|Budget Amount *help
¥7,500,000 (Direct Cost : ¥7,500,000)
Fiscal Year 1995 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1994 : ¥6,000,000 (Direct Cost : ¥6,000,000)
The present project was designed to develope an artificial pancreas. The artificial pancreas was prepared by culturing transkaryotic artificial beta-cells in diffusion chamber with microcarrier beads, and artificial beta-cells were produced by transfection of human proinsulin gene into Chinese hamster ovary (CHO) cells. Total RNA was prepared from human pancreas, and human proinsulin cDNA was prepared from RNA by reverse transcriptase-polymerase chain reaction. After digesting with restriction enzymes cDNA was ligated into cloning vector and inserted into competent E.coli by heat shock. Transformed E.coli was selected by X-gal colony formation method, proliferated, and lysed. Separated proinsulin cDNA was digested with restriction enzymes, ligated with mammalian expression vector having Neomycin resistance gene, and transfected into CHO cells by Lipofectin method. Transfected CHO cells were then cultured with medium including Neomycin, and 8 clones (CHO/I) were separated, and they released immunoreactive insulin (IRI) into culture supernatant. CHO/I cells were cultured in diffusion chamber (DC) with microcarrier beads and then transplanted into the peritoneal cavity of 90% pancreatectomized Wistar rats, of which the fasting serum IRI was not detected and the fasting blood glucose (FBG) ranged between 250 and 450 mg/dl. After transplantation of DCs, the fasting serum IRI increased to the level between 5 and 25 mu U/ml and the FBG decreased to the level between 100 and 200 mg/dl, respectively, for 12 weeks, after when DCs were removed from rats, and immuno-peroxidase staining also demonstrated the proinsulin in cytoplasm of CHO/I cells.
These results demonstrate that human proinsulin gene can be transfected into mammalian cells and work for more than 3 months, and suggest that this kind of gene technology may be applicable for development of artificial pancreas.