The Establishment And Application Of Screening Model For Protein Tyrosine Phosphatase Inhibitor

Protein tyrosine phosphatases are important regulators for cell signal transduction,closely related to human disease. SHP-1is an important member of SH2domain containing ahighly conserved protein tyrosine phosphatase sub-family, studies have shown that PTPs hasa close relationship with blood glucose control in patients with diabetes mellitus and diabeticvascular complications. Highly active inhibitors for protein tyrosine phosphatase has broadapplication prospect in the treatment of diabetes and obesityIn this paper we extracted mRNA from human brain tissue and built protein tyrosinephosphatase SHP-1-pEASY-E1recombinant plasmid by RT-PCR amplification of the targetcDNA. The recombinant plasmid was transformed into E.coli competent cells of TOP10, thepositive clones were verified, converting correct sequencing of plasmid in E.coli Transettacompetent cells to express SHP-1. In order to make SHP-1efficient and stable expression invitro we optimize the expressive condition of SHP-1protein. The results showed thatthe optimal induction concentration of IPTG was0.4mmol/L and the best temperature forinduction was20℃. Established a screening model for protein tyrosine phosphatasehigh-throughput by screening of various compounds with SHP-1model and we found highlyactive inhibitors of SHP-1, The results is that the sample J11310are active compoundsscreened.Human hepatocellular carcinoma cell line HepG2cells and rat skeletal myoblast L6cellswere selected as the research object and we selected the insulin30nM as positive controlgroup, and evaluated the effect of J11310on glucose metabolism in cells. The results showedthat sample J11310are non-toxic on HepG2cells and l6cells, the HepG2and L6cells thatcan be dose-dependent glucose consumption, there are significant effect on promoting consumption of sugar when concentration was1μg/ml. Mechanism of the sample of J11310has played the role of glucose lowering through the promotion of the liver and peripheralutilization of glucose. In HepG2cells by adding insulin, presence or absence of controlsamples, taking rosiglitazone10-5M as the positive control, comparison of glucoseconsumption in order to evaluate samples with and without insulin cells synergies. Resultsshow that, J11310has better insulin coordination in HepG2cells, a stronger effect thanrosiglitazone,1μg/ml J11310had the strongest effect with the decrease of concentration,sample synergistic action on glucose consumption is gradually weakened, but there is nosynergistic effect in10-4μg/ml,10-5μg/ml. The damage of RINm5F cells with differentconcentration of Alloxan, samples of different concentrations of J11310on the damage ofislet cell RINm5F has no protective effect. Using cell immunofluorescence method fordetecting cells in PGC-1α, Pi-AMPK, expression of Pi-IRS, J11310effect on HepG2resultsshowed, sample cells, phosphorylated AMPK and phosphorylated IRs expression increasedand there is no obvious effect on the expression of PGC-1α. This suggests that J11310mayplay a regulation effect of glucose and lipid metabolism by activating AMPK activity, whilealso improving IRS phosphorylation levels and activate downstream signal transduction ofpancreatic islet in order to promote the transport of glucose in the blood to the cells andreduce blood sugar levels.The above research for finding high activity of SHP-1inhibitors, and evaluation ofbiological activity of compounds in the body laid the foundation. It provides us research ideasof medicines for diabetes and obesity, has important research value and application prospectat the same time.