| [Background]In recent years, with the rapid development of economic, people’s living standards have been improved gradually with a sharp rise in the global prevalence of diabetes, diabetes has become the third killer of threat to human health. In the just concluded10th National Conference of endocrinology, the President of the General Assembly, Professor Guang Ning introduced 《Investigation of2010Diabetics Topics》. The survey shows that9.7percent of the2010prevalence of diabetes mellitus on the2007survey is similar to the males and10.24%,9.05%of women. Therefore, it is imperative to curb the epidemic of diabetes. However, diabetes has become increasingly prominent as the progress of the traditional treatment of oral hypoglycemic drugs, surgery, insulin and other defects of various treatments can no longer prevent the progressive failure of pancreatic β cells.With the progress of treatment traditional oral antidiabetic drugs and insulin of various defects has become increasingly prominent and could not prevent the progressive failure of pancreatic β cells.Similarly, because islet transplantation is lack of source of islet cells, activity decreased after transplantation immune rejection has limited islet cell transplantation. Therefore, to explore to promote islet cell proliferation, enhance islet cell function, and inhibit apoptosis of islet cells of the active substance has become hot and difficult spot in the field of diabetes research.In recent years, the glucagon-like peptide-1in promoting pancreatic β cell proliferation and reducing apoptosis of are concerned. It can promote islet cell proliferation, reduce apoptosis and increase the number of islet cells, but it has more adverse reactions in clinical application, the most common gastrointestinal reactions, and they are expensive and difficult to popularity. Therefore, the development of a new drup which promote islet cell proliferation, inhibit apoptosis in islet cells, fewer adverse reactions, the production process is simple, inexpensive has more significance.In this study,we use the source organ of diabete-spancreas as target,select the animal pancreas as raw materials,take modern processing technology from animal pancreas extract-pancreatic small molecular active peptides as object,it is small molecule active material solution extracted from the young fresh pigpancreas, extract the clear liquid PH value of6.0-7.0,1mL extraction solution containing peptides less than15mg, and insulin-free,which has been used for pancreatic β cellstrains, the experimental results show that pancreatotrophin at certain concentrations can promote the proliferation of islet cells, this experiment will be used in SD rat primary islet cells, a preliminary discussion will be detected on the following aspects:1.to observe pancreatotrophin for largerat islet cell proliferation and function.2. to understand pancreatotrophin on hydrogen peroxide induced apoptosis of rat islet cells identification of apoptosis and Bcl-2, Bax, Caspase3mRNA expression, observe the related protein Bcl-2and Bax expression and explore its mechanism. Part1The effects of pancreatic small molecular active peptides on the apoptosis of the rat islet cells induced by hydrogen peroxide[Objective]Observe the influence of pancreatotrophin pancreatic small molecular active peptides on rat islet cell apoptosis induced by hydrogen peroxide.[Methods](1) Seperation, purification and culture of rat pancreatic islet cells:after the perfusion of collagenase P digestion through the common bile duct,purify the SD rat islets with Ficoll-400density gradient, then culture and observe at37℃in the containing with5%CO2.(2) The experiment group:separaed, cultured of the islet cell were divided into group A-E:A is normal control group:add culture medium containing20%fetal bovine serum RPMI-1640; The group B-E in the culture medium were added to final the concentration of100μg/mL,200μg/mL,300μg/mL,400μg/mL pancreatic small molecular active peptides, each group of cells supposed were set4wells (n=4).(3) Morphological observation of hydrogen peroxide induced apoptosis in rat pancreatic islet cells:cells grouped in the same (2). Each sample randomly selected four vision, stimulate cell apoptosis happened, Hoechst33258nuclear staining and morphological observation.(4) The effects that pancreatic small molecular active peptides to hydrogen peroxide induced apoptosis in rat pancreatic islet cells:cells are grouped with part(2). Cells were apoptosis by100μmmol/L hydrogen peroxide,then cells were staied by Annexin-FITC/PI,and flow cytometry was used to detect apoptosis ratio. (5) The effects from pancreatic small molecular active peptides to hydrogen peroxide induced rat pancreatic islet cells Bax, Bcl-2and Caspase3gene:Cells are grouped with part(2). Cells were apoptosis by100μmmol/L hydrogen peroxide,then islet cells Bax, Bcl-2and Caspase3mRNA expression were detected by qPCR after intervention for5d.(6) Effects of pancreatic small molecular active peptides to hydrogen peroxide induced rat pancreatic islet cells Bax, Bcl-2protein expression:Cells are grouped with (2) part. Islet cells Bax and Bcl-2protein expression were detected by Westerm-blot after intervention for5d.[Results](1) The effects of pancreatic small molecular active peptides against the apoptosis of rat islet induced by hydrogen peroxide:Cell apoptotic rates of rat islet induced by hydrogen peroxide are detected by the AnnexinV-FITC/PI kit and flow cytometry:there are significant differences among the apoptotic rates after various intervention (P=0.000),multiple comparison demonstrated that besides the100μg/mL and200μg/mL groups, the rate of the other two groups are both significantly lower than the control group(P<0.01), but the difference between these two groups is of no statistical significance.