| IntroductionData from the WHO showed that, in the mid-1980s of 20th century, the number of patients with diabetes was around 30 million and increases almost four times when reaching the mid-1990s during 10 years, up to 120 million people suffer from diabetes. Moreover, it is pridicted that the number of patients with diabetes in the world will double increase and reach 240 million. The mean annual rate of increase is around 10 percent. China's diabetes prevalence rate increased rapidly during recent 10 years, in 1980 was 6.09‰, in 1993 up to 2.5 percent which was four times increase compared with 10 years ago. The death rate has risen to the third after cancer, cardiovascular disease. So the health and economic problems that diabetes causes have become tough problems for peope and society.The main pathogenesis of Type 2 diabetes is insulin secretion defects and insulin resistance. Although insulin receptor has always been a main candidate gene for study of insulin resistance and type 2 diabetes, however, the role of insulin receptor in insulin resistance remains unclear. Mutation of insulin receptor encoding gene downregulate the insulin effect. The down regulation of insulin receptor gene and protein expression or the increase of insulin receptor degradation lead to insulin secretion defects. It has been confirmed in the 1980s that insulin receptor and four insulin receptor substrates,(IRS)-1, 2, 3, 4 as well as the downstream transduction protein expressed inβcell. The insulin signaling transduction protein could been activated by glucose or direct insulin stimulation, and theβcell of islet is the target cell of insulin effect. Thus the theory of insulin resistance inβcell was proposed. The insulin resistance result from the the expression abnormality of insulin receptor affect the first phase insulin secretion stimulated by glucose, however, little has been performed in this field. Here we take advantage of RNAi technology to promote the down regulation of insulin receptor mRNA inβTC-3 cell. Then the effect of the first phase insulin secretion stimulated by glucose was observed and the dose-effect relationship was analysed which provided the information for the research of pathogenesis of Type 2 diabetes.ObjectiveTo establish a mouse insulin receptor lowered model with mouse insulinomaβTC-3 cells, then incubated with different concentrations of glucose for 0,3,5,10 minutes and the insulin secretion were detected at each time point, for the further approach ofβcells insulin receptor mRNA expression on the impact of the first phase insulin secretion and its dose-effect relationship.MethodsFour pairs of insulin receptor specific siRNAs (experimental group: siRNA-1 group, siRNA-2 group, siRNA-3 group, siRNA-4 group) and one pair of non-specific siRNA (NC) were constructed by chemical synthesis. SiRNAs were transfected intoβTC-3 cells with cationic liposome at different concentration, then the transfection efficiency were observed with fluorescent microscope after 24 h, mean while RT-PCR were used to detect mRNA expression of insulin receptor and RIA used to test the first phase insulin secretion when cells treated with high glucose.Results①Twenty four hours after siRNA transfection ofβTC-3 cells with concentration gradient (50, 100, 150, 200 nmol/L), the transfection efficiencies were (73.1±4.07)%, (92.9±3.35)%, (90.8±4.02)%, (93.9±2.81)% respectively. The 100 nmol/L was used as the final siRNA concentration in our study.②Compared with control group, the expression ofβTC-3 cells'insulin receptor mRNA in experimental groups(siRNA-1, 2, 3 and 4 group) was reduced by 62%, 78.8%, 0%, 7.4% respectively.③RIA used to detect the first phase insulin secretion peak in five minutes indicted that siRNA-1 group decreased about 27.7% than the control group, the siRNA-2 group decreased about 28.6%, the siRNA-3 group decreased about 7.3%, and the siRNA-4 group decreased about 0.8%.Conclusions①The expression of insulin receptor mRNA could be inhibited whenβTC-3 cells were transfected with insulin receptor specific siRNAs.②The first phase insulin secretion stimulated by glucose inβTC-3 cells rely on the expression of insulin receptor. |
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