Study Of Effects Of Type 1 Parathyroid Hormone Receptor On INS-1 Cell Under High Glucose

BackgroundWith the development of global economy and improvement of people's material living standard, those result in rapid increasing trend of the incidence rate of diabetes mellitus, and it has become one of chronic non-communicable diseases damaged human health seriously. Diabetes mellitus will be prevented and treated early, which maybe bring the best way to reduce the incidence rate of diabetes mellitus, but our endocrine doctor will face a pressing study task of diabetes mellitus.Insulin resistant and dysfunction of beta cells take part in duration of diabetes mellitus, are two of the basic pathogenesis of diabetes mellitus. Therefore, at the molecular and genetic level studies on the mechanism causing dysfunction of beta cells, contribute to our deeper understanding of the occurrence, development process and basic pathophysiology of diabetes mellitus, and provide effective ways for clinical prevention, diagnosis and treatment with diabetes mellitus. In recent years, with the development of molecular biology, It is possible for the treatment of disease, to provide a new target, thereby enhancing the accuracy and success rate of treatment. Recent studies have shown that the new drugs research such as GLP-1 receptor agonist prove an effective molecular target in the treatment of diabetes. GLP-1 considered as hormone-like peptide and gastric inhibitory polypeptide (GIP) comprise the intestinal "incretin" hormones released from the intestine in response to feeding and augment glucose-stimulated insulin secretion from the beta-cells of the islets of Langerhans. GLP-1 also enhances insulin-stimulated glucose uptake in peripheral tissues (muscle, fat, liver), suppresses glucagon secretion, induces satiety, and promotes the growth and differentiation of new beta-cells in the pancreas. These antidiabetic properties of GLP-1 depend on GLP-1 receptor, which belong to G protein couple receptor. G protein couple receptors have prompted considerable interest in the potential of function and regulation for metabolic organ and tissue, and are conceived as metabolic receptors. Proliferation of beta cell and insulin secretion are often dependent on the majority of G protein couple receptor regulating the same time, following the interaction between various G protein couple receptor also affectβ-cell function.Just like GLP-1, parathyroid hormone-related protein (PTHrP) also is known as hormone-like protein, and whose receptor Type 1 receptor parathyroid hormone-related protein (PTH1R) belongs to G protein couple receptor. PTHrP and PTH1R discovered in recent years the role of pancreatic beta cell function as a target related to the gradual attention. Epidemiological studies found that diabetes significantly increased serum PTHrP, recently our research group in diabetic nephropathy also showed significantly high glucose-induced renal tubular epithelial cells and the expression of PTHrP and the PTH1R, but recent studies have shown that the expression of PTHrP were found almost all body organs and tissues.The four kinds of islet endocrine cells (α,β,δ, PP) cells can express PTHrP. At the same time, in beta cells PTH1R as the only role of the PTHrP receptor, is considered in the study of PTH1R in beta cells may be more representative than PTHrP. PTH1R relate to signal pathway activation and cell proliferation related to a variety of tissues and cells in the past has been confirmed, particularly studies have found that PTH1R activation of signal transduction and the existence of cross-cutting role in insulin secretion. but so far, with regard to PTHrP and the PTH1R in diabetes occurrence and development of the relationship is not very clear,Diabetes mellitus arises following the body's inability to normally respond to high blood glucose levels. This inability can reflect different pathological changes provoking the failure of the pancreatic beta cell to secrete enough insulin to normalize blood glucose levels. hyperglycemia exerts deleterious effects on beta cell function, as shown in primary cultured rat and human islet cells as well as beta cell lines. One of the hallmarks of such glucotoxicity is reduced insulin gene expression caused by decreased insulin promoter activity. The transcriptional activity of the insulin gene is mainly regulated by transcription factors that are prominently expressed in beta cells. These include pancreatic duodenal homeobox factor 1 (PDX-1), BETA/NeuroD, and GLUT-2, and it has been demonstrated that impaired insulin gene expression resulting from prolonged exposure to elevated glucose levels is associated with diminished