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

本研究的目的是在体外将活化的癌基因导入体外分离的啮齿动物肝细胞，然后将其移植到同种动物的肝脏或身体其他部位，从而建立一种肝癌变模型。癌基因被插入到白蛋白基因启动子的下游，这可能有助于转基因在肝细胞内的表达。1)用聚合酶链式反应扩增白蛋白基因核心启动子片段，克隆到载体上。利用该DNA，我们构建了将ELA插入到白蛋白启动子下游的pAlbElA载体。当该载体被导入分离的肝细胞时，该基因在细胞中瞬时表达。2)为了有效地将该基因导入分离的肝细胞，以β-半乳糖基因为报告基因，对逆转录病毒方法进行了检测。我们可以成功地将β-半乳糖基因导入培养的新生大鼠肝细胞。3)用F344大鼠和F344同源同种异型白蛋白血症大鼠检测分离的肝细胞的可移植性。在门静脉注射F344大鼠肝细胞30-90天后，通过肝内白蛋白mRNA和白蛋白阳性细胞的增加以及血清白蛋白的升高，我们证实了F344大鼠肝脏内存在F344大鼠肝细胞。4)对培养的肝细胞进行肝、脾移植实验。原代分离的F344大鼠肝细胞经球体培养后移植到模拟蛋白血症大鼠的肝和脾内。我们证实，移植的肝细胞在受体的器官内保持了正常的结构和功能。将β-半乳糖基因导入分离的F344大鼠肝细胞，移植后在脾内有暂时性表达。

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.