高脂饮食引起肌少症的机制研究

发布时间：2018-02-28 16:00

本文关键词： 高脂饮食 11-βHSD1 棕榈酸肌肉干细胞氧耗率线粒体　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:随着社会经济的发展,膳食结构逐渐西方化,代谢综合征及心脑血管疾病发病率急剧上升,高脂饮食(High fat diet,HFD)被认为是威胁人类健康的重要危险因素之一。近年来,因高脂饮食对老化的九个标志都有影响,能促进老化相关性疾病的发生,如肌少症,被认为是老化加速器。肌少症(Sarcopenia)作为以骨骼肌质量及其力量的下降为特征的新的老年综合征,是老年人躯体功能障碍,生活质量下降及跌倒、衰弱、失能及死亡的重要危险因素,逐渐受到重视。随着老化,肌蛋白平衡倾向肌肉降解一方,更为重要的是,骨骼肌的再生、修复能力明显减退。目前认为负责骨骼肌再生主要为骨骼肌干细胞,通过增殖、分化修复老化过程中的慢性损伤。随着研究的进展,有效增强骨骼肌干细胞的增殖及分化功能,可成为延缓肌少症的有效手段。方法:体内研究方面:我们建立高脂喂养C57/B6 5月小鼠模型,进一步分析西方生活式(高脂饮食)对小鼠肌纤维量及力量的影响。使用双能X线仪检测小鼠体内肌肉、脂肪组织,并计算其含量;使用小鼠抓力仪测定小鼠抓力;分离腓肠肌(Gastrocnemius,Gas)及胫骨前肌(Tibialis anterior,TA),用苏木精-伊红(HE)染色观察比较肌纤维横截面积大小;荧光定量PCR测定老化基因肿瘤抑制基因(P53、P16),肌降解蛋白泛素蛋白连接酶E3s(muscle atrophy F-box,Atrogin-1 和 muscle-specific RING finger protein 1,MuRF-1),炎症相关指标(IL-6、TNFα)及11β-羟基类固醇脱氢酶(11β-Hydroxysteroid Dehydrogenase,11 β-HSD1)。体外研究方面:使用不同浓度棕榈酸(Palmitic acid,PA)干预肌肉干细胞分化,分析游离脂肪酸对肌肉干细胞分化及功能的影响。在小鼠骨骼肌干细胞诱导分化后,同时予PA及BVT干预,用荧光染色法、荧光定量PCR测定成肌基因肌球蛋白重链(Myosin heavy chains,MyHC)表达;seahorse能量代谢仪检测细胞的氧耗增加率;荧光定量PCR检测线粒体融合基因线粒体融合素基因1(mitofusin1,Mfn1)、视神经萎缩症蛋白基因(opticatrophy 1,Opa1)和裂解基因发动相关因子基因(dynamin 1-like,Drp1)、线粒体分裂素基因1(fission 1,Fis1)的相对表达量。结果:动物实验中发现高脂饮食组小鼠,脂肪组织增加明显,肌间出现脂肪组织沉积,骨骼肌纤维变细,而且小鼠抓力相对减少。荧光定量PCR测定老化基因肿瘤抑制基因P16升高(P0.05),肌降解蛋白泛素蛋白连接酶E3s(Atrogin-1和MuRF-1)均增加,高脂诱发IL-6、TNFα为代表的炎症指标增加,11-βHSD1基因表达量增加。体外实验发现肌肉干细胞给予不同浓度PA刺激后,PA100umol/l时MyHC表达量少,11β-HSD1含量较高;给予PA刺激后MyHC荧光表达量下降,肌小管融合率减低,用seahorse能量代谢仪检测细胞的氧耗增加率Oxygen consumption rates(OCR)发现细胞线粒体氧化呼吸功能受抑制,且线粒体融合/裂解比值下降,裂解基因增加。而加入11β-HSD1特异性抑制剂BVT.2733后,肌小管融合率增加;细胞氧化呼吸能力改善;线粒体融合/裂解比值增加,裂解基因表达下降(P0.05)。结论:高脂饮食促使身体成分改变,脂肪组织明显增加,骨骼肌纤维变细,肌肉力量减退。高脂饮食加重骨骼肌萎缩、老化,增强肌肉组织内炎症反应。而且抑制肌肉干细胞分化、影响线粒体氧耗功能,增加线粒体裂解。11β-HSD1特异性抑制剂BVT.2733可以改善肌肉干细胞分化,为延缓肌少症的发生发展提供有效治疗手段。
[Abstract]:Objective: with the development of social economy, the dietary structure gradually westernized, and the incidence of cardiovascular and cerebrovascular disease rate of a sharp rise in metabolic syndrome, high fat diet (High fat, diet, HFD) is considered to be one of the important risk factor of threatening human health. In recent years, due to the high fat diet had a great influence on the nine signs of aging that can promote the aging related diseases, such as sarcopenia, is considered to be the aging accelerator. Sarcopenia (Sarcopenia) as a decline in skeletal muscle mass and strength for the characteristics of the new syndrome in the elderly, is the avoidance of somatic function of the elderly because of the decline in the quality of life, and fall, weakness, disability and factors an important risk of death, has been paid more attention. With the aging tendency, muscle protein balance muscle degradation, more importantly, regeneration of skeletal muscle, significantly impaired ability to repair. It is mainly responsible for skeletal muscle regeneration of skeletal muscle stem cells The cell proliferation, differentiation, aging and chronic injury repair process. With the progress of the study, effectively enhance the proliferation and differentiation of skeletal muscle stem cell function, can become the effective means of delaying sarcopenia. Methods: in vivo study: we establish the model of high fat fed rats C57/B6 May, further analysis of the Western life style (high fat diet) on mice weight and muscle fiber strength. The use of dual energy X-ray detector in mice muscle, adipose tissue, and calculate the content; using mouse grip grip tester