Proinsulin-Secreting Hybrid Skin That Can Be Used to Treat Diabetic Animals

Abstract

The present study attempted to utilize the hybrid-type artificial skin (hybrid skin) as an effective transgene carrier for somatic gene therapy. We have elaborated a proinsulin-secreting hybrid skin that can be used to treat diabetic animals. Rat skin fibroblasts (RSFs) were transfected with human proinsulin cDNA-containing plasmid vectors, pBMG-Neo-Ins.¹ The fibroblasts were then maintained for 2 weeks in the culture medium containing G-418 to select transgenic cells (RSF308 cells). Fibroblasts transfected with pBMG-Neo without the insert cDNA were also prepared and used as control cells (RSF1518 cells). An immuno-reactive insulin (IRI) level in the culture medium of RSF308 cells was 319 ± 22.8 µIU/10₆ cells/h (mean ± SD). A hybrid skin was reconstituted in a 35-mm plastic dish by incorporating 2 x 10⁶ rat fibroblasts into 2 ml of three-dimensional lattices of type I collagen (collagen gel) and then placing 106 rat keratinocytes on the gel. We made three types of hybrid skin: (a) a hybrid skin prepared with 2 x 10⁶ nontransfected fibroblasts (HS/RSF); (b) with 1.5 x 106 proinsulin-producing RSF308 cells and 5 x 10⁵ parent fibroblasts (HS/308); and (c) with 1.5 x 10⁶ fibroblasts transfected with pBMG-Neo and 5 x 10⁵ parent fibroblasts (HS/1518). HS/308 produced 178 µlU proinsulin/h at 3 days of culture. To investigate a therapeutic effect of the reconstituted skins on diabetic animals, they were transplanted to the back of streptozotocin-induced diabetic nude mice. At the day of transplantation, the average of blood glucose levels of the experimental animals was 293 ± 48.7 mg/dl. Blood glucose levels of the animals with HS/308 significantly improved to 169 ± 99.4 mg/dl at 28 days, while those with HS/RSF and HS/1518 were 516 ± 64.8 mg/dl and 505 ± 75.8 mg/dl, respectively. The average of plasma IRI levels of animals with HS/308 was 2044 ± 594.6 µlU/ml at 4 to 5 weeks after the transplantation, while those with control HSs were undetectable. These results indicate that the proinsulin-producing hybrid skin may have the potential to treat diabetes mellitus patients and show that the hybrid skin is a useful and effective transgene carrier for somatic gene therapy.