胰高血糖素通过cAMP信号通路对胰岛β细胞胰岛素分泌的调节作用及机制的研究

发布时间：2018-03-03 17:52

本文选题：胰高血糖素　切入点：MIN6细胞　出处：《石河子大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过测定不同浓度葡萄糖(Glucose Glu)环境下不同浓度的胰高血糖素(Glucagon Gc)对小鼠胰岛β细胞系MIN6细胞胰岛素(Insulin Ins)分泌及环磷酸腺苷(Cyclic adenosine monophosphate c AMP)信号通路的影响来进一步探讨Gc在MIN6细胞Ins分泌的c AMP信号通路中的作用及机制。方法:1.体外培养小鼠胰岛β细胞系MIN6细胞,细胞生长达到一定密度后传代分组进行实验;2.电穿孔法转染ICUE3(c AMP表达的质粒)及阴性对照质粒PCDNA3.1进入MIN6细胞,在无Glu(0mmol/L)、低Glu(2.8mmol/L)、高Glu(16.7 mmol/L)的Glu环境下,分别用无Gc(0 ng/L)、低Gc(500ng/L)、高Gc(1000ng/L)的Gc浓度,然后添加盐酸异丙肾上腺素(Isoprenaline Hydrochloride ISO)对MIN6细胞进行处理,采用基于荧光生物传感器(Biosenser)的荧光共振能量转移(Fluorescence resonance energy transfer FRET)技术法检测不同处理阶段细胞内c AMP水平;3.在不同的Glu浓度及不同的Gc浓度环境下添加或不添加ISO后收集细胞上清液,酶联免疫吸附剂测定(ELISA)法检测细胞Ins释放量和细胞内c AMP水平;4.数据整理统计分析。结果:1.FRET技术实时定量检测在不同的Glu浓度及不同的Gc浓度环境下的MIN6活细胞内c AMP含量的结果:无Glu时:ISO组CFP/YFP比值较无Gc组增高(P0.05);低Glu时:ISO组CFP/YFP比值较无Gc组、低Gc组和高Gc组增高(P0.05),高Gc组较无Gc组增高(P0.05);高Glu时:ISO组CFP/YFP比值较无Gc组、低Gc组和高Gc组增高(P0.05),高Gc组较无Gc组增高(P0.05);低Gc组较无Gc组增高(P0.05);不同浓度Gc在不同浓度Glu条件下对c AMP生成影响的比较的结果:无Glu时:高Gc加ISO组比高Gc组和低Gc组高(P0.05),低Gc加ISO组比高Gc组和低Gc组高(P0.05);低Glu时:高Gc加ISO组比高Gc组和低Gc组高(P0.05);低Gc加ISO组比高Gc组和低Gc组高(P0.05);高Gc组比低Gc组高(P0.05),高Glu时:高Gc加ISO组比;低Gc加ISO组、高Gc组和低Gc组(P0.05);低Gc加ISO组比高Gc组和低Gc组高(P0.05)。2.ELISA法检测不同处理组MIN6细胞内c AMP水平结果:在同一Glu环境和同一Gc浓度处理因素下ISO组c AMP水平均高于无ISO组(P0.05),高Gc组c AMP水平均高于低Gc组和无Gc组(P0.05),低Gc组c AMP水平均高于无Gc组(P0.05)。3.ELISA法检测不同处理组MIN6细胞Ins分泌量的结果:在同一Glu环境和同一Gc浓度处理因素下ISO组Ins分泌量均高于无ISO组(P0.05),高Gc组Ins分泌量均高于低Gc组和无Gc组(P0.05),低Gc组Ins分泌量均高于无Gc组(P0.05)。4.MIN6细胞内c AMP含量与Ins分泌量的相关性分析的结果:相关系数r=0.901(R~2=0.813 P0.05),MIN6细胞内c AMP含量与Ins分泌量之间具有正相关关系。结论:1.Gc以浓度梯度的形式增加小鼠胰岛β细胞系MIN6细胞内c AMP浓度的同时来促进Ins分泌;2.在加入ISO刺激后这种作用越发明显,说明Gc通过c AMP信号通路促进Ins分泌;3.Gc通过c AMP信号通路促进Ins分泌的作用具有一定的Glu依赖性。
[Abstract]:Objective: to investigate the effects of glucagon Gcc at different concentrations of glucose on insulin Ins secretion and cyclic adenosine monophosphate c AMP signaling pathway in murine islet 尾 cell line MIN6 cells. Methods: the effect of Glucagon Gcc on insulin secretion in mouse islet 尾 cell line MIN6 cell line was studied by measuring the effects of different concentrations of glucagon Gluus on insulin secretion and cyclic adenosine monophosphate c AMP signaling pathway in mouse islet 尾 cell line. One step was to investigate the role and mechanism of GC in c AMP signaling pathway secreted by Ins of MIN6 cells. Methods: 1. Murine islet 尾 cell line MIN6 cells were cultured in vitro. After cell growth reached a certain density, the cells were transfected with ICUE3(c AMP expression plasmid by electroporation) and the negative control plasmid PCDNA3.1 was transfected into MIN6 cells. Under the condition of no Glun 0 mmol / L, low Gluo 2.8 mmol / L, high Glu(16.7 mol / L), the Gc concentration of no Gc(0 ngr / L, low GcCN 500ngL / L, high GcN 1000ngL / L, respectively, was used in the environment of Glu with no Gc(0 ngr / L, low GcN 500ngL / L, and high GcN 1000ngL / L, respectively. Then MIN6 cells were treated with isoprenaline Hydrochloride ISO. Fluorescence resonance resonance energy transfer fret technique based on fluorescence biosensor was used to detect c AMP level in cells at different treatment stages. After adding or not adding ISO at different Glu concentration and different GC concentration, Collect cell supernatant, Enzyme linked immunosorbent assay (Elisa) was used to detect the release of Ins and the level of c AMP in cells. Statistical analysis was made. Results 1. FRET real-time quantitative detection