不同时间持续缺氧对胰岛β细胞自噬的影响

发布时间：2018-04-02 12:01

  本文选题：胰岛细胞　切入点：缺氧　出处：《天津医科大学》2017年硕士论文

【摘要】：目的观察不同时间持续缺氧孵育83213胰岛β细胞后,自噬相关蛋白Beclin1、LC3BII、P62的表达情况,从而判断自噬的活性变化。方法1.将符合实验条件的83213胰岛细胞作为研究对象,随机分为常氧组(对照组)、缺氧组(处理组),对照组用常氧(21%O_2)培养,缺氧组为持续缺氧(1%O_2,5%CO_2)2h、3h、6h、12h、24h、48h胰岛细胞,提取各组胰岛细胞的总蛋白并检测其浓度。2.采用Western Blot蛋白印记方法测定对照组和缺氧组缺氧诱导因子HIF-1a蛋白的表达,以反映常氧组及缺氧组各时相点HIF-1α的动态变化。3.采用Western Blot蛋白印记方法,检测常氧组、缺氧组自噬相关蛋白Beclin1、LC3BⅡ、P62表达情况,以反映对照组及缺氧处理后各时相点自噬水平的动态变化。结果1.与常氧组比较,缺氧2h、3h、6h、12h、24h、48h组HIF-1α蛋白表达均升高,在24h达高峰,且差异均有统计学意义(P0.05)2.与常氧组比较,缺氧2h胰岛细胞的Beclin1水平升高,于缺氧3h达高峰,缺氧6h时有下降趋势,但仍高于常氧组,差异有统计学意义(P0.05);缺氧12h、24h、48h组Beclin1水平均低于常氧组,差异有统计学意义(P0.05)。与常氧组比较,自噬相关蛋白LC3BⅡ在缺氧2h、3h、6h升高,差异有统计学意义(P0.05),并在3h达峰,自缺氧12h后LC3BⅡ逐渐下降,且低于常氧组,差异有统计学意义(P0.05)。与常氧组比较,缺氧2h、3h组P62蛋白降低,缺氧3h与之相比差异有统计学意义(P0.05),于缺氧6h逐渐升高,缺氧12h、24h、48h组差异有统计学意义(P0.05),并在缺氧24h达高峰。结论1.在持续缺氧情况下,胰岛β细胞的HIF-1α表达水平增加。2.在一定时间缺氧情况下,自噬相关蛋白Beclin1和LC3BⅡ表达水平增加,P62表达水平下降,反映胰岛β细胞自噬增强;但超过某个时间点,Beclin1和LC3BⅡ表达水平下降,P62表达水平上升,反映随着缺氧时间进一步延长,自噬不再增强,反而减弱。细胞的生存状态与缺氧的时间和强度有关。在短时间缺氧条件下,自噬可能参与对胰岛β细胞的保护。
[Abstract]:Objective to observe the expression of autophagy associated protein Beclin1, LC3BIIP62, and determine the change of autophagy activity after incubating 83213 islet 尾 cells with hypoxia for different periods. Methods 1. 83213 islet cells, which meet the experimental conditions, were selected as the research objects. They were randomly divided into normoxic group (control group) and hypoxic group (treatment group, control group) cultured with normoxic oxygen 21 and O _ 2. Hypoxia group was treated with continuous hypoxia of 1O _ 2 and 5 cells. The total protein of islet cells in each group was extracted and its concentration was determined. The expression of hypoxia inducible factor (HIF-1a) protein in control group and hypoxia group was determined by Western Blot protein imprinting method. To reflect the dynamic changes of HIF-1 伪 in normoxic group and hypoxia group, the expression of HIF-1 伪 was detected by Western Blot protein imprinting method in normal oxygen group and hypoxia group. Results 1.Compared with the normoxic group, the expression of HIF-1 伪 protein in the hypoxia group (2 h ~ 3 h ~ 6 h ~ 6 h ~ 12 h ~ 24 h ~ 48 h) increased and reached its peak at 24 h, and the difference was statistically significant compared with the normoxic group. 2. The level of Beclin1 in islet cells increased at 2h after hypoxia, peaked at 3h, decreased at 6h, but still higher than that in normoxic group (P 0.05), and the Beclin1 level was lower in 12h ~ 24h ~ 48h hypoxia group than in normoxic group (P < 0.05). Compared with the normoxic group, the level of autophagy associated protein LC3B 鈪，

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