Experiment Of B10 Site-directed Mutagenesis On Enhancing The Expressive Efficiency Of Human Proinsulin Gene Recombinants In Diabetic Rat

ObjectiveInsulin injection is major treatment of type 1 diabetes and seriouse type 2 diabetes , In spite of the development and application of various insulin formulations and insulin pump, insulin injection can not achieves the same degree of glycemic control as that provided by endogenous insulin , nor prevents the long -term complications associated with type 1 diabetes. With the development of gene recombination and gene transfer technique , insulin gene therapy has being a hot study spot of type 1 diabetes treatment. Its major strategy is to make other cells can produce insulin physiologically with the transfection of insulin cDNA and to control insulin production, thereby reducing the elevated blood glucose levels to a normal range without causing hypoglycemia.To achieve this objective, there are at least three crucial issues that must be adequately addressed: (1) the effectiveness of INS transgene expression for glycemic control. (2) the adequate regulaton of insulin production. (3) safe and effective vector associated with INS transgene expression. We have constructed high titer retrovirus - expressed recombinants of the two - site - mutated or three - site - mutated human proinsulin gene which was colligated with regulatory elements at the upstream. The recombinants was integrated into the genome of the hepatocarcinoma cells, which may secret mature insulin regulated physiologically by glucose and the expressive efficiency was enhanced significantly.By now , in vivo there have no report about the effect of the two mutated in-sulin genes which were colligated with regulatory elements. We have experiment them ex vivo before . This article is a further study about them in vivo.MethodsMake STZ - treated diabetic rat model.Transfer the retrovirus - expressed mutated human insulin gene into the STZ— treated rats.Then tissue expression of the retrovirus - expressed mutated human insulin gene were determined by RT - PCR, at the same time , blood glucose , body weigh ,and blood insulin were observed.Statistical analysis;all data were presented as x ± s, analysis of variance (One -way ANOVA) was used to test the differences between four groups,and paired T test was used for self compare.ResultsBlood glucose of the diabetic rat reduced rapidly on the first day after the injection of the two retrovirus - expressed mutated human insulin gene. After 14 days the effections are most significientThe glucose of the rats transfected with the three - site - muted proinsulin was reduced to normal level . While the glucose level transfected with two - site- mutated human proinsulin gene recombinants is still above the normal level. Diabetic control rats injected with DMEM maintained hyperglycemia during the whole experiment.In the IPGTT test, the glucal line of the rats transfected with the three site muted proinsulin is like that of the normal control rats.ConclusionThe retrovirus - expressed mutated human Insulin gene recombinants were transfered into STZ - treated rats successfully.The retrovirus - expressed three - site - mutated human insulin gene recom-binants enhance the expressive efficiency significantly.In vivo , the retrovirus - expressed mutated human Insulin gene recombi-nants secret mature insulin which was regulated physiologically by glucose and insulin in blood.