Expression Of Proinsulin Gene In Rat Ntestinal Epithelial Cell

Objective: Diabetes mellitus is caused by defects in insulin secretion or its biological function is impaired,or both lead to a group characterized by hyperglycemia and metabolic diseases.Previous studies have found that the small intestine is capable of secreting insulin,which plays a role in the pathogenesis of diabetes.To further study the secretion of insulin in the small intestine of the rat,we extracted the cells of rat small intestine epithelial in vitro by this study.then determine the insulin gene in rat intestinal epithelial cell,and whether the rat intestinal epithelial cell can secrete proinsulin insulin and C peptide.To define the function of the insulin secretion of rat small intestine epithelial cells.Methods: The wistar pregnant rats were killed by cervical dislocation,split the uterus and get the small intestine of fetal rat,the epithelial cells of fetal rat intestine and islet cells were cultured in glucose concentrations were 100mg/dL,200mg/dL,300mg/dL,400mg/dL,islet cells group for comparison.After passage,we obtained the total RNA from the cells,reverse transcription into cDNA,PCR to detect the insulin of rat intestinal epithelial cells in primary mRNA,and then use Quantitative Real-time PCR to detect the relative expression of mRNA in rat intestinal epithelial cells and pancreatic cells.And use statistical methods to compare expression of insulin mRNA in two cells at the same concentration.We use the method of immunohistochemistry to detect insulin and proinsul in cells.Use the method fo protein chromatography to separate and purificat protein,then use Western Blot to identify the activity of proinsulin.Results: After PCR to detect the rat small intestinal epithelial cell have the expression of insulin gene.Data displayed from Quantitative Real-time PCR show that in the normal circumstances the rat intestinal epithelial cell insulin mRNA expression was lower than pancreatic cells,but under the condition of high concentration of glucose insulin mRNA expression level was improved,in the glucose concentration when 200mg/dL reached the highest,nearly the pancreatic cells in rats.Through determination,can determine the rat intestinal epithelial cells can secrete proinsulin,but without insulin and C peptide.Conclusion: The experiment proved that the small intestine of rats can express the gene of insulin.And there was no significant difference in the expression of insulin mRNA between the small intestine epithelial and the islet cells at a glucose concentration of 200mg/dL(P>0.05).This experiment shows that rat intestinal epithelial cells have the function to secrete proinsulin.And has a close relationship with the concentration of glucose,may be related to the pathogenesis of diabetes.