those proteins activity, which pathway mostly depend on G protein couple receptors. The research suggests that PTH1R may also participate in the islet beta-cell proliferation and secretion process, but increased expression of PTH1R in diabetes and its mechanism of action is for the protection factor, or pathogenic factor? we will conduct a preliminary study,which were not reported at home and abroad.The topics to rat islet cell tumor cell line INS-1 to study the carrier, with high sugar state of the entry point for the study to explore cytokine PTH1R and islet beta cell function in relationships and their functions PTH1R-mediated mechanism. With a view to prevention and treatment of diabetes, to provide more theoretical support. Specific study consists of three chapters Chapterl Study of Small RNA interference PTH1R gene vector construction, identification and transfection in INS-1 cellObjectiveBy constructing a small RNA interference PTH1R gene vector and identified, PTH1R gene transfected in INS-1 cells were detected by transfection efficiency, in order to clarify the role of PTH1R in the latter part of mechanism of INS-1 cells under high glucose condition, and provide technical assuranceMethods1. Online search for suitable PTH1R interference target, three positive clones were selected and a negative clone used in the experiment after the synthesis of interference fragment.2. The establishment of interfering with PTH1R gene carriers:The synthetic oligodeoxynucleotides annealed to conduct pSUPERretro-GFP/Neo vector digestion, annealing the formation of DNA double-stranded and double-digested vector by a certain percentage of the connection will connect the product was transformed into DH5αCompetent in the colony PCR, an initial positive clones were selected and sequenced to finalize positive clones. The use of Lipofectamine2000 transfected INS-1 cells..3. Realtime-PCR and Western blotting were performed to assay the expressions of PTH1R mRNA and protein. The transfection effieieney were detected by counting the rate of transfected cells cultured.4. Statistical analysis:Statical analyses were performed using the SPSS 13.0 package. The results are presented as the mean±S.D of at least three-independentexperiments. Data were analyzed by one-way ANOVA. The level of significant was set at P<0.05.Results1. The positive clone of PTH1R screening and identification:online search for suitable targets PTH1R gene disruption, synthesis, back to form double-stranded DNA and by double digestion pSUPERretro-GFP/Neo connection, colony PCR, an initial positive clones were selected and sequenced, The positive clones were finally identified with the theoretical expectations.2. Transfection of small RNA interference PTH1R gene in INS-1 cells:Transfection of small interfering PTH1R vector pSUPERretro-GFP/Neo- into INS-1 cells by Lipofectamine2000. Fluorescent green fluorescence could be seen under the fluorescence microscope to detect the three positive clones and one negative clone transfection efficiency,which is the best in 48h. Realtime-PCR and Western blotting were used to be proof of incorporates a small interfering RNA of the PTH1R in INS-1 cells.Conclusions It was successfully constructed and found out INS-1 cell with the specificsilencing of rat parathyroid hormone receptor 1 gene pSUPERretro-GFP/Neointerfere with retroviral vector.Chapter2 Study of the effect of PTH1R on the cells function under high glucose in INS-1 cellsObjectiveTo observed the cell proliferation, apoptosis and insulin synthesis and secretion of capacity in INS-1 under high glucose, and clarify the effect of SiPTHlR. the function of glucose stimulation of insulin secretion (GSIS) capability and intracellular insulin levels in the situation of PTH1R gene silencing was assessed under high concentrations of glucose, following the expression of GLUT-2 was detected.Methods1. According to the literature, the glucose concentration intruduced 25mmol/L glucose, Control group, mock vector group, SiPTH1R-negative control group and SiPTHIR group were observed, whose glucose-stimulated insulin secretion eapability and intracellular insulin content were measured by radioimmunoassay in INS-1 cell.2. GLUT-2 mRNA and protein expression were detected by Realtime PCR and Western blotting in INS-1 cells under high glucose for 48h. Which experiment was divided into 4 groups under high glucose, the first group was Control group, the second group was SiPTH1R-NC group, the third group was 20nmol/L PTHrP treatment group and the forth group was SiPTHIR group.3. Statistical analysis:Statical analyses were performed using the SPSS 13.0 package. The results are presented as the mean±S.D of at least