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
目的研究在非β细胞中表达成熟胰岛素,探索替代胰岛素注射或胰岛移植治疗1型糖尿病的方法 .方法采用重叠区扩增基因拼接法(gene splicing by overlap extention, gene SOEing)自胰岛素原基因组基因中扩增胰岛素原的cDNA,并于C肽的两端引入蛋白酶furin的识别和切割位点Arg-Xaa-Lys/Arg-Arg.将获得的突变体重组表达载体PcDNA3.1/C,通过脂质体法转染体外培养的Hela,293,和L02细胞,G418筛选L02细胞.同时用放射免疫和免疫荧光的方法监测细胞内外胰岛素的表达水平.结果成功地对胰岛素原cDNA的三个位点进行了突变,空载体转染的细胞无胰岛素表达.而在转染突变后胰岛素原基因cDNA的细胞培养液中胰岛素的表达量各不相同:Hela 和293细胞的暂态表达量分别为8.45~188.00?μIU/2.0×106 cells*d-1和159.88~242.14 ?μIU/2.0×106 cells*d-1, 筛选出的L02各细胞株的表达量为2.56~61. 95?μIU/2.0×106 cells*d-1;免疫荧光显示L02细胞浆内有囊泡状分泌颗粒 .结论突变的胰岛素原cDNA能成功转染非胰岛β细胞并表达胰岛素,为进一步开展糖尿病的基因治疗研究奠定了基础.
更多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.
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