發(fā)布時(shí)間：2018-08-29 17:11

【摘要】：糖尿病是影響全身各個(gè)器官的內(nèi)分泌系統(tǒng)疾病,以血液循環(huán)中葡萄糖濃度升高為特點(diǎn),在肌肉、脂肪、肝臟組織的胰島素抵抗為主要原因。骨骼肌是胰島素作用的重要部位及人體調(diào)節(jié)血糖代謝的主要器官,可以消耗人體近80%的葡萄糖,但是高血糖環(huán)境也可以損害骨骼肌細(xì)胞,骨骼肌病變又使外周胰島素抵抗加重,所以治療糖尿病的藥物對骨骼肌的生長發(fā)育及損傷后修復(fù)的作用受到越來越廣泛的關(guān)注。利拉魯肽(LG)是一種胰高血糖素樣肽-1(GLP-1)類似物,與人體天然的GLP-1氨基酸序列具有97%的同源性,通過激活胰高血糖素樣肽-1受體(GLP-1R)作用于胰島β細(xì)胞產(chǎn)生葡萄糖依賴性的促進(jìn)胰島素分泌及其抑制胰島a細(xì)胞分泌胰高血糖素的作用已應(yīng)用于2型糖尿病的臨床治療。GLP-1還可作為促分化因子促進(jìn)前脂細(xì)胞的分化以及胰島細(xì)胞的增生。然而GLP-1在對骨骼肌細(xì)胞分化及功能的作用及調(diào)控機(jī)制尚未闡明,因此我們首先研究了 GLP-1R激動(dòng)劑LG能否促進(jìn)骨骼肌細(xì)胞的分化,以改善糖尿病病人肌細(xì)胞的萎縮與凋亡。另外骨骼肌細(xì)胞胰島素抵抗是2型糖尿病發(fā)生的一個(gè)重要因素,研究報(bào)道LG可能通過促進(jìn)糖原的合成及葡糖糖轉(zhuǎn)運(yùn)體4(GLUT4)的轉(zhuǎn)運(yùn)增強(qiáng)骨骼肌細(xì)胞對葡萄糖的利用,改善胰島素抵抗,但其機(jī)制尚未完全清楚。鳶尾素(irisin)是一種新發(fā)現(xiàn)的由肌肉細(xì)胞分泌的細(xì)胞因子,其前體物質(zhì)為III型纖連蛋白結(jié)構(gòu)域(FNDC5)。有研究表明Irisin作為AMPK激活后PGC-1 a的表達(dá)產(chǎn)物,可能參與BCL-2所調(diào)控的骨骼肌細(xì)胞內(nèi)自噬作用,從而改善骨骼肌的胰島素的敏感性,所以一些臨床研究已經(jīng)證實(shí)的2型糖尿病病人的Irisin分泌會(huì)減少可能是肌肉組織發(fā)生胰島素抵抗的一個(gè)原因,因此我們進(jìn)一步研究了 LG是否通過調(diào)控Irisin的合成來發(fā)揮改善胰島素抵抗的作用。第一部分利拉魯肽對骨骼肌成肌細(xì)胞C2C12分化的影響研究目的:探討LG對骨骼肌成肌細(xì)胞分化的影響研究內(nèi)容:1.于C2C12細(xì)胞誘導(dǎo)分化過程中第0、2、4、6天收集樣品,提取mRNA和蛋白,分別用熒光定量PCR和Western blot檢測肌管細(xì)胞分化標(biāo)志物以及GLP-1R的基因和蛋白水平的表達(dá)。2.于C2C12細(xì)胞誘導(dǎo)分化過程中,給予100nM濃度GLP-1受體激動(dòng)劑LG處理,分別于第0、2、4、6天收集樣品,提取mRNA和蛋白,分別用熒光定量PCR和Western blot檢測肌管細(xì)胞分化標(biāo)志物基因蛋白水平的表達(dá)。3.于C2C12細(xì)胞誘導(dǎo)分化過程中,給予100 nM LG和10 nM GLP-1R拮抗劑exendin(9-39)處理,于第6天收取樣品,提取蛋白,用Western blot檢測肌管細(xì)胞分化標(biāo)志物蛋白水平的表達(dá)。研究結(jié)果:1.Western blot及熒光定量PCR的結(jié)果顯示,GLP-1R在骨骼肌成肌細(xì)胞C2C12中表達(dá),且在分化成熟肌管細(xì)胞中的表達(dá)水平明顯高于未分化成肌細(xì)胞。2.C2C12細(xì)胞分化過程中,用LG進(jìn)行處理,結(jié)果顯示成肌細(xì)胞決定基因1(MYOD1)及肌球蛋白輕鏈3(MYL3)的蛋白表達(dá)水平明顯高于對照組;同樣MYOD1及肌球蛋白(Myosin)的基因表達(dá)水平明顯高于對照組。3.C2C12細(xì)胞分化過程中,用LG及exendin(9-39)進(jìn)行處理,結(jié)果顯示分化第六天,LG組細(xì)胞MYOD1及MYL3蛋白表達(dá)顯著高于對照組;與LG組細(xì)胞比較,Ex組細(xì)胞的MYOD1及MYL3蛋白表達(dá)受到抑制,顯著降低,而LG + Ex組細(xì)胞的MYOD1及MYL3蛋白表達(dá)未受到Ex的抑制,與LG組持平。研究結(jié)論:1.GLP-1R存在于肌管細(xì)胞中2.在C2C12成肌細(xì)胞分化成熟為肌管細(xì)胞的過程中,LG發(fā)揮促進(jìn)作用。第二部分利拉魯肽對肌管細(xì)胞合成FNDC5的影響及其機(jī)制研究研究目的:探討LG對肌管細(xì)胞合成Irisin前體物質(zhì)FNDC5的影響及其機(jī)制研究內(nèi)容:1.小鼠骨骼肌成肌細(xì)胞C2C12經(jīng)誘導(dǎo)分化為肌管細(xì)胞后,給予梯度濃度LG(1-1000 nm/L)處理不同時(shí)間(0-24 h),觀察LG對FNDC5表達(dá)及磷酸化腺苷酸活化蛋白激酶(cAMP-activated protein kinase,AMPK)和絲裂原活化激酶(mitogen-activated protein kinase,MAPK)信號(hào)通路活性的影響2.分別應(yīng)用GLP-1R拮抗劑Exendin(9-39),AMPK的抑制劑Compound c及MAPK的抑制劑SCH772984預(yù)處理肌管細(xì)胞,應(yīng)用Western blot方法觀察FNDC5蛋白表達(dá)的改變,檢測AMPK及MAPK信號(hào)通路的的活性。研究結(jié)果:1.LG可促進(jìn)肌管細(xì)胞FNDC5的蛋白表達(dá),并具有時(shí)間-劑量依賴性。2.LG可激活A(yù)MPK與MAPK信號(hào)通路,呈時(shí)間-劑量依賴性。3.Compound c抑制LG誘導(dǎo)的AMPK表達(dá),顯著降低LG誘導(dǎo)的FNDC5蛋白表達(dá),SCH772984抑制LG誘導(dǎo)的MAPK表達(dá),并顯降低著LG誘導(dǎo)的FNDC5蛋白表達(dá)。4.Exendin(9-39),可抑制LG激活的MAPK及AMPK磷酸化水平信號(hào)通路;LG促進(jìn)的FNDC5蛋白表達(dá)也受到抑制。研究結(jié)論:LG通過GLP-1R激活A(yù)MPK和MAPK信號(hào)通路促進(jìn)肌管細(xì)胞FNDC5的表達(dá)
