摘要
目的 研究胰淀素短时间条件下对格列苯脲刺激INS-1细胞胰岛素分泌的影响及机制.方法 分别采用全细胞膜片钳技术、激光共聚焦显微镜技术及ELISA分析不同浓度胰淀素短时间孵育前后格列苯脲刺激下INS-1细胞ATP敏感性钾通道(KATP通道)电生理学特性、细胞内钙离子浓度([Ca2+]i)及胰岛素含量变化.结果 (1) ELISA结果显示:与1μmol/L格列苯脲刺激下胰岛素释放量(11.43 ±1.22) μg/L比较,0.1μmol/L胰淀素预处理组[(11.45 ±1.20) μg/L]无明显差异,而1 μmol/L及10 μmol/L胰淀素预处理组均显著下降,分别为(9.40±0.87)μg/L和(7.11±0.85) μg/L,且呈剂量相关性(P<0.05);(2)激光共聚焦显微镜测钙结果显示:1μmol/L格列苯脲刺激后[Ca2+]i荧光强度变化的AUC为427.78±2.32,0.1μmol/L胰淀素预处理组(424.09 ±2.21)与之差异无统计学意义;而1 μmol/L及10 μmol/L胰淀素预处理组均可使其AUC显著降低(分别为380.59 ±1.49和246.53±8.41),并呈剂量相关性(P<0.05);(3)全细胞膜片钳结果显示:在1μmol/L、10 μmol/L胰淀素预处理组中,1μmol/L格列苯脲刺激下KATP通道半数激活电压(V0.5)分别为(28.75 ±0.77)mV和(46.95 ±1.81)mV,均显著高于单纯格列苯脲组(14.59±0.69)mV,并呈剂量相关性(P<0.05).结论 高浓度胰淀素短时间作用可抑制格列苯脲对KATP通道的关闭,进而抑制[Ca2+]i增加,推测这种作用是INS-1细胞胰岛素分泌减少的机制之一.
Objective To observe the effects,and study the mechanism of islet amyloid polypeptide (IAPP) on insulin secretion in INS-1 cells stimulated by glibenclamide.Methods Whole cell patch clamp technique was employed to study the influences of short exposure to IAPP on electrophysiological characteristics of ATP-sensitive K+ channel(KATP channel) upon sulfonylurea stimulation.Intracellular free calcium changes in this process was observed by laser scanning confocal microscope.Insulin was measured by enzyme-linked immunoassay.Results (1)Insulin secretion stimulated by 1 μmol/L glibenclamide was significantly decreased from (11.43 ± 1.22) μg/L to (9.40 ± 0.87) μg/L and to (7.11 ± 1.85) μg/L after 1 μmol/L and 10 μmol/L IAPP incubation,respectively.(2)Glibenclamide-stimulated calcium influx was dose dependently inhibited by IAPP from 1 μmol/L to 10 μmol/L,with the AUC of fluorescence intensity-time reduced from 427.78 ± 2.32 to 380.59 ± 1.49,and to 246.53 ± 8.41,respectively.(3) Compared with that in control cells (14.59 ± 0.69) mV,the half activation voltage of KATP channel in response to glibenclamide was significantly increased to (28.75 ±0.77) mV and to (46.95 ± 1.81) mV in cells pretreated with 1 μmol/L and 10 μmol/L IAPP,implicating an inhibitory effect of IAPP on activation of KATP channel.Conclusion Short-term exposure to high concentration of IAPP inhibited glibenclamideinduced closure of KATP,channels and decreased calcium influx,which may ultimately lead to the reduction of insulin secretion in INS-1 cells.
出处
《中华内科杂志》
CAS
CSCD
北大核心
2015年第3期214-218,共5页
Chinese Journal of Internal Medicine
基金
国家自然科学基金(81241030)
天津市应用基础与前沿技术研究计划(14JcYBJc24600)