游泳运动调控miR-34a介导的细胞自噬在脑衰老中的作用

发布时间：2018-06-13 00:28

本文选题：衰老 + 游泳运动　；参考：《武汉体育学院》2017年硕士论文

【摘要】：目的:探究游泳运动干预对mi R-34a介导的细胞自噬在D-半乳糖诱导大鼠衰老进程中的调控作用。方法:1.雄性Sprague-Dawley(SD)大鼠30只,随机分为对照组(Control)、D-gal衰老模型组(D-gal)、衰老模型+运动干预组(D-gal+EXE),每组10只。一周的适应性喂养后,D-gal+EXE组大鼠进行适应性游泳训练:第一天游泳10 min,之后每天增加10 min,直至增加到60 min。一周的适应性训练后,开始正式实验。每天对D-gal组和D-gal+EXE组大鼠按照200 mg/kg的剂量颈背部皮下注射D-半乳糖诱导衰老,同时D-gal+EXE组大鼠进行60 min游泳训练,每周5天。药物注射和游泳训练在均在每天同一时间进行。6周的实验后,Morris水迷宫检测各组大鼠学习记忆能力。水迷宫结束24小时后处死所有大鼠并取材。试剂盒检测大鼠血清超氧化物歧化酶(SOD)活性及海马组织中丙二醛(MDA)含量,Real-time-PCR检测miR-34a的表达量,透射电镜观察海马中线粒体形态变化及自噬小体,Western blot检测细胞自噬及线粒体动力学相关蛋白表达情况。2.培养SH-SY5Y细胞,当细胞融合度达到50-70%时接种6孔板,转染miR-34a抑制剂并用D-半乳糖刺激细胞,转染后48 h收集细胞样品,Real-time-PCR检测miR-34a的表达情况,Western blot检测细胞自噬及线粒体动力学相关蛋白表达量。结果:1.Morris水迷宫结果显示,与对照组相比,皮下注射D-半乳糖6周后,大鼠学习记忆能力明显下降,而经过6周游泳训练的D-gal+EXE组大鼠则有明显改善。D-gal诱导的衰老大鼠穿越靶象限次数少于对照组大鼠,同时其潜伏期则高于对照组;而游泳运动干预组大鼠穿越靶象限次数明显多于D-gal诱导模型组大鼠,其潜伏期则明显短于衰老模型组。同时与对照组相比,D-gal模型组大鼠血清中SOD活性显著下降,海马组织中MDA含量显著上升,运动干预后的D-gal+EXE组大鼠SOD则呈现显著性提高,海马中的MDA含量则呈现非常显著性的下降。透射电镜观察发现,D-gal模型组大鼠海马中线粒体肿胀、融合增多,而D-gal+EXE组大鼠海马组织中线粒体形态明显趋于正常且有较多自噬小体。Real-time-PCR结果显示:D-半乳糖诱导的衰老大鼠海马组织中和D-半乳糖处理后的SH-SY5Y细胞中miR-34a的表达都显著地升高,游泳干预和miR-34a抑制剂处理后其表达量展现为显著性的下降。Western blot结果显示,与正常对照组相比,D-gal模型组大鼠海马中细胞自噬相关蛋白Atg7、Beclin1、LC3II/I均显著下降,p62则显著升高;线粒体动力学相关蛋白Drp1和Mfn2表达量都显著升高,D-gal+EXE组大鼠则Atg7、Beclin1、LC3II/I显著升高,p62的表达量显著下降;Drp1和Mfn2表达量也明显下降。2.D-半乳糖处理后的SH-SY5Y细胞中自噬相关蛋白Atg7、Beclin1、LC3II/I均显著下降,p62则显著升高,线粒体动力学相关蛋白Drp1和Mfn2表达量都显著升高;mi R-34a抑制剂转染后Atg7、Beclin1、LC3II/I显著上升,p62的表达量显著下降;Drp1和Mfn2表达量也都明显下降。结论:在D-半乳糖诱导的大鼠脑衰老模型以及细胞衰老模型中,miR-34a通过调节细胞自噬以及线粒体动力学相关蛋白的表达参与调控大鼠脑衰老进程,游泳运动可以通过激活mi R-34a介导的细胞自噬改善线粒体功能,延缓脑衰老进程。
[Abstract]:Objective: To explore the effect of swimming exercise intervention on MI R-34a mediated cell autophagy in the aging process of D- galactose induced rats. Methods: 1. male Sprague-Dawley (SD) rats were randomly divided into control group (Control), D-gal aging model group (D-gal), aging model + exercise intervention group (D-gal+EXE), 10 rats in each group. After the D-gal+EXE group, the rats of group D-gal+EXE were trained for adaptive swimming: swimming 10 min on the first day, and then increasing 10 min every day, until the adaptive training was increased to 60 min. a week, and the formal experiment began. Every day, the D-gal and D-gal+EXE group rats were injected with D- galactose under the neck of the neck of 200 mg/kg to induce aging, while the D-gal+EXE group was large. Rats were trained with 60 min swimming training, 5 days a week. After the drug injection and swimming training were performed at the same time of.6 weeks, the learning and memory ability of rats in each group was detected by the Morris water maze. After the water maze ended 24 hours, all rats were killed and selected. The serum superoxide dismutase (SOD) activity and hippocampus tissue of rats were detected by the reagent box. The content of MDA (MDA) and the expression of miR-34a were detected by Real-time-PCR. The morphologic changes of mitochondria in the hippocampus and autophagosomes in the hippocampus were observed by transmission electron microscope. Western blot was used to detect the expression of autophagy and mitochondrial dynamics related protein in.2. culture SH-SY5Y cells. When the degree of fusion reached 50-70%, the 6 pore plates