Mature insulin production by engineered non-beta cells / 中华医学杂志(英文版)

ABSTRACT

<p><b>OBJECTIVE</b>To pursue insulin and islet-transplantation replacement therapy for type 1 diabetes based on engineered human non-beta cells which secrete mature insulin.</p><p><b>METHODS</b>Human proinsulin cDNA was cloned from its genomic gene and mutated by overlap extension PCR, introducing furin consensus cleavage sequences (Arg-Xaa-Lys/Arg-Arg). An expression vector encoding a genetically modified human proinsulin cDNA was generated and transduced to Hela, 293, and L02 cells by lipofectin-mediated DNA transfection. Following G418 screening, the surviving L02 cells were selected and enriched. Insulin levels in the supernatant and cells were evaluated using radioimmunoassay and immunofluorescence staining.</p><p><b>RESULTS</b>Three sites in the insulin gene were mutated simultaneously. Insulin gene modified cells were able to express insulin at different levels 8.45 - 188.00 microIU/24 h/2.0 x 10(6) Hela cells and 159.88 - 242.14 microIU/24 h/2.0 x 10(6) 293 cells for transient expression, and 2.56 - 61.95 microIU/24 h/2.0 x 10(6) from several L02 clones screened with G418. No insulin was released by control cells. Furthermore, immunofluorescence staining confirmed that proinsulin was stored as vacuoles in the cytoplasm of L02 cells.</p><p><b>CONCLUSION</b>A correctly mutated human proinsulin cDNA was obtained successfully, transfected and expressed efficiently in non-beta cells, lending support to the study of somatic gene therapy in diabetes mellitus.</p>