three-independent experiments. Data were analyzed by one-way ANOVA. The level of significant was set at P<0.05.Results1. The effect of SiPTHIR on glucose-stimulated insulin secretion(GSIS) capability under high glucose:Compared with Control group, vector group and SiPTH1R-NC group, the GSIS capability under high glucose after 48h of SiPTHIR were decreased by 24.1%(P<0.05),30.84%(P<0.01)及25.26%(P <0.05), respectively.2. The effect of SiPTH1R on intracellular insulin levels under high glucose:Control group, mock vector group, SiPTH1R-NC group and SiPTH1R group have similar mtracellular insulin levels (P>0.05).3. Effect of PTH1R on the expression of GLUT-2 in INS-1 cell under high glucose: Compare with Control group, SiPTH1R-NC group and PTHrP group, the expression of NeuroD mRNA and protein were decreased, and PTH1R was activation by PTHrP induced higher the expression of GLUT-2 than Control and SiPTH1R-NC group.Conclusions1. SiPTHIR had no significant effect on intracellular insulin levels of INS-1 cells, but significantly attenuated GSIS capability in INS-1 cells. PTH1R could take part in GSIS capability in INS-1 cell.2. PTH1R activation may be one of the mechanisms in PTH1R-induced the change of insulin secretion of beta cell under high glucose and it participated in the formation of insulin secretion by GLUT-2. There is a closely relationship between PTH1R activation and insulin secretion, which dependent on GLUT-2 partily.Chapter3 Study of the effect of PTH1R on the cells viability under high glucose in INS-1 cellsObjectiveTo observed the cell viability in INS-1 under high glucose, and clarify the effect of SiPTH1R. the cell growth activity, cell apoptosis, cell-cycle and the expression of various apoptosis and proliferation of factors in INS-1 under high concentrations of glucose, following the expression of NeruoD was detected.Methods1. According to the literature, the glucose concentration intruduced 25mmol/L glucose, Control group, vector group, SiPTH1R-negative control group and SiPTH1R group were observed by using MTT to detect cell proliferation, Annexin V detection of cell apoptosis, cell cycle detected by DNA Flow Cytometry and immunocytochemical detection of pro-apoptotic protein Bad and anti-apoptotic protein Bcl-2 expression.2. NeuroD mRNA and protein expression were detected by Realtime PCR and Western blotting in INS-1 cells under high glucose for 48h. Which experiment was divided into 4 groups under high glucose, the first group was Control groups, the second group was SiPTH1R-NC group, the third group was 20nmol/L PTHrP treatment group and the forth group was SiPTH1R group.3. Statistical analysis:Statical analyses were performed using the SPSS 13.0 package. The results are presented as the mean±S.D of at least three-independent experiments. Data were analyzed by one-way ANOVA. The level of significant was set at P<0.05.Results1. Effect of SiPTHIR on cell activity in INS-1 cell under high glucose:Compare with Control group, vector group and SiPTH1R-NC group, the cell activity under high glucose after 48h of SiPTHIR were decreased by 22.11%(P<0.01),18.90%(P<0.01),29.88%(P<0.01), respectively.2. The effect of SiPTH1R on cell apoptosis under high glucose:Under high glucose, the rate of cell apoptosis in SiPTHIR was 19.57±1.18%. which was higher than Control group, vector group and SiPTH1R-NC group(P<0.01).3. The effect of SiPTHIR on cell apoptosis under high glucose:SiPTHIR resulted in inhibiting the cell cycle from G0/G1 to S, compared with Control, Mock and SiPTH1R-NC(P<0.01)..4. The effect of SiPTH1R on the expression of Bad and Bcl-2 in INS-1 under high glucose:SiPTHIR result in up-regulated the expression of Bad and down-regulated the expression of Bcl-2 compared with Control group, vector group, and SiPTH1R-NC group under high glucose.5. Effect of PTH1R on the exprsssion of NeuroD in INS-1 under high glucose: Compare with Control group, SiPTH1R-NC group and PTHrP group, the expression of NeuroD mRNA and protein were decreased, but there are not significant change in Control group, SiPTH1R-NC and PTHrP group.Conclusions1. SiPTHIR affect the cell activity rate in INS-1 cells under high glucose, which result in cells apoptosis. PTH1R could take a role in INS-1 viability.2. SiPTHIR can reduce the INS-1 cell NeuroD protein expression. It indicates that the reduction expression of NeuroD protein may be one of the mechanisms in PTH1R-induced the change of proliferation or apoptosis of beta cell under high glucose. The maintain of expression of PTHIR can completely keep the expression levels of NeuroD protein in INS-1. It shows that inactivation of PTHIR is involved in the PTH1R-mediated reduction of cell apoptosis.