The Intervention Effect Of α-Tocopherol On Pancreatic Beta Cells Damaged By Streptozotocin And Its Relevant Mechanism

PART ONE:THE MORPHOUS AND FUNCTION IN VITRO. CULTURE OF PRIMARY PANCREATIC CELLS OF NEONATE RATSObjective:To investigate the function of pancreatic beta cells from neonate rats cultured in vitro.Methods:The pancreatic tissues of neonate rats underwent repeated digestion for short durations with collagenase,and the cell growth was observed under inverted microscope l8h after cell inoculation。Then the cultured cells were transferred to a new culture plate。The pancreatic beta cells were identified by dithizone and immunohistochemical staining,and insulin secretion stimulated by high-glucose were examined by radioimmunoassay.72h after cell inoculation。Results: the ratio of dithizone-stained cells were more than 80%with the cell viability exceeding 90% as assessed by trypan blue staining.The secretion function of the cultured cells remained normal. Conclusion: Repeated digestion of the pancreatic tissues for short durations with collagenase and timely cell transfer to new plate may help achieve highly purified viable islet cells that are well applicable for experimental studies.PART TWO: THE INTERVENTION EFFECT OFα-TOCOPHEROL ON PANCREATICβCELLS DAMAGED BY STREPTOZOTOCINObjective:To investigate the intervention effect ofα-tocopherol on pancreatic beta cells damaged by STZ.Methods: 1.Groups: Isolated and cultured beta cells from pancreas of newborn Wistar rats were divided into four groups:(1)control group; (2)α-T group: onlyα-T was added to the culture medium at the concentration of 20 mg/L for 24 hours;(3)STZ group: only STZ was added to the culture medium at the concentration of 0.58 mg/L for 30 minutes;(4) intervention group: Islet cells were treated for 24 hours with 20mg/Lα-T,then 0.58 mg/L STZ was added for 30 minutes.Culture medium was removed at specific time and the cells were resuspended with normal culture medium, then the following procedures were undertaken after 18 hours of recovery period.2. Measurements: the viability of islet cells was evaluated by MTT; insulin contents of cell supernatant were examined by radioimmunoassay; the apoptotic cells were identified by transmission electron microscope and Hoechest33258 fluorescence staining under fluorescent microscope as well as quantified by TUNEL.Results: In STZ group, the insulin contents and the viability of islet cells were lower, the apoptosis rate was higher than those in control group (p﹤0.05).The apoptotic beta cells were identified by transmission electron microscope and Hoechest33258 fluorescence staining under fluorescent microscope. In intervention group, the insulin contents, the activity of islet cells and the apoptosis rate did not return to the normal levels, but they were inhibited significantly byα-T (compared with STZ group, p﹤0.05).Conclusion:α-T can inhibite the apoptosis of pancreatic beta cells induced by STZ.PART THREE: THE INTERVENTION MECHANISM OFα-TOCOPHEROL ON PANCREATICβCELLS DAMAGED BY STREPTOZOTOCINObjective:To investigate the intervention mechanism ofα-tocopherol on pancreatic beta cells damaged by STZ.Methods: 1.Groups: the same as the part two.2.Measurements: the T-AOC level,the SOD and NOS activity,the MDA and NO levels were evaluated by spectrometer respectively;the activity of caspase-3 and caspase-8 were also measured by spectrometer; The expression of bcl-xl and bax protein were evaluated by immunohistochemical staining.Results: In STZ group, the T-AOC level, SOD activity, the expression of bcl-xl were lower, the activity of caspase-3 and caspase-8 ,the levels of MDA and NO, the expression of bax were higher than those in control group(p﹤0 .05); In intervention group, the above indexes did not return to the normal levels, but they were inhibited significantly byα-T (compared with STZ group, p﹤0.05).Conclusion: The inhibition ofα-T on the apoptosis of pancreatic beta cells induced by STZ is probably correlated with its capability of increasing antioxidation of islet cells, down-regulating the activity of caspase-3 and caspase-8 as well as the proportion of bax/bcl-xl.PART FOUR: THE DOSE-EFFECT RELATIONSHIP OFα-TOCOPHEROL ON PANCREATICβCELLS DAMAGED BY STREPTOZOTOCINObjective:To investigate the dose-effect relationship ofα-tocopherol on pancreaticβcells damageded by STZ.Methods: 1.Groups: Isolated and cultured beta cells from pancreas of newborn Wistar rats were divided into four groups:(1)control group; (2)α-T group: onlyα-T was added to the culture medium at the concentration of 80 mg/L for 24 hours;(3)STZ group: only STZ was added to the culture medium at the concentration of 0.58 mg/L for 30 minutes;(4) intervention groups: Islet cells were treated for 24 hours with5,10,20,40,80mg/Lα-T respectively,then 0.58 mg/L STZ was added for 30 minutes.Culture mediu- m was removed at specific time and the cells were resuspended with normal culture medium, then the following procedures were undertaken after 18 hours of recovery period.2.Measurements: the viability of islet cells was evaluated by MTT; insulin contents of cell supernatant were examined by radioimmunoassay;the T-AOC level was evaluated by spectrometer.Results:Compared with in STZ group, In different intervention groups(the concentrations ofα-T from 5 to 80 mg/L ), the viability of islet cells and the insulin contents as well as the T-AOC level were higher(p﹤0.05), the effects ofα-T enhanced along with increased with its concentration increased,showing a certain dose-dependent trend (compared in different intervention groups, p﹤0.05).Conclusion: The protective effect ofα-T on the pancreaticβcells damaged by STZ showed a dose-dependent trend.