[Abstract]:Diabetes mellitus is an endocrine disorder affecting all organs of the body. It is characterized by elevated glucose concentration in the blood circulation. Insulin resistance is the main cause in muscle, fat and liver tissues. Skeletal muscle is an important part of insulin and the main organ of regulating blood glucose metabolism. It can consume nearly 80% of human glucose. Hyperglycemic environment can also damage skeletal muscle cells, skeletal muscle lesions and aggravate peripheral insulin resistance, so the role of diabetic drugs on skeletal muscle growth and development and repair after injury has received more and more attention. Liraglutide (LG) is a glucagon-like peptide-1 (GLP-1) analogue, and the human body's natural GLP-1. Glucagon-like peptide-1 receptor (GLP-1R) stimulates glucose-dependent insulin secretion by islet beta cells and inhibits glucagon secretion by islet a cells. GLP-1 can also be used as a differentiation-promoting factor to promote preadipheresis in type 2 diabetes mellitus. However, the role of GLP-1 in the differentiation and function of skeletal muscle cells has not been clarified. Therefore, we first studied whether GLP-1R agonist LG could promote the differentiation of skeletal muscle cells to improve the atrophy and apoptosis of skeletal muscle cells in diabetic patients. LG may enhance glucose utilization and improve insulin resistance by promoting glycogen synthesis and glucose transporter 4 (GLUT4) transport in skeletal muscle cells, but the mechanism is not fully understood. Iris is a newly discovered cytokine secreted by muscle cells. Irisin, as an expression product of PGC-1a activated by AMPK, may be involved in the autophagy of skeletal muscle cells regulated by BCL-2, thus improving the insulin sensitivity of skeletal muscle. Therefore, Irisin secretion in type 2 diabetic patients has been confirmed by some clinical studies. Reduction may be one of the causes of insulin resistance in muscle tissue, so we further investigated whether LG can improve insulin resistance by regulating the synthesis of Irisin. Part I Effect of liraglutide on the differentiation of skeletal myoblasts C2C12 Objective: To investigate the effect of LG on the differentiation of skeletal myoblasts. Contents: 1. Samples were collected on the 0th, 2nd, 4th and 6th day of the induction of differentiation of C2C12 cells, and the mRNA and protein were extracted. The expression of differentiation markers and GLP-1R gene and protein were detected by fluorescence quantitative PCR and Western blot respectively. 