發(fā)布時(shí)間：2018-03-03 17:52

  本文選題：胰高血糖素　切入點(diǎn)：MIN6細(xì)胞　出處：《石河子大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:通過測定不同濃度葡萄糖(Glucose Glu)環(huán)境下不同濃度的胰高血糖素(Glucagon Gc)對小鼠胰島β細(xì)胞系MIN6細(xì)胞胰島素(Insulin Ins)分泌及環(huán)磷酸腺苷(Cyclic adenosine monophosphate c AMP)信號通路的影響來進(jìn)一步探討Gc在MIN6細(xì)胞Ins分泌的c AMP信號通路中的作用及機(jī)制。方法:1.體外培養(yǎng)小鼠胰島β細(xì)胞系MIN6細(xì)胞,細(xì)胞生長達(dá)到一定密度后傳代分組進(jìn)行實(shí)驗(yàn);2.電穿孔法轉(zhuǎn)染ICUE3(c AMP表達(dá)的質(zhì)粒)及陰性對照質(zhì)粒PCDNA3.1進(jìn)入MIN6細(xì)胞,在無Glu(0mmol/L)、低Glu(2.8mmol/L)、高Glu(16.7 mmol/L)的Glu環(huán)境下,分別用無Gc(0 ng/L)、低Gc(500ng/L)、高Gc(1000ng/L)的Gc濃度,然后添加鹽酸異丙腎上腺素(Isoprenaline Hydrochloride ISO)對MIN6細(xì)胞進(jìn)行處理,采用基于熒光生物傳感器(Biosenser)的熒光共振能量轉(zhuǎn)移(Fluorescence resonance energy transfer FRET)技術(shù)法檢測不同處理階段細(xì)胞內(nèi)c AMP水平;3.在不同的Glu濃度及不同的Gc濃度環(huán)境下添加或不添加ISO后收集細(xì)胞上清液,酶聯(lián)免疫吸附劑測定(ELISA)法檢測細(xì)胞Ins釋放量和細(xì)胞內(nèi)c AMP水平;4.數(shù)據(jù)整理統(tǒng)計(jì)分析。結(jié)果:1.FRET技術(shù)實(shí)時(shí)定量檢測在不同的Glu濃度及不同的Gc濃度環(huán)境下的MIN6活細(xì)胞內(nèi)c AMP含量的結(jié)果:無Glu時(shí):ISO組CFP/YFP比值較無Gc組增高(P0.05);低Glu時(shí):ISO組CFP/YFP比值較無Gc組、低Gc組和高Gc組增高(P0.05),高Gc組較無Gc組增高(P0.05);高Glu時(shí):ISO組CFP/YFP比值較無Gc組、低Gc組和高Gc組增高(P0.05),高Gc組較無Gc組增高(P0.05);低Gc組較無Gc組增高(P0.05);不同濃度Gc在不同濃度Glu條件下對c AMP生成影響的比較的結(jié)果:無Glu時(shí):高Gc加ISO組比高Gc組和低Gc組高(P0.05),低Gc加ISO組比高Gc組和低Gc組高(P0.05);低Glu時(shí):高Gc加ISO組比高Gc組和低Gc組高(P0.05);低Gc加ISO組比高Gc組和低Gc組高(P0.05);高Gc組比低Gc組高(P0.05),高Glu時(shí):高Gc加ISO組比;低Gc加ISO組、高Gc組和低Gc組(P0.05);低Gc加ISO組比高Gc組和低Gc組高(P0.05)。2.ELISA法檢測不同處理組MIN6細(xì)胞內(nèi)c AMP水平結(jié)果:在同一Glu環(huán)境和同一Gc濃度處理因素下ISO組c AMP水平均高于無ISO組(P0.05),高Gc組c AMP水平均高于低Gc組和無Gc組(P0.05),低Gc組c AMP水平均高于無Gc組(P0.05)。3.ELISA法檢測不同處理組MIN6細(xì)胞Ins分泌量的結(jié)果:在同一Glu環(huán)境和同一Gc濃度處理因素下ISO組Ins分泌量均高于無ISO組(P0.05),高Gc組Ins分泌量均高于低Gc組和無Gc組(P0.05),低Gc組Ins分泌量均高于無Gc組(P0.05)。4.MIN6細(xì)胞內(nèi)c AMP含量與Ins分泌量的相關(guān)性分析的結(jié)果:相關(guān)系數(shù)r=0.901(R~2=0.813 P0.05),MIN6細(xì)胞內(nèi)c AMP含量與Ins分泌量之間具有正相關(guān)關(guān)系。結(jié)論:1.Gc以濃度梯度的形式增加小鼠胰島β細(xì)胞系MIN6細(xì)胞內(nèi)c AMP濃度的同時(shí)來促進(jìn)Ins分泌;2.在加入ISO刺激后這種作用越發(fā)明顯,說明Gc通過c AMP信號通路促進(jìn)Ins分泌;3.Gc通過c AMP信號通路促進(jìn)Ins分泌的作用具有一定的Glu依賴性。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1

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