| Objective: With the development of economy and people lifestyle changes, the trend of aging increase rapidly in modern society. The morbidity of Diabetes Mellitus(DM) is rising year after year and influences on quality of life seriously.International Diabetes Federation reported that in2011 there are at least 366 million patients with diabetes, the number of patients with diabetes will grow to 566 millions in 2030. Diabetic patients with multiple metabolic disorders leading to vascular endothelial dysfunction, which caused a series of diabetic vascular complications.Diabetic vascular complications has become a major cause of disability and death, therefore the protection of vascular endothelial cells has important significance in diabetic patients.The pathogenesis of diabetic vascular complications include the formation of the polyol pathway, protein kinase C activation, advanced glycation end products and its receptor activation, hexosamine pathway and mitochondrial reactive oxygen species(ROS) generation increased etc.The increase in mitochondrial reactive oxygen species produced by oxidative stress is believed to be the initiating step and key factor.Glucagon like peptide-1(Glucagon-like peptide 1, GLP-1) is a peptide hormone secreted by intestinal L cells, with the Glucose dependent effect on pancreatic beta cells, promote insulin gene transcription, increase insulin biosynthesis and secretion.Research shows that GLP-1 can stimulate the proliferation and differentiation of islet beta cells, inhibit beta cell apoptosis, so as to increase the number of beta cells, and can also inhibit glucagon secretion, suppressing appetite and food intake, delay the emptying of gastric contents etc.These features are beneficial to reduce postprandial blood glucose and keep blood glucose at a constant level, therefore in the treatment of type 2 diabetes is paid more and more attention.In recent years, studies have shown that GLP-1 can reduce the inflammatory injury in blood vessels, the receptor agonists can stimulate endothelial cell proliferation, repair the damaged vessel, but whether the damage to endothelial cells induced by H2O2 has a protective effect is not clear.H2O2 is a stable and active oxygen free radical, and vascular endothelial cell injury is an important characteristic of diabetes regardless of macrovascular or microvascular complications.Therefore, human umbilical vein endothelial cell(HUVECs) preparation of cellular oxidative stress model in this study H2O2 in cultured GLP-1 after treatment, observe whether can reduce the injury of endothelial cells induced by H2O2.Method: HUVECs culture: Isolation of HUVECs by collagenase perfusion digestion method,inoculated to the culture flask coated with polylysine 25cm2.After 24 h culture,culture medium is changed.When the primary cell fusion is more than 90%, with 0.05% trypsin digestion of-0.02%EDTA solution, we can subculture.The fourth generation of endothelial cells are used for determination of cell viability.Identification of HUVECs: Application of Rabbit anti human factor VIII monoclonal antibody immunocytochemistry staining was performed in HUVECs.Cell viability was measured by CCK-8 assay:Under the microscope, HUVECs which growed to about 70% were randomly divided into 5 groups,H2O2 were added in each group,which concentrations respectively were 0μM, 100μM, 200μM, 400μM, 800μM.the human umbilical vein endothelial cells were cultured for 8,16,24 hours, when arrived at the specified time,testing the absorbance of each pore in the 450 n M with the multifunctional enzyme mark instrument.According to the survival rate of each group cells, we selected appropriate stimulus concentration and time.after GLP-1(7-36)intervention,cell viability was measured by CCK-8 method:cells were randomly divided into control group and stimulating group,the stimulation group addedthe 200μM H2O2 were also addedthe 0n M, 200 n M, 400 n M, 800 n M GLP-1(7-36)and then were incubated for 24 hours.After 24 hours,we valued the absorbance and the cell survival rate.Statistical analysis: All data were presented as mean±SD. Statistical analysis was determined by the independent Student t test and one-way ANOVA using SPSS 13.0 software. A P value cut-off 0.05 was considered significant.Results:1 The first generation of HUVECs were spindle shaped or polygonal cells, whose cell body is full,surface is smooth and boundary is clear.2 CCK-8 assay revealed loss of cell viability significantly after stimulating with H2O2 for 24 h. Cell viability assay also showed the dose-dependent and time-dependent excitotoxicity of H2O2 on primary cultured HUVECs.3 After the treatment of GLP-1(7-36), the viability of endothelial cells increased significantly(P < 0.01).Conclusion:1 The results of CCK-8 assay suggested that the cell viability was significantly reduced after H2O2 stimulation.Our study demonstrated that the injury of HUVECs induced by oxidative stress may be one of the most important pathgenesis of Diabetes complications.2 The results of CCK-8 assay suggested that the cell viability was significantly increased after the intervention of GLP-1(7-36). |