of MIN6 living cells at different Glu concentration and different GC concentration. The results of c AMP content: the ratio of CFP/YFP to CFP/YFP in the group without Glu was higher than that in the group without GC, and the ratio of CFP/YFP in the group with low Glu was higher than that in the group without GC. In low GC group and high GC group, the ratio of CFP/YFP in high GC group was higher than that in no GC group, the ratio of CFP/YFP in high GC group was higher than that in no GC group, the ratio of CFP/YFP in high GC group was higher than that in no GC group. The effect of different concentrations of GC on c AMP production in low GC group and high GC group was higher than that in high GC group compared with no GC group, P 0.05 in low GC group was higher than that in no GC group, and the effect of different concentrations of GC on c AMP production under different concentrations of Glu: without Glu: high GC + ISO group was higher than high GC group. In low Glu group, high Gc + ISO group was higher than high GC group and low GC group, low GC + ISO group was higher than high GC group and low GC group, high P0.05A was higher in high GC + ISO group than in high GC group and low GC group, and in high GC + ISO group, high GC + ISO group ratio was higher than that in high GC group and low GC group, and high Gc + ISO group in high GC group was higher than that in low GC group and low GC group, and high Gc + ISO group was higher in high GC + ISO group than in high GC group and low GC group, and high Gc + ISO group was higher in high GC + ISO group than in high GC group and low GC group. Low GC + ISO group, The level of c AMP in MIN6 cells in low GC + ISO group was higher than that in high GC group and low GC group by Elisa. 2. The results showed that the level of c AMP in ISO group was higher than that in ISO group without ISO under the same Glu environment and same GC concentration. 2. The results showed that the level of c AMP in ISO group was higher than that in high GC group and low GC group. 2. Elisa results showed that the level of c AMP in ISO group was higher than that in ISO group without ISO. The level of c AMP in high GC group was higher than that in low GC group and no GC group, and the level of c AMP in low GC group was higher than that in no GC group. 3. The result of Elisa for detecting Ins secretion of MIN6 cells in different treatment groups: under the same Glu environment and same GC concentration treatment factor. Ins secretion in ISO group was higher than that in ISO group, Ins secretion in high GC group was higher than that in low GC group and no GC group, and Ins secretion in low GC group was higher than that in no GC group. The correlation between c AMP content and Ins secretion in low GC group was higher than that in no GC group. There was a positive correlation between c AMP content and Ins secretion in the cells of 0.901g ~ 0.901g ~ (-1) ~ 0.901p ~ (0.05) P ~ (0.813) P ~ (0.05). Conclusion: 1. GC can increase the concentration of c AMP in MIN6 cells of mouse islet 尾 cell line in the form of concentration gradient, and promote the secretion of Ins ~ (2) at the same time. ISO is added to stimulate the secretion of Ins. This effect is becoming more and more obvious. The results showed that GC promoted the secretion of Ins through c AMP signaling pathway. 3. The effect of GC on Ins secretion through c AMP signaling pathway was Glu dependent.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

【参考文献】