磷脂酶C在INS-1细胞胰岛素分泌信息传导途径中的作用

发布时间：2018-01-01 03:22

本文关键词：磷脂酶C在INS-1细胞胰岛素分泌信息传导途径中的作用　出处：《重庆医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:初步探讨六类磷脂酶C(Phospholipase c)在营养物质、离子、神经递质和激素刺激INS-1细胞胰岛素分泌的信息传导途径中的作用。方法:①分别设计五组刺激因素:葡萄糖、L-谷氨酸、KCL、八肽胆囊收缩素、氯化乙酰胆碱(分别属于营养物质、离子、神经递质和激素类),②设计五组刺激因素作用的时间梯度和浓度梯度,③收集刺激后的INS-1细胞培养上清液, ELISA法检测细胞上清液中胰岛素的含量,④最适刺激条件作用于INS-1细胞后,RT-PCR检测六类PLC的表达。结果:①PLCε在INS-1细胞中没有表达;②葡萄糖刺激INS-1细胞,RT-PCR检测刺激前后表达量的变化大小顺序为:PLCδPLCβPLCγPLCηPLCζ;L-谷氨酸作用后:PLCβPLCδPLCζPLCηPLCγ;KCL作用后:PLCηPLCδPLCγPLCζPLCβ;八肽胆囊收缩素作用后:PLCδPLCγPLCηPLCζPLCβ;氯化乙酰胆碱作用后:PLCδPLCβPLCγPLCηPLCζ 结论:葡萄糖、L-谷氨酸、KCL、八肽胆囊收缩素、氯化乙酰胆碱均可刺激INS-1细胞显著分泌胰岛素;六类PLC在胰岛素分泌信息传导途径中表达上调,但各类PLC的增加量不同。第二章过表达PLCβ1对β细胞葡萄糖刺激胰岛素分泌的影响目的:探讨磷脂酶Cβ1(phospholipase Cβ1,PLCβ1)在葡萄糖刺激胰岛素分泌(glucose-stimulated insulin secretion GSIS)信息传递中的作用。方法:①以最适葡萄糖浓度刺激INS-1细胞适当时间后,RT-PCR检测PLCβ1表达变化;②构建PLCβ1真核表达载体(PCMV-HA-PLCβ1),转染INS-1细胞,Western-blot检测INS-1细胞中PLCβ1蛋白的表达;③收集转染后INS-1细胞培养上清液,检测胰岛素含量。结果:①半定量RT-PCR检测葡萄糖刺激后PLCβ1表达显著升高。②过表达PLCβ1的INS-1细胞培养上清液中胰岛素含量为1.906±0.080 ng/mL,较对照组(0.740±0.091 ng/mL)显著升高(P㩳0.01)。结论:过表达PLCβ1显著增加INS-1细胞的胰岛素分泌,提示PLCβ1可能参与GSIS信息传递。
[Abstract]:Objective: to study the effect of phospholipase c6 kinds of phospholipase on nutrients and ions. Role of neurotransmitters and hormones in the signaling pathway of insulin secretion in INS-1 cells. Methods five stimulation factors were designed: glucose L-glutamate KCL, cholecystokinin octapeptide, acetylcholine chloride (which belong to nutrients, ions, neurotransmitters and hormones, respectively). 2 the time gradient and concentration gradient of stimulation factors were designed to collect the culture supernatant of INS-1 cells after stimulation, and the insulin content in the supernatant was detected by ELISA method. 4 the expression of six kinds of PLC was detected by RT-PCR after the optimal stimulation condition was applied to INS-1 cells. Results: 1 PLC 蔚 was not expressed in INS-1 cells. (2) the order of the change of expression before and after glucose stimulation in INS-1 cells was: INS-1 未 PLC 尾 PLC 纬 PLC 畏 PLC 味; PLC 尾 PLC 未 PLC 味 PLC 畏 PLC 纬; After the action of KCL, KCL 畏 PLC 未 PLC 纬 PLC 味 PLC 尾; After the action of cholecystokinin octapeptide, PLC 未 PLC 纬 PLC 畏 PLC 味 PLC 尾; PLC 未 PLC 尾 PLC 纬 PLC 畏 PLC 味 after acetylcholine chloride Conclusion: glucose L-glutamate, cholecystokinin octapeptide, acetylcholine chloride can significantly stimulate the secretion of insulin in INS-1 cells. The expression of PLC was up-regulated in insulin secretory signaling pathway, but the increase of PLC was different. Effect of overexpression of PLC 尾 1 on glucose-stimulated insulin secretion in 尾 -cells Objective: to study the phospholipase C 尾 1 of phospholipase C 尾 1. PLC 尾 1 in glucose-stimulated insulin secretion. The role of GSIS in the transmission of information. Methods the expression of PLC 尾 1 was detected by RT-PCR after INS-1 cells were stimulated with the optimal glucose concentration. 2 PLC 尾 1 eukaryotic expression vector was constructed and transfected into INS-1 cells. The expression of PLC 尾 1 protein in INS-1 cells was detected by Western-blot. 3 the supernatant of INS-1 cell culture after transfection was collected and the insulin content was detected. Results:. 1 the expression of PLC 尾 1 was significantly increased by semi-quantitative RT-PCR. 2. The insulin content in the culture supernatant of INS-1 cells with overexpression of PLC 尾 1 was 1.906 卤0.080. Ng/mL. Compared with the control group (0.740 卤0.091 ng / mL), the P was significantly higher than that in the control group. 0.01g. Conclusion: overexpression of PLC 尾 1 significantly increases insulin secretion in INS-1 cells, suggesting that PLC 尾 1 may be involved in GSIS information transmission.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

【共引文献】