The Biological Activity In Pancreatic Nit-1Cell Regulated By Foxo1and Torc2Transfection And The Relationship Between The Two Genes

Objective: Using small molecules interfering RNA (small interfering of RNA of SiRNA) transfection NIT-1Pancreatic cell line under different concentrations of glucose, FoxOl and TORC2gene expression were measured in The NIT-1cells. Pancreatic β cells Proliferation and apoptosis were detected and gene expression production and relationship of the both of two genes were tested.The results will provide a pivotal theoretical to diabetic treatment.Methods:1By optimize transfection conditions, the effective of SiRNA were measured. With the manual design synthesis of three Pairs T0RC2SiRNA (TORC2SiRNA-1TORC2SiRNA-2, TORC2SiRNA-3), optimize transfection conditions with fluorescently was determined. In accordance with the best three Pairs T0RC2SiRNA transfected by Nanotechnology-based of SBI’s PureFection? Transfection Reagent. LiPosomes to NIT-1cells24hours and then detected by Western blot. NIT-1cells cultured for48hours in1×106/well in6-well culture Plates.2. In accordance different concentrations of glucose, then divided into four groups:5.6mmol/L,11.1mmol/L, and16.7mmol/L,27.6mmol/L and then cultured for120hours.3Intervented with FoxO1siRNA and TORC2SiRNA respectively. Intervention in24hours and48hours of TORC2of small interfering RNA.4the application of four methods of experimental monitoring and assessment:①cell survival and growth determined by MTT assay;②determination of the level of insulin secretion measured with radioimmunoassay;③apoptosis were tested by immunofluorescent asssy with Hoechst33258staining;④AKT, FoxO1, TORC2gene protein expression in NIT cell were deteced by Western-blot "technique.Result:(1) Transfection Reagent. Transfection reagent in accordance with volume (v:v) ratio of4:1mixed transfection is the best cell viability by transfected with fluorescently labeled negative control of SiRNA concentration of160pmol/L after24hours.(2) Cell Proliferationsignificantly higher than the normal control group by FoxO1siRNA inhibition; the contrary, the application of high concentrations of glucose intervention in high-expression the FoxO1genes will significantly inhibits cell Proliferation (P<0.05).(3) The FoxO1aPPlication FoxO1siRNA inhibition of gene exPression levels of insulin secretion was significantly higher than the normal control grouP; the contrary, high exPression of FoxO1gene, the inhibition of insulin secretion (P<0.05).(4) Application FoxO1siRNA inhibition of the the FoxO1gene expression, decreased cell apoptosis was significantly lower than the normal control group; the contrary, high exPression of FoxO1gene, Promotes cell aPoPtosis by immunofluorescent assay with nuclear staining (P<0.05).(5)①Application FoxO1siRNA inhibit FoxO1gene expression, the TORC2Protein expression was reduced with decreasing FoxO1expression, lower than the untransfected FoxO1siRNA group; AKT, Protein expression was no significant change.②The TORC2SiRNA inhibits TORC2gene exPression, The FoxO1protein expression with the TORC2Protein expression was reduced to decrease. All date above are tested with Western blotting.Conclusion:Inhibit the FoxO1gene expression, promote cell proliferation. apoptosis. and stimulate insulin secretion, but also can reduce TORC2gene exPression; high expression of FoxO1can inhibit NIT-1cells Proliferation and Promote apoptosis and reduce insulin secretion. Inhibit TORC2gene expression, the FoxO1gene expression was also reduced.