AIM:To study the expression of human insulin gene in gastrointestinal tracts of diabetic rats. METHODS: pCMV.Ins, an expression plasmid of the human insulin gene, wrapped with chitosan nanoparticles, was transfected t...AIM:To study the expression of human insulin gene in gastrointestinal tracts of diabetic rats. METHODS: pCMV.Ins, an expression plasmid of the human insulin gene, wrapped with chitosan nanoparticles, was transfected to the diabetic rats through lavage and coloclysis, respectively. Fasting blood glucose and plasma insulin levels were measured for 7 d. Reverse transcription polymerase chain reaction (RT-PCR) analysis and Western blot analysis were performed to confirm the expression of human insulin gene. RESULTS: Compared with the control group, the fasting blood glucose levels in the lavage and coloclysis groups were decreased significantly in 4 d (5.63 ± 0.48 mmol/L and 5.07 ± 0.37 mmol/L vs 22.12 ± 1.31 mmol/L, respectively, P < 0.01), while the plasma insulin levels were much higher (32.26 ± 1.81 μIU/mL and 32.79 ± 1.84 μIU/mL vs 14.23 ± 1.38 μIU/mL, respectively, P < 0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and coloclysis groups. CONCLUSION: Human insulin gene wrapped with chitosan nanoparticles can be successfully transfected to rats through gastrointestinal tract, indicating that chitosan is a promising non-viral vector.展开更多
To pursue insulin and islet transplantation replacement therapy for type 1 diab etes based on engineered human non β cells which secrete mature insulin Methods Human proinsulin cDNA was cloned from its genomic ge...To pursue insulin and islet transplantation replacement therapy for type 1 diab etes 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 e xtension PCR, introducing furin consensus cleavage sequences (Arg Xaa Lys/Arg Arg) An expression vector encoding a genetically modified human proinsulin c DNA was generated and transduced to Hela, 293, and L02 cells by lipofectin medi ated DNA transfection Following G418 screening, the surviving L02 cells were s elected and enriched Insulin levels in the supernatant and cells were evaluate d using radioimmunoassay and immunofluorescence staining Results Three sites in the insulin gene were mutated simultaneously Insulin gene m odified cells were able to express insulin at different levels: 8 45-188 00? μIU/24 h/2 0×10 6 Hela cells and 159 88-242 14?μIU/24 h/2 0×10 6 293 cells for transient expression, and 2 56-61 95?μIU/24 h/2 0×10 6 from se veral L02 clones screened with G418 No insulin was released by control cells Furthermore, immunofluorescence staining confirmed that proinsulin was stored a s 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 s omatic gene therapy in diabetes mellitus展开更多
文摘AIM:To study the expression of human insulin gene in gastrointestinal tracts of diabetic rats. METHODS: pCMV.Ins, an expression plasmid of the human insulin gene, wrapped with chitosan nanoparticles, was transfected to the diabetic rats through lavage and coloclysis, respectively. Fasting blood glucose and plasma insulin levels were measured for 7 d. Reverse transcription polymerase chain reaction (RT-PCR) analysis and Western blot analysis were performed to confirm the expression of human insulin gene. RESULTS: Compared with the control group, the fasting blood glucose levels in the lavage and coloclysis groups were decreased significantly in 4 d (5.63 ± 0.48 mmol/L and 5.07 ± 0.37 mmol/L vs 22.12 ± 1.31 mmol/L, respectively, P < 0.01), while the plasma insulin levels were much higher (32.26 ± 1.81 μIU/mL and 32.79 ± 1.84 μIU/mL vs 14.23 ± 1.38 μIU/mL, respectively, P < 0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and coloclysis groups. CONCLUSION: Human insulin gene wrapped with chitosan nanoparticles can be successfully transfected to rats through gastrointestinal tract, indicating that chitosan is a promising non-viral vector.
基金agrantfromtheShanghaiMunicipalGovernment (No 9841190 2 4)
文摘To pursue insulin and islet transplantation replacement therapy for type 1 diab etes 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 e xtension PCR, introducing furin consensus cleavage sequences (Arg Xaa Lys/Arg Arg) An expression vector encoding a genetically modified human proinsulin c DNA was generated and transduced to Hela, 293, and L02 cells by lipofectin medi ated DNA transfection Following G418 screening, the surviving L02 cells were s elected and enriched Insulin levels in the supernatant and cells were evaluate d using radioimmunoassay and immunofluorescence staining Results Three sites in the insulin gene were mutated simultaneously Insulin gene m odified cells were able to express insulin at different levels: 8 45-188 00? μIU/24 h/2 0×10 6 Hela cells and 159 88-242 14?μIU/24 h/2 0×10 6 293 cells for transient expression, and 2 56-61 95?μIU/24 h/2 0×10 6 from se veral L02 clones screened with G418 No insulin was released by control cells Furthermore, immunofluorescence staining confirmed that proinsulin was stored a s 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 s omatic gene therapy in diabetes mellitus
