MicroRNA-126 Regulates Insulin Signal Pathway In Rat Pancreatic Islet Beta Cell

Objective: To screen out an optimal method and condition of isolating and culturing pancreatic islet cells for the sake of providing active and functional SD rat pancreatic islet cells for research on the role and mechanism of micro RNA-126( mi R-126) in Type 2 diabetes mellitus(DM2).Method: After successfu Lanesthesia of SD rats via intraperitoneal injection of chloraldurat, 8 m L Collagen V containing 100 U DNase I was administered by retrograde perfusion via the common bile duct. After in situ digestion, SD rat pancreatic islet cells were isolated and purified with gradient centrifugation in Hitopaque-1077. The cultured islet cells were digested with trypsin and then dispersed into individual beta cells for culture.Results: The mean number of pancreatic islet cells isolated from a SD rat was 372±45.The purity of isolated pancreatic islet cells was higher than 90 % with more than 90 %viability.Conclusion: Retrograde perfusion with Collagen V plus DNase I could effectively avoid the production of colloidal substances during pancreatic islet cell digestion, thus avoiding the failure of isolation. Gradient centrifugation in Hitopaque-1077 is an effective method to isolate and purify SD rat pancreatic islet cells due to simplicity.Research background with the change in the human life style and living environment, the number of diabetic patients has been increasing yearly.Over 95 percent of diabetics have type 2 diabetes mellitus(DM2). Insulin resistance plays an impotent role in DM2.It was found in our previous study that mi R-126 played a role in the regulation of insulin signaling pathway, and there was a certain correlation between mi R-126 and insulin resistance.To facilitate further study on the role of mi R-126 in the regulation of insulin signaling pathway and apoptosis in pancreatic islet beta cells, it is necessary to establish a favorite pancreatic islet beta cell model.Objective: The current study aims to investigate the regulatory effects of mi R-126 on beta cell insulin signaling pathway and apoptosis of SD rat beta cells.Method: SD rat pancreatic islet beta cells were transfected with mi R-126 mimic, mi R-126 inhibitor and negative control for 48 h and then stimulated withinsulin(100nm) for 12 h. The transfection efficiency and apoptosis were detected by flow cytometry.RNA was extracted, and the relative expression of mi R-126 and IRS-1/2 was detected by q PCR. The U6 and β-actin expression was used for m RNA-level normalization. IRS1/2, Akt and PIK3R2 protein expressions were detected by Western blot.The GAPDH expression was used as internal reference.Results Thetransfection efficiency of mi R-126 mimics, mi R-126 inhibitor and negative control was high as shown by flow cytometry. Artificial synthetic mi RNA effectively regulated mi RNA expression in beta cells. It was found that the number of apoptotic pancreatic islet beta cells was increased in the overexpression group.q PCR results showed that the IRS-1/2 expression was down-regulated in mi R-126-overxpressed pancreatic islet beta cells. Western blot showed that IRS-1/2, Akt and PIK3R2 protein expressions were down-regulated in mi R-126-overxpressed pancreatic islet beta cells.Conclusion: Overexpression of mi R-126 may be involved in insulin resistance via regulation of IRS-1/2,Akt and PIK3R2 protein expressions, and induce pancreatic islet beta cell apoptosis.