本文關(guān)鍵詞： 高脂飲食 11-βHSD1 棕櫚酸 肌肉干細胞 氧耗率 線粒體　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:隨著社會經(jīng)濟的發(fā)展,膳食結(jié)構(gòu)逐漸西方化,代謝綜合征及心腦血管疾病發(fā)病率急劇上升,高脂飲食(High fat diet,HFD)被認為是威脅人類健康的重要危險因素之一。近年來,因高脂飲食對老化的九個標志都有影響,能促進老化相關(guān)性疾病的發(fā)生,如肌少癥,被認為是老化加速器。肌少癥(Sarcopenia)作為以骨骼肌質(zhì)量及其力量的下降為特征的新的老年綜合征,是老年人軀體功能障礙,生活質(zhì)量下降及跌倒、衰弱、失能及死亡的重要危險因素,逐漸受到重視。隨著老化,肌蛋白平衡傾向肌肉降解一方,更為重要的是,骨骼肌的再生、修復(fù)能力明顯減退。目前認為負責(zé)骨骼肌再生主要為骨骼肌干細胞,通過增殖、分化修復(fù)老化過程中的慢性損傷。隨著研究的進展,有效增強骨骼肌干細胞的增殖及分化功能,可成為延緩肌少癥的有效手段。方法:體內(nèi)研究方面:我們建立高脂喂養(yǎng)C57/B6 5月小鼠模型,進一步分析西方生活式(高脂飲食)對小鼠肌纖維量及力量的影響。使用雙能X線儀檢測小鼠體內(nèi)肌肉、脂肪組織,并計算其含量;使用小鼠抓力儀測定小鼠抓力;分離腓腸肌(Gastrocnemius,Gas)及脛骨前肌(Tibialis anterior,TA),用蘇木精-伊紅(HE)染色觀察比較肌纖維橫截面積大小;熒光定量PCR測定老化基因腫瘤抑制基因(P53、P16),肌降解蛋白泛素蛋白連接酶E3s(muscle atrophy F-box,Atrogin-1 和 muscle-specific RING finger protein 1,MuRF-1),炎癥相關(guān)指標(IL-6、TNFα)及11β-羥基類固醇脫氫酶(11β-Hydroxysteroid Dehydrogenase,11 β-HSD1)。體外研究方面:使用不同濃度棕櫚酸(Palmitic acid,PA)干預(yù)肌肉干細胞分化,分析游離脂肪酸對肌肉干細胞分化及功能的影響。在小鼠骨骼肌干細胞誘導(dǎo)分化后,同時予PA及BVT干預(yù),用熒光染色法、熒光定量PCR測定成肌基因肌球蛋白重鏈(Myosin heavy chains,MyHC)表達;seahorse能量代謝儀檢測細胞的氧耗增加率;熒光定量PCR檢測線粒體融合基因線粒體融合素基因1(mitofusin1,Mfn1)、視神經(jīng)萎縮癥蛋白基因(opticatrophy 1,Opa1)和裂解基因發(fā)動相關(guān)因子基因(dynamin 1-like,Drp1)、線粒體分裂素基因1(fission 1,Fis1)的相對表達量。結(jié)果:動物實驗中發(fā)現(xiàn)高脂飲食組小鼠,脂肪組織增加明顯,肌間出現(xiàn)脂肪組織沉積,骨骼肌纖維變細,而且小鼠抓力相對減少。熒光定量PCR測定老化基因腫瘤抑制基因P16升高(P0.05),肌降解蛋白泛素蛋白連接酶E3s(Atrogin-1和MuRF-1)均增加,高脂誘發(fā)IL-6、TNFα為代表的炎癥指標增加,11-βHSD1基因表達量增加。體外實驗發(fā)現(xiàn)肌肉干細胞給予不同濃度PA刺激后,PA100umol/l時MyHC表達量少,11β-HSD1含量較高;給予PA刺激后MyHC熒光表達量下降,肌小管融合率減低,用seahorse能量代謝儀檢測細胞的氧耗增加率Oxygen consumption rates(OCR)發(fā)現(xiàn)細胞線粒體氧化呼吸功能受抑制,且線粒體融合/裂解比值下降,裂解基因增加。而加入11β-HSD1特異性抑制劑BVT.2733后,肌小管融合率增加;細胞氧化呼吸能力改善;線粒體融合/裂解比值增加,裂解基因表達下降(P0.05)。結(jié)論:高脂飲食促使身體成分改變,脂肪組織明顯增加,骨骼肌纖維變細,肌肉力量減退。高脂飲食加重骨骼肌萎縮、老化,增強肌肉組織內(nèi)炎癥反應(yīng)。而且抑制肌肉干細胞分化、影響線粒體氧耗功能,增加線粒體裂解。11β-HSD1特異性抑制劑BVT.2733可以改善肌肉干細胞分化,為延緩肌少癥的發(fā)生發(fā)展提供有效治療手段。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R685

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