mice; separation of gastrocnemius muscle (Gastrocnemius, Gas) and anterior tibial muscle (Tibialis, anterior, TA) with hematoxylin eosin (HE) staining to observe and compare the cross-sectional area of muscle fiber size; fluorescence quantitative PCR determination of aging gene of tumor suppressor genes (P53, P16), muscle protein degradation of ubiquitin protein ligase E3s (muscle atrophy F-box, Atrogin-1 muscle-specific and RING Finger protein 1, MuRF-1), inflammation related indicators (IL-6, TNF) and 11 hydroxysteroid dehydrogenase (11 P -Hydroxysteroid Dehydrogenase 11 beta -HSD1). In vitro study: palmitic acid with different concentrations (Palmitic, acid, PA) muscle stem cell differentiation intervention, analysis of the effect of free fatty acid on muscle stem cells the differentiation and function. In mouse skeletal muscle stem cells after differentiation, at the same time with PA and BVT staining method for intervention, fluorescence, fluorescence quantitative PCR determination of muscle myosin heavy chain gene (Myosin heavy chains, MyHC) expression; increase the consumption rate of energy metabolism of seahorse cells were detected by oxygen; fluorescence quantitative PCR detection of mitochondrial fusion mfn-2 gene 1 (mitofusin1, Mfn1), optic nerve atrophy protein gene (opticatrophy 1, Opa1) and lytic gene related factor gene (dynamin launched 1-like, Drp1), mitochondrial fission gene 1 (fission 1, Fi The relative expression of S1). Results: high fat diet group were found in animal experiments, adipose tissue increased significantly, appear fat deposition in muscle, skeletal muscle fibers became thinner, and the grip strength of mice decreased. Fluorescence quantitative PCR determination of aging gene of tumor suppressor gene P16 increased (P0.05), muscle protein ubiquitin protein degradation E3s ligase (Atrogin-1 and MuRF-1) were increased, induced by high lipid IL-6, increased inflammatory markers as the representative of the TNF alpha, 11- beta HSD1 gene expression increase. In vitro experiments showed that the muscle stem cells treated with different concentrations of PA after stimulation, PA100umol/l MyHC expression amount, high content of -HSD1 11 beta MyHC expression decreased fluorescence; after PA stimulation, myotubule fusion rate decreased with energy metabolism of seahorse cells were detected by the increase in oxygen consumption rate of Oxygen consumption rates (OCR) found that the oxidation of mitochondrial respiratory function was inhibited, and the mitochondrial fusion / cleavage ratio Decreased lytic gene increased. While adding 11 beta -HSD1 specific inhibitor BVT.2733, myotubule fusion rate increase; improve the oxidative respiratory capacity; mitochondrial fusion / cracking ratio increase, decreased lytic gene expression (P0.05). Conclusion: high fat diet prompted a change in body composition, fatty tissue increased, skeletal muscle fibers fine, loss of muscle strength. High fat diet increased skeletal muscle atrophy, aging, enhance inflammatory reaction and inhibit muscle tissue. Muscle stem cell differentiation, affecting mitochondrial oxygen consumption, increased mitochondria.11 beta -HSD1 specific inhibitor BVT.2733 can improve muscle stem cell differentiation, provide effective treatment to delay the progression of sarcopenia.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R685

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