were inoculated and the miR-34a inhibitors were transfected. The cells were stimulated with D- galactose, and the cells were collected at 48 h after transfection. The expression of miR-34a was detected by Real-time-PCR. Western blot was used to detect the expression of autophagy and mitochondrial dynamics related protein. Results: the results of 1.Morris water maze showed that the learning and memory ability of rats decreased significantly after 6 weeks of subcutaneous injection of D- galactose to the control group. The D-gal+EXE group after 6 weeks of swimming training had obviously improved.D-gal induced aging rats to cross the target quadrant times less than the control group, while the incubation period was higher than the control group, while the swimming exercise intervention group was more than the D-gal induced model group, and the incubation period was significantly shorter than that of the aging model rats. At the same time, compared with the control group, the activity of SOD in the serum of the D-gal model rats decreased significantly, the content of MDA in the hippocampus increased significantly. The SOD in the D-gal+EXE group of the group of D-gal+EXE rats showed significant improvement, and the content of MDA in the hippocampus decreased significantly. Transmission electron microscope observation found that the median grain of hippocampus in the D-gal model group was found in the rat model group. The body swelling and fusion increased, while the mitochondria in the hippocampus of D-gal+EXE rats tended to be normal and there were more autophagic bodies.Real-time-PCR results showed that the expression of miR-34a in the hippocampus of D- galactose induced aging rats and the SH-SY5Y cells treated with D- galactose were significantly increased, swimming intervention and miR-34a inhibitors were observed. After treatment, the expression amount showed a significant decrease in.Western blot results. Compared with the normal control group, the autophagy related protein Atg7, Beclin1, LC3II/I in the hippocampus of the D-gal model rats decreased significantly, and the p62 increased significantly; the mitochondrial kinetic related proteins Drp1 and Mfn2 increased significantly, and the D-gal+EXE group rats were Atg7, Beclin1, LC3II/I significantly increased, the expression of p62 decreased significantly, and the expression of Drp1 and Mfn2 also significantly decreased the autophagy associated protein Atg7, Beclin1, LC3II/I in SH-SY5Y cells after.2.D- galactose treatment. P62 was significantly increased, the mitochondrial kinetic related proteins Drp1 and expressions were significantly increased. TG7, Beclin1, LC3II/I significantly increased, the expression of p62 decreased significantly, and the expression of Drp1 and Mfn2 also decreased significantly. Conclusion: in the rat brain senescence model and cell aging model induced by D- galactose, miR-34a regulates the aging process of rat brain by regulating autophagy and the expression of mitochondrial dynamics related egg white. Activation can improve mitochondrial function and delay brain aging by activating mi R-34a mediated autophagy.
【学位授予单位】：武汉体育学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：G861.1;G804.2

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