摘要
自上世纪50年代初磺脲类降糖药(Sulfonylurea Drug,SuD/SUs)用于临床以来,每年约有5%～10%接受其治疗的糖尿病患者产生药物继发失效,五年失效率达50%,其发生原因及机制还未明确,对此学者们从不同角度进行了研究,其中有关胰岛β细胞淀粉样蛋白沉积导致胰岛素分泌障碍,使SUs继发失效的学说引起关注。胰淀素(Amylin)的生理功能尚不完全清楚,但已发现在高糖刺激下其分泌量远远超过胰岛素,局部胰淀素浓度增高成为胰岛内淀粉样蛋白形成的前提,有报道高浓度胰淀素抑制大鼠胰岛细胞分泌胰岛素,使SUs促胰岛细胞分泌胰岛素作用减弱,因此认为胰淀素分泌异常是导致胰岛β细胞分泌功能障碍、SUs继发失效的原因之一。本研究应用全细胞膜片钳技术观察了胰淀素对胰岛βTC3细胞ATP敏感性钾通道(ATP-sensitive Potassium Channels,K~+-ATP channels)的影响,探讨其影响胰岛素分泌及SUs作用的机制。
目的:应用全细胞膜片钳技术,在单个细胞水平上观察胰岛βTC3细胞K~+-ATP通道的电学特性、胰淀素对葡萄糖和SUs对该通道作用的影响。
方法:胰岛βTC3细胞系复苏后,选择有光泽、形态良好的直径约10-15μm的细胞进行膜片钳实验。当封接电阻达1GΩ以上时吸破细胞膜,补偿电容、电流及串联电阻,形成全细胞记录模式,设置钳制电压为—40mV,每隔1分钟给予细胞一系列不同去极化电压刺激后记录电流及图形,在不同实验条件观察电流及图形的变化。电刺激脉冲的给出及数据采入等均通过Pulse软件包和DAQ-2B数据采集接口装置一起构成的IBBDigitizer数字化系统完成,采集信号均经5kHz滤波后输入计算机处理,所有电流数据均经P/N漏减处理,用t检验进行统计学分析,以均数±标准差((?)±S)表示,P＜0.05为差异显著,P＜0.01为差异极显著。
结果:
1.建立了单个胰岛βTC3细胞膜上K~+-ATP通道的检测方法:选择出适合检测胰岛βTC3细胞膜上K~+-ATP通道的细胞外液及电极内液,确定了全细胞膜片钳实验的参数。
2.观察了5mmol/L葡萄糖和16.7mmol/L葡萄糖时胰岛βTC3细胞膜上K~+-ATP通道电流的情况以及优降糖作用后该通道的变化:在5mmol/L葡萄糖作用下,胰岛βTC3细胞膜上K~+-ATP通道电流呈弱的内向整流性质,其电流存在时间依赖性失活,16.7mmol/L葡萄糖可使电流降低;3nmol/L优降糖可使K~+-ATP通道电流减小。
3.0.1、1、10μmol/L胰淀素对5mmol/L葡萄糖条件下胰岛βTC3细胞膜上K~+-ATP通道的影响与对照组比无明显变化。10μmol/L胰淀素使16.7mmol/L葡萄糖作用下的K~+-ATP通道最大电流减低程度降低,与对照组相比具有统计学意义;10μmol/L胰淀素使3nmol/L优降糖作用下的K~+-ATP通道最大电流减低程度降低,与对照组相比具有统计学意义。
结论:
1.葡萄糖,优降糖是使胰岛βTC3细胞膜上K~+-ATP通道关闭的物质。
2.高浓度胰淀素抑制高糖和优降糖刺激胰岛素分泌的作用与抑制K~+-ATP通道关闭有关。
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.
Result
1.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.
Conclusions
1.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.
引文
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