(P>0.05)(2) The effects on Bax、Bcl-2and Caspase3of pancreatic small molecular active peptides against the apoptosis of rat islet induced by hydrogen peroxide:The qPCR of the rat islet intervened by pancreatic small molecular active peptides:There are significant differences in Bax mRNA among the intervened groups (P=0.000).Besides the100μg/mL and200μg/mL groups, the expression of Bax in the other two groups are both significantly lower than the control group(P<0.05), but the difference between these two groups is of no statistical significance.(P>0.05).At the same time,There are significant differences in Bcl-2mRNA among the intervened groups (P=0.000).Besides the100μg/mL and200μg/mL groups, the expression of Bcl-2in the other two groups are both significantLy higher than the control group(P<0.05), but the difference between these two groups is of no statistical significance.(P>0.05).In addition,There are significant differences in Caspase mRNA among the intervened groups (P=0.000).Besides the100μg/mL and200μg/mL groups, the expression of Caspase in the other two groups are both significantly lower than the control group(P<0.05), but the difference between these two groups is of no statisticaL significance.(P>0.05).(3) The effects on Bax and Bcl-2protein expression of pancreatic small molecular active peptides against the apoptosis of rat islet induced by hydrogen peroxide:after the intervention with pancreatic small molecular active peptides for5days,the islet cells Bax, Bcl-2protein expression quantitative results are shown:there are significant differences in Bax protein expression among the intervened groups (P=0.001).Besides the100μg/mL and200μg/mL groups, the expression of Bax in the other two groups are both significantly lower than the controlgroup(P<0.05), but the difference between these two groups is of no statistical significance.(P>0.05).At the same time,There are significant differences in Bcl-2protein expression among the intervened groups (P=0.000).Besides the100μg/mL and200μg/mL groups, the expression of Bcl-2in the other two groups are both significantly higher than the control group(P<0.05), but the difference between these two groups is of no statistical significance.(P>0.05).[ConcLusion]Pancreatic small molecular active peptides reveals an obvious protection for the rat islet cells in the high concentration. Part2The effects of pancreatic small molecular active peptides on proliferation and function of rat pancreatic islet cells in vitro[Objective]Observe the effects of pancreatic small molecular active peptides on pancreatic islet cell proliferation and function of cultured rat.[Methods](1) Rats islet cells separation, purification and training:the specific operations are same with the first part (1).(2) The experiment group and processing:islet cells will be divided into blank control group and the experimental group, join common culture medium into blank control group, join different quality concentration (100、200、300、400μg/mL) pancreatic small molecular active peptides element into the medium.(3) pancreatic small molecular active peptides effecs on the proliferation of rat pancreatic islet cells:cells are grouped in the same (2).CCK-8method was used to detect cells proliferation after incubation for1d、3d or5d.(4) The effects of pancreatic small molecular active peptides on function of rat pancreatic islet cells:cells are grouped as the (2) section.Cells were simulated with KRBH contained3.3mM or16.7mM glucose respectly for2h radio-immunity assay method was used to detect insulin secretion after incubation for5d.[Results](1) The effects of pancreatic small molecular active peptides on rat islet cells proliferation activity:rat islet cells proliferation activity had significantly different with various concenteation of pancreatic small molecular active peptides for ld,3d or 5d(P=0.000;P=0.000; P=0.000). Besides the100μg/mL and200μg/mL groups, the proLiferation activity of islet cells for for ld,3d or5d in the other two groups are both significantly higher than the control group(P<0.05), but the difference between any two groups is of no statistical significance.(P>0.05). There are significant differences in the different treatment proliferative activity (P=0.000).Except for the400μg/mL pancreatic small molecular active peptides group, the other four groups in the different intervention time islet cells proliferative activity have no significant difference (P=0.694; P=0.772; P=0.417;P=0.801). The concentration of pancreatic small molecular active peptides in400μg/mL cells proliferative activity cultured for5d significantly higher than of the Id and3d(P<0.05),and there were no significant difference between1d and3d (P>0.05).(2) The effects of pancreatic small molecular active peptides on rat islet cells insulin secretion:After pancreatic small molecular active peptides intervention5d low sugar stimulate the secretion of insulin among the five groups were significantly different (P=0.000).Besides the100μg/mL and200ug/mL groups, the secretion of insulin in the other two groups are both significantly higher than the control group(P<0.05), but the difference between these two groups is of no statistical significance.(P>0.05). High sugar stimulate the secretion of insulin among the five groups were significantly different (P=0.008).Besides the100μg/mL and200μg/mL groups, the secretion of insulin in the other two groups are both significantly higher than the control group(P<0.05), but the difference between these two groups is of no statistical significance.(P>0.05). Insulin stimulation index among the five groups were significantly different (P=0.000).Besides the100μg/mL and200μg/mL groups, the secretion of insulin in the other two groups are both significantly higher than the control group(P<0.05), but the difference between these two groups is of no statistical significance.(P>0.05). [ConcLusion]The high concentrations of pancreatic small mollecular active peptides can not only promote islet cells proliferation, but also can significantly enhance the function of islet cells to secrete insulin. |
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