2. During the induction of differentiation of C2C12 cells, 100nM concentration of GLP-1 receptor agonist LG was given. Samples were collected on the 0th, 2nd, 4th and 6th day, and mRNA and protein were extracted. The expression of gene protein, a marker of myotube differentiation, was detected by fluorescence quantitative PCR and Western blot respectively. Western blot and fluorescence quantitative PCR showed that GLP-1R was expressed in C2C12 of skeletal myoblasts, and the expression level of GLP-1R in differentiated mature myoblasts was significantly higher than that in undifferentiated myoblasts. The expression of MYOD1 and myosin light chain 3 (MYL3) in myoblasts was significantly higher than that in control group. The expression of MYOD1 and myosin was also significantly higher than that in control group. Compared with LG group, MYOD1 and MYL3 protein expression in Ex group was inhibited and significantly decreased, while MYOD1 and MYL3 protein expression in LG + Ex group was not inhibited by Ex, which was the same as that in LG group. Conclusion: 1. GLP-1R was present in myotube cells 2. MYOD1 and MYL3 protein expression in C2C12 myoblast differentiated into myotube cells. In the second part, the effect of liraglutide on the synthesis of FNDC5 by myotube cells and its mechanism were studied. Objective: To investigate the effect of LG on the synthesis of Irisin precursor FNDC5 by myotube cells and its mechanism: 1. After induction and differentiation of mouse skeletal muscle myoblast C2C12 into myotube cells, the gradient concentration of LG (LG) was given. Effects of LG on FNDC5 expression and signal pathway activity of cAMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) were observed at 1-1000 nm/L for different time (0-24 h). 2. GLP-1R antagonist Exendin (9-39), AMPK inhibitors UNDC and M PK inhibitors Exendin (9-39), respectively. APK inhibitor SCH772984 was used to pretreat myotubal cells. Western blot was used to observe the expression of FNDC5 protein and detect the activity of AMPK and MAPK signaling pathway. 3. Compound C inhibited LG-induced AMPK expression, significantly decreased LG-induced FNDC5 protein expression, SCH772984 inhibited LG-induced MAPK expression, and significantly decreased LG-induced FNDC5 protein expression. 4. Exendin (9-39) inhibited LG-activated MAPK and AMPK phosphorylation signaling pathway; LG-promoted FNDC5 protein expression was also inhibited. Activation of AMPK and MAPK signaling pathway through GLP-1R promotes FNDC5 expression in myotube cells
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1

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