中华医学杂志（英文版）2002，Vol.115Issue(4)：532-535,4.
基因工程改造非β细胞分泌成熟胰岛素的研究
Mature insulin production by engineered non-β cells
摘要
Abstract
Objective To pursue insulin and islet-transplantation replacement therapy for type 1 diabetes based on engineered human non-β cells which secrete mature insulin.Methods 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. Results Three sites in the insulin gene were mutated simultaneously. Insulin gene modified cells were able to express insulin at different levels: 8.45-188.00μIU/24 h/2.0×106 Hela cells and 159.88-242.14μIU/24 h/2.0×106 293 cells for transient expression, and 2.56-61.95μIU/24 h/2.0×106 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.Conclusion 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.关键词
人胰岛素原基因/1型糖尿病/基因治疗/胰岛Key words
human proinsulin gene/type 1 diabetes/gene therapy/islets分类
医药卫生引用本文复制引用
沈坤堂,秦新裕,肖华胜,张新,许相儒,韩泽广..基因工程改造非β细胞分泌成熟胰岛素的研究[J].中华医学杂志（英文版）,2002,115(4):532-535,4.基金项目
This study was supported by a grant from the Shanghai Municipal Government (No. 984119024). （No. 984119024）
