运动疲劳状态下抗增殖蛋白Prohibitin1与ATP合酶相互作用研究

发布时间：2018-05-02 20:45

本文选题：PHB1 + ATP合酶　；参考：《天津体育学院》2017年硕士论文

【摘要】：目的:研究运动疲劳状态下骨骼肌线粒体中PHB1与F_0F_1-ATP合酶是否存在相互作用,以及与其具体产生作用的亚单位。方法:选取雄性SD大鼠80只(8周龄),先适应性喂养2天,称重后随机分为两个运动程序进行动物跑台运动实验:1.急性运动训练程序(1)对照组:20只,常规饲养,不加干涉;(2)急性力竭组:20只,常规饲养,满8周后让大鼠一次性力竭活动;判别力竭状况的标准为:大鼠的跑姿由蹬地式转变为伏地式,滞留在跑道尾部不再连续跑动,且电刺激也不能驱使大鼠再继续跑动。2.长期运动训练程序:(1)对照组:20只,常规饲养,不加干涉。(2)中等强度训练组:20只,每周6天,第一周以坡度为10%,10m/min的速率跑10min;第二周以坡度为10%,15m/min的速度跑10min每天增加10min之第二周结束为止,从第三周开始维持这速度和时间跑到第8周完为止;(3)过度疲劳训练组:20只,每周练习6天,歇息1天,采取强度递增式跑台活动。从练习的第1周起,逐步增添速率、坡度和跑步时长。跑台的速率从15m/min,每周增添5m/min,增至30m/min,后再也不增添;坡度从0°起,每周增添5°,增添至15°后再也不增添;训练时间从30min,每周增加20min增加到110min后稳定训练时间,不再增加。训练周数直至第8周。收集各组模型动物的骨骼肌组织样品,一部分留样,一部分用研磨管进行组织匀浆,匀浆组织用线粒体蛋白试剂盒提取线粒体蛋白,质谱鉴定分析出与PHB1相互作用的ATP合酶亚单位。然后采取免疫共沉淀方式验证不同运动形式大鼠线粒体ATP合酶中与PHB1相互作用的亚单位。结果:1.质谱分析检测出了ATP合酶亚单位,四组不同运动形式的大鼠骨骼肌线粒体中都含有的与PHB1结合的ATP合酶蛋白:ATP合酶α亚单位和ATP合酶亚单位γ。其中以过度训练组ATP合酶蛋白种类最多,对照组ATP合酶蛋白种类最少。与对照组相比,中等训练组大鼠又检测出ATP合酶亚单位d;过度训练组大鼠又检测出ATP合酶F_0复合体亚单位B1、ATP合酶亚单位d、ATP合酶亚单位O、ATP合酶亚单位β。与中等强度训练组相比,过度训练组又多检测出ATP合酶F_0复合体亚单位B1、ATP合酶亚单位β、ATP合酶亚单位O。与对照组相比,一次性力竭组又多检测出ATP合酶亚单位β。2.免疫共沉淀发现PHB1与ATP合酶的α、γ亚单位相互作用。结论:1.骨骼肌线粒体中PHB1与ATP合酶存在相互作用。2.大鼠骨骼肌线粒体中PHB1与ATP合酶的α、γ亚单位存在相互作用。
[Abstract]:Aim: to study the interaction between PHB1 and F_0F_1-ATP synthase in mitochondria of skeletal muscle under exercise fatigue and the specific subunits. Methods: 80 male Sprague-Dawley rats were selected and fed adaptively for 2 days. Acute exercise training program (1) the control group: 20 rats were fed with routine feeding without interference. 20 rats in acute exhaustion group were fed with routine exercise. After 8 weeks, the rats were given a one-time exhaustive exercise. According to the criteria of exhaustion, the running posture of the rats changed from pedaling to volley, and the rats did not run continuously in the tail of the runway, and the electrical stimulation could not drive the rats to continue to run. 2. Long-term exercise training program: 1) the control group: 20 rats, conventional rearing, without interference...) moderate intensity training group: 20 mice, 6 days a week, The first week ran at a slope of 10 to 10 m / min for 10 minutes; the second week at a gradient of 10 to 15 m / min 10min increased 10min daily until the end of the second week, and from the third week to the end of week 8) the overfatigue training group: 20. Practice 6 days a week, rest for 1 day, and take a step-by-step treadmill. From the first week of practice, step by step increase in speed, slope, and running time. From 15 m / min, 5 m / min per week to 30 m / min, then no further increase; from 0 掳to 5 掳per week, to 15 掳per week, no more; from 30 minutes, the increase of 20min per week to the stable training time after 110min, no longer increase. (3) the speed of the running stage is increased from 15 m / min to 30 m / min, and then increases to 30 m / min, and increases to 30 m / min per week. Number of training weeks until 8 th week. The skeletal muscle tissue samples of each group were collected, some samples were retained, some tissues were homogenized with grinding tubes, and mitochondrial proteins were extracted from homogenate tissue by mitochondrial protein kit. The ATP synthase subunits interacting with PHB1 were identified by mass spectrometry. Then the immunoprecipitation method was used to identify the interaction subunits between PHB1 and mitochondrial ATP synthase in rat mitochondria in different exercise forms. The result is 1: 1. The ATP synthase subunits were detected by mass spectrometry. The ATP synthase protein (ATP synthase 伪 subunit) and ATP synthase subunit 纬 (ATP synthase subunit) binding to PHB1 were found in the mitochondria of four different exercise forms of rat skeletal muscle. The types of ATP synthase protein were the most in the overtraining group and the least in the control group. Compared with the control group, the ATP synthase subunit d was detected in the middle training group, and the ATP synthase F0 complex subunit B1 + ATP synthase subunit, OG ATP synthase subunit 尾, was detected in the overtraining group. Compared with medium intensity training group, ATP synthase F0 complex subunit B1 + ATP synthase subunit 尾 -tATP synthase subunit O was detected in overtraining group. Compared with the control group, the ATP synthase subunit 尾. 2. The interaction between PHB1 and 伪, 纬 subunits of ATP synthase was found by immunoprecipitation. Conclusion 1. There is interaction between PHB1 and ATP synthase in mitochondria of skeletal muscle. There is interaction between PHB1 and 伪, 纬 subunits of ATP synthase in mitochondria of rat skeletal muscle.
【学位授予单位】：天津体育学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：G804.7

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