胰岛素对小鼠MIN6细胞FoxO1细胞核-细胞质穿梭定位的影响

发布时间：2018-04-10 23:12

  本文选题：Fox + O1　； 参考：《第四军医大学》2015年硕士论文

【摘要】：研究背景:Fox O1是胰岛β细胞的转录因子之一,在细胞内参与多种信号转导通路,能够调节细胞分化、增殖和凋亡,对胰岛β细胞的功能具有重要影响。Fox O1-EGFP实时成像研究显示,在小鼠胰岛β细胞或MIN6细胞,用3mmol/L葡萄糖孵育时,Fox O1一直在细胞核显著分布。当分别以16.7mmol/L、30mmol/L葡萄糖或116.16ng/m L、1161.6ng/m L胰岛素孵育细胞10-20min时,可检测到胞质荧光增多而细胞核荧光减少。另有研究表明,用25mmol/L葡萄糖刺激MIN6细胞和小鼠胰岛β细胞30min时Fox O1发生急剧磷酸化并且由细胞核转位至细胞质,这一效应具有时间(0.5-2h)和剂量(2.5-20mmol/L)依赖性。而分别以0.58ng/m L、5.80ng/m L、580ng/m L和1161.6ng/m L胰岛素刺激MIN6细胞和小鼠胰岛β细胞同样能够观察到Fox O1发生磷酸化改变,但却未对Fox O1的胞质-胞核转位进行观察。为探讨胰岛素对Fox O1表达部位的影响,本实验拟通过给予不同浓度胰岛素孵育小鼠MIN6细胞,观察不同浓度胰岛素在不同时间对β细胞Fox O1细胞核-细胞质穿梭定位的影响。目的:采用激光扫描共聚焦显微镜对MIN6细胞胰岛素作用后免疫化学染色观察Fox O1在不同浓度刺激不同时间后表达位置的变化。方法:1.小鼠胰岛素瘤MIN6细胞用DMEM(high glucose)培养基(含有25mmol/L葡萄糖、15%FBS、100U/m L青霉素、100μg/m L链霉素)于25ml培养瓶放置在37℃、5%CO2浓度的细胞孵箱中培养。2.分别以含25mmol/L和5.5mmol/L葡萄糖培养基孵育12小时并检测培养上清液胰岛素浓度。3.在含5.5mmol/L葡萄糖培养基基础上给予50μIU/m L(1.75ng/m L)、100μIU/m L(3.5ng/m L)和150μIU/m L(5.25ng/m L)浓度胰岛素分别作用12、24和48小时。4.细胞免疫化学染色结合激光扫描共聚焦显微镜观察Fox O1的细胞内定位。结果:1.25mmol/L葡萄糖培养MIN6细胞12小时胰岛素分泌浓度为74.37ng/m L,Fox O1主要在细胞质表达;5.5mmol/L葡萄糖培养MIN6细胞12小时胰岛素分泌浓度为37.46ng/m L,Fox O1主要在细胞核表达。2.与对照组相比,胰岛素浓度50μIU/m L组分别在刺激12、24、48小时后Fox O1荧光强度的胞质/胞核比增加,12小时组差异不具有统计学意义(P=0.084),24小时和48小时组存在统计学差异(P0.05);胰岛素浓度100μIU/m L组分别在刺激12、24和48小时后Fox O1荧光强度胞质/胞核比增加,差异具有统计学意义(P0.05);胰岛素浓度150μIU/m L组分别在刺激12、24和48小时后Fox O1荧光强度胞质/胞核比增加,均存在统计学差异(P0.05)。50μIU/m L胰岛素组、100μIU/m L胰岛素组和150μIU/m L胰岛素组分别在不同时间的组内比较存在统计学差异(P0.05)。结论:1.25mmol/L葡萄糖水平下Fox O1主要表达于细胞质;5.5mmol/L葡萄糖水平下Fox O1主要表达于细胞核。2.外源性胰岛素作用使Fox O1出细胞核转位至细胞质,且与胰岛素浓度和刺激时间的递增成正比。
[Abstract]:Background: 1: Fox O1 is one of the transcription factors of islet 尾 cells. It participates in many signal transduction pathways in cells and can regulate cell differentiation, proliferation and apoptosis, which has an important effect on the function of islet 尾 cells.In mouse islet 尾 cells or MIN6 cells, 3mmol/L glucose was used to incubate 3mmol/L O1 in the nucleus.When 10-20min was incubated with 16.7 mmol / L 30 mmol / L glucose or 116.16ng/m L 1161.6 ng / mL insulin, the cytoplasmic fluorescence increased and the nuclear fluorescence decreased.Other studies showed that Fox O1 was phosphorylated sharply and translocated from nucleus to cytoplasm when 25mmol/L glucose was used to stimulate 30min in MIN6 cells and mouse islet 尾 cells. This effect was time-dependent and dose-dependent (2.5-20 mmol / L).However, the phosphorylation of Fox O 1 was also observed in MIN6 cells and 尾 cells of mouse islets stimulated with 0.58ng/m L 5.80 ng / m L and 1161.6ng/m L insulin 580ng / mL, respectively, but the cytoplasmic nuclear translocation of Fox O 1 was not observed.In order to investigate the effect of insulin on the expression site of Fox O1, the effect of different concentrations of insulin on the localization of Fox O1 nucleus-cytoplasmic shuttle in 尾 cells was observed by incubating MIN6 cells with different concentrations of insulin at different times.Aim: to observe the expression of Fox O1 in MIN6 cells after insulin treatment with laser scanning confocal microscope (LSCM).Method 1: 1.Mouse insulinoma MIN6 cells were cultured on DMEM(high glucose medium (containing 25mmol/L glucose, 100 渭 g / mL penicillin 100 渭 g / m L streptomycin) in 25ml culture flask and cultured in a cell incubator with CO 2 concentration at 37 鈩，

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