PTP1B Targets Using RNAi Technology Preliminary Study Of The Treatment Of Diabetes

Diabetes and diabetes complication are the third killer of human lives in modern society. The most widely spreading subtype, type 2 diabetes, is associated with insufficient insulin secretion and impaired insulin response. It was reported that defects of post-receptor signaling are related with type 2 diabetes and PTPIB, a negative regulator of insulin signaling pathway is highlighted. Inhibition of PTP1B sensitizes insulin action, and improves symptoms of type 2 diabetes.The work about this thesis was focused on PTPIB. We use RNA interference techniques to inhibit PTPIB gene and investigate the effect on up-regulating insulin signaling in cells and improving of diabetic symptoms in animal models.The main purpose of the first chapter is to select one siRNA sequence with best inhibitory effect against PTPIB. We constructed 5 short hairpin RNA expression plasmids, pSilencer-MPl~5, each containing one piece of five distinct regions of mouse PTPIB open reading frame. The PTPIB ORF was inserted in front of EGFP gene in plasmid pEGFP-N2 to express PTP1B-EGFP fusion protein (named plB-EGFP). When both pSilencer-MP and plB-EGFP were co-transfected, the fluorescence of fusion protein was weakened by RNA interference against PTPIB. According to this method, one fragment was screened out with best inhibitory effect. We synthesized siRNA against this sequence, and also prepared endonuclease-hydrolyzed PTPIB siRNA (esiMP). Both siRNAs were proven to be with more than 90% inhibitory efficiency.The main content of the second chapter is to investigate the effect of PTP1B siRNA in vivo. We constructed a reporter system in which secretory HBsAg is driven by insulin controlled mouse fatty acid synthase promoter. By this reporter system, we can monitor insulin signaling by measuring HBsAg secretion. The hydrodynamic injection was used to transfer pSilencer-MP plasmid and siRNA into mice livers, and an up-regulated HBsAg expression level was observed. Alloxan induced diabetic mouse model was used to determine the anti-diabetic effects of siRNAs against PTPIB. In siRNA treated group, decreased plasma glucose was observed in the next day post-injection.In the third chapter we assayed expression profile of other phosphates in response to reduction of PTPIB expression using RT-PCR. Although mRNA level of SHP2...