| Since the the beginning of 50s at the last century when the sulfonylurea drug was used clinically,5%～10%of DM patients have been found secondary failure of sulfonylureas each year.Both the cause and mechanism need to be disclosed.The researchers have studied on it from different angles.The hypothesis that increased deposit of amyloid peptide on pancreaticβcells,which will cause insulin secretion deficiency and secondary failure of sulfonylureas,has received much concern.The physiological function of amylin was unclear now,while its secretion was found much more than the insulin secretion after the stimulation of high level glucose.The increased secretion of amylin had become the premise of the deposit of amyloid peptide in local area of islet.It was reported that high level of amylin inhibited the secretion of insulin from pancreaticβcells,meanwhile weakened the function of sulfonylureas on insulin secrition.It was hypothesized that the abnormal amylin secretion was one of the reasons that caused insulin secretion deficiency and secondary failure of sulfonylureas.In this study,we used whole-cell patch clamp technique to observe the influence of amylin on insulin secretion and the effect of sulfonylureas inβTC3 cells.Objective Using whole-cell patch clamp technique to study the characteristics of K~+-ATP channels and the influence of amylin on K~+-ATP channels on plasma membrane of singleβTC3 cells.And also study the effects of glucose and glibenclamide on the K~+-ATP channels with and without the pretreatment of amylin.Method After pancreaticβTC3 cells were resuscitated,selected bright and good shaped cells with diameter between 10-15μm as experiment object.After sealing resistance was over 1GΩ,we sucked the plasma membrane and made it brocken,then the whole-cell recording mode became.The clamp voltage was kept to—40mV.Give a series of stimulation to the cells every one minute,we would record the current and figure.We would observe the change of the current and figure in diffrent experimental conditions.The giving of the electrical stimulation pulse and the obtaining of the data were completed through the IBBDigitizerdigital system which were composed by pulse software package and DAQ-2B data acquisition interface.After the collection signal was filtered at 5kHz,it was disposed by the computer.All the current data was disposed by P/N leak subtraction.statistical analysis was conducted by using t-test,P＜0.05 showed significant difference,P＜0.01 showed extremely significant difference.Result1.Establish the detection method for K~+-ATP channels on plasma membrane of singleβTC3 cells:select suitable extracellular solution and intracellular solution for the detection,determine the experimental parameters for the whole-cell patch clamp.2.The condition of 5mmol/L and 16.7mmol/L concentration glucose on K~+-ATP channels and the variation of the channels after the effect of glibenclamide were observed and found:on the condition of 5mmol/L glucose,the current of K~+-ATP channels presented a weak inword rectifier current,the current had time-dependent deactivate manner.16.7mmol/L concentration glucose could decrease the current;Glibenclamide could decrease the current of the K~+-ATP channels.3.On the condition of 5mmol/L glucose,the effect of amylin with different concentrations(0.1,1,10μmol/L) on the K~+-ATP channels was observed and found:On the condition of 5mmol/L glucose,amylin had not obviousely change the current.After 10μmol/L amylin acted,16.7mmol/L concentration glucose made the degree of the maximum current decreased lower than without amylin,and had statistical significance compared with control group.After 10μmol/L amylin acted,3nmol/L glibenclamide made the degree of the maximum current decreased lower than without amylin,and had statistical significance compared with control group.Conclusions1.Glucose and glibenclamide were the substances who could close the K~+-ATP channels on pancreaticβcells.2.High concentration amylin inhibited the function of glucose and glibenclamide on stimulating insulin secretion was related to inhibiting the closing of K~+-ATP channels.
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