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目的：通过激光扫描共聚焦显微镜对小鼠胰岛素瘤min6 细胞免疫化学染色后观察外源性高胰岛素对FoxO1 胞质- 胞核穿梭定位的影响。方法：小鼠胰岛素瘤min6 细胞用DMEM( low glucose )培养基（含有15%FBS 、100 U/mL青霉素、100 U/mL链霉素）于25 mL培养瓶放置在37 ℃、5%CO2浓度的细胞孵箱中培养。细胞爬片后给予100 uIU/mL 浓度胰岛素分别刺激12、24 和 48 小时。细胞免疫化学染色后激光扫描共聚焦显微镜观察FoxO1 的表达位置变化，用Image pro plus 软件对FoxO1 荧光强度进行半定量分析。结果：与低糖孵育的对照组相比，胰岛素孵育12 h、24 h和48 h时胞质内FoxO1 荧光强度逐渐增强，而细胞核 FoxO1 荧光强度减弱（P＜0.05）。结论：高浓度胰岛素孵育min6细胞使FoxO1 出细胞核转位至细胞质，并且具有时间依赖性，提示FoxO1是高胰岛素血症对beta细胞功能影响的机制之一。

英文摘要:

Objective:To explore the effect of exogenous high insulin on FoxO1 nucleus--cytoplasmic shutting in beta cells.Methods:1. The min6 cells were cultured with DMEM( low glucose ) medium ( containing 15 %FBS, 100 U/mL penicillin, 100U/mL streptomycin ) in a 25 mL culture flask under the condition of 37℃, 5 %CO2. 2. Making min6 cells growing on glass coverslips, then stimulating the cells with 100 uIU/mL insulin in 12 h, 24 h and 48 h separately. 3. The expression of FoxO1 in min6 cells in different incubation time exposure to 100 uIU/mL insulin was analyzed by immune fluorescence cytochemistry combined with laser scanning confocal microscopy. Semi-quantitative analysis of fluorescence intensity of FoxO1 was determined by Image ProPlus 6.0 software.Results:Compared with the control group, the fluorescence intensity of FoxO1 which incubated with insulin for 12 h, 24 h and 48 h of min6 cells was gradually increased in cytoplasm and decreased in nucleus (P＜0.05).Conclusion:High concentration of insulin could induce FoxO1 translocation fromnucleus to the cytoplasmin a time-dependent manner, suggesting that FoxO1 may be one of the mechanisms of the impact of hyperinsulinemia on beta-cell function.

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