructed a pAlbElA vector in which ElA was inserted downstream to the albumin promoters. When this vector was introducted into isolated hepatocytes, the gene was transiently expressed in the cells. 2)To efficiently introduce the gene into isolated hepatocytes, a retroviral method was tested using the beta-gal gene as a reporter. We could successfully introduce the beta-gal gene within the cultured newborn rat hepatocytes. 3)Transplantability of isolated hepatocytes was tested using F344 rats and F344 congenic analbuminemic rats. We confirmed the presence of F344 rat hepatocytes within the liver of analbuminemic rats by increase of albumin mRNA and albumin-positive cells within the liver, and elevation of serum albumin 30-90 days after infusion of the cells into the portal vein. 4)Transplantability of the cultured hepatocytes into the livers and spleens was then tested. Primarily-isolated hepatocytes of F344 rats were spheroidally-cultured and transplanted into the livers and spleens of analbuminemic rats. We confirmed that the transplanted hepatocytes maintained normal structures and functions within the organs of the recipients. The beta-gal gene transfected into the isolated F344 rat hepatocytes was temporally expressed within the spleen after transplantation.