| Aim To study the effect of proinsulin gene therapy on diabetic rats, and construct pseudo- β cell line.Methods ( 1 ) Total RNA was extracted from rat pancrease and the desired cDNA was obtained by RT-PCR. After enzyme reaction with EcoR I and Hind III, rat proinsulin gene was oriently cloned into eukaryotic cell expression vector pCMV by recombinant gene and molecular cloning technique. ( 2 ) Recombinant eukaryotic cell expression plasmid of rat proinsulin gene pCMV/proinsulin was transferred into streptozotocin-induced diabetic rats by intraportal infusion and intramuscular injection, respectively, to observe the effect of rat proinsulin gene therapy on diabetic rats' blood glucose. The treatment group by intraportal infusion (Group A) was injected with pCMV/proinsulin naked plasmid DNA 100 μ g , while the corresponding control group (Group B) was treated with similar amount of pCMV DNA. The treatment group by intramuscular injection (Group C) was injected with pCMV/proinsulin naked plasmid DNA 100 μg, whereas the corresponding control group (Group D) was treated with similar amount of pCMV DNA. Normal group and DM group were also observed at the same time. Blood glucose level was tested and plasma insulin was determined by hypersensitive radioimmunoassay. RT-PCRwas used to detemine the alteration of proinsulin mRNA expression in liver, skeletal muscles and pancrease. Sections of livers, skeletal muscles and pancrease in each group were observed by immunohistochemistry and stained with haematoxylin and eosin for morphological studies. ( 3 ) Recombinant eukaryotic cell expression plasmid of rat proinsulin gene pCMV/proinsulin was introduced into mouse intestinal epithelial cells (IEC-6) by lipofetin-mediated DNA transfection. RT-PCR was used to detect the expression of proinsulin gene mRNA in gene modified IEC-6 cells and hypersensitive radioimmunoassay was applied to detect insulin levels produced by the IEC-6 cells containing proinsulin gene. Results ( 1 ) The recombinant eukaryotic cell expression plasmid of rat proinsulin gene pCMV/proinsulin was obtained. ( 2 ) The blood glucose levels in two treated group were both decreased. In Group A, the level of blood sugar decreased about 7mmol/L and the phenomenon of glycemic control maintained for 3 ~ 4 weeks. The serum concentration of insulin step up significantly after pCMV/proinsulin gene therapy. The blood glucose in Group A was significantly lower than that of Droup B and DM Group (P<0.05 ) , while serum proinsulin was higher than that of two groups (P<0.05 ) . In Group C, the blood glucose level decreased about 4mmol/L and glycemic control maintained for 1~2 weeks. Meanwhile, the serum concentration of insulin increased markedly after gene therapy. The blood glucose in Group C was significantly lower than that of Group... |
