本文關(guān)鍵詞：GLP-1對3T3-L1前脂肪細(xì)胞分化的影響及其調(diào)控機(jī)制研究，由筆耕文化傳播整理發(fā)布。

        研究背景：目前,肥胖及2型糖尿病已經(jīng)成為我國面臨的嚴(yán)重公共衛(wèi)生問題。作為代謝異常性疾病二者存在相近的發(fā)病基礎(chǔ),脂質(zhì)異常沉積與胰島素抵抗是其共同土壤[1]。盡管多種遺傳因素引起肥胖及胰島素抵抗,但環(huán)境因素中最主要的是長期能量攝入超過消耗,導(dǎo)致體內(nèi)脂肪過多蓄積,引起肥胖及脂質(zhì)代謝紊亂。肥胖在細(xì)胞層面上主要表現(xiàn)為已存在的脂肪細(xì)胞體積的肥大和由前脂肪細(xì)胞分化而來的脂肪細(xì)胞數(shù)量的增多。研究表明,脂肪細(xì)胞體積的肥大以及由前脂肪細(xì)胞分化而來的脂肪細(xì)胞數(shù)量的增多在成人肥胖的發(fā)生發(fā)展過程中均發(fā)揮著重要作用。然而,這兩種變化對脂質(zhì)及糖代謝的作用有著顯著的差別。脂肪細(xì)胞體積的肥大與脂質(zhì)代謝紊亂及胰島素抵抗密切相關(guān)；而脂肪細(xì)胞數(shù)量的增多(小體積脂肪細(xì)胞)則有助于改善高脂血癥,增加胰島素敏感性。胰高糖素肽-1(glucagon-like peptide-1, GLP-1)是由腸L細(xì)胞產(chǎn)生的目前己知作用最強(qiáng)的腸促胰島素分泌肽,除了調(diào)控血糖之外,還具有調(diào)節(jié)食欲、血壓、血脂,改善心功能、保護(hù)血管內(nèi)皮功能等多重生物學(xué)功能。研究證明,GLP-1能夠上調(diào)脂肪細(xì)胞表面胰島素受體(IR-β)及胞內(nèi)胰島素底物(IRS-1)的數(shù)量,促進(jìn)葡萄糖轉(zhuǎn)運(yùn)子4(GLUT-4)的表達(dá),抑制脂肪細(xì)胞中炎癥通路激活等改善脂肪細(xì)胞體積的肥大,進(jìn)而改善胰島素抵抗；然而,GLP-1對前脂肪細(xì)胞的分化的影響尚有爭議,其能否促進(jìn)小體積脂肪細(xì)胞的形成尚缺乏足夠研究。本實(shí)驗(yàn)以3T3-L1前脂肪細(xì)胞為實(shí)驗(yàn)對象,在其分化過程中給予重組人GLP-1進(jìn)行干預(yù),研究GLP-1對前脂肪細(xì)胞分化的影響及其調(diào)控機(jī)制,以期為肥胖的防治提供新的作用靶點(diǎn)。研究目的：1.觀察GLP-1對3T3-L1前脂肪細(xì)胞分化過程中脂肪標(biāo)志性蛋白LPL、 aP2及轉(zhuǎn)錄因子PPAR-γ、C/EBPa表達(dá)的影響。2.觀察GLP-1對3T3-L1前脂肪細(xì)胞分化過程中脂質(zhì)聚集以及脂肪細(xì)胞形態(tài)的影響。3.初步探討GLP-1發(fā)揮上述作用的分子機(jī)制。研究方法：1.3T3-L1前脂肪細(xì)胞的培養(yǎng)與分化：在無菌條件下培養(yǎng)3T3-L1前脂肪細(xì)胞,采用傳統(tǒng)“雞尾酒”法來誘導(dǎo)前脂肪細(xì)胞分化。2.給予3T3-L1前脂肪細(xì)胞不同濃度的GLP-1進(jìn)行干預(yù),用MTT法檢測其對細(xì)胞活力的影響,選取合適的濃度范圍。3.RT-PCR及Western-blot檢測脂肪細(xì)胞脂肪標(biāo)志性蛋白LPL、aP2及轉(zhuǎn)錄因子PPAR-γ、C/EBPa的表達(dá)。4.油紅O染色檢測3T3-L1前脂肪細(xì)胞誘導(dǎo)分化第8天細(xì)胞內(nèi)脂質(zhì)聚集,用Image Pro plus5.02軟件分析脂肪細(xì)胞體積和數(shù)量的變化。5. RT-PCR檢測誘導(dǎo)分化第8天時解偶聯(lián)蛋白-1(UCP-1)和肉堿脂酰轉(zhuǎn)移酶-1(CPT-1A)的mRNA表達(dá)。6.Western-blot檢測Akt、P38、ERK1/2信號通路的變化。研究結(jié)果：1.GLP-1顯著促進(jìn)脂肪細(xì)胞標(biāo)志性蛋白LPL、aP2和轉(zhuǎn)錄因子PPAR-γ、 C/EBPa的mRNA水平,呈濃度依賴性及時間依賴性(P<0.05)。2.與對照組相比,GLP-1顯著促進(jìn)轉(zhuǎn)錄因子PPAR-γ和脂肪細(xì)胞標(biāo)志性蛋白aP2的蛋白表達(dá),呈濃度依賴性及時間依賴性(P<0.05)。3.與對照組相比,GLP-1組且pAkt的蛋白表達(dá)水平顯著增加(P<0.05),而pP38和pERK1/2的蛋白水平未見顯著變化。4.油紅O染色結(jié)果顯示,100nM GLP-1顯著增加小體積脂肪細(xì)胞的形成(P<0.05)。5.與對照組和其他濃度組相比,100nM GLP-1組脂肪細(xì)胞中CPT-1A的mRNA表達(dá)水平顯著增加(P<0.05),而UCP-1的mRNA表達(dá)無顯著變化。研究結(jié)論：1.在3T3-L1前脂肪細(xì)胞分化過程中,GLP-1呈劑量及時間依賴性的促進(jìn)PPAR-γ、C/EBPα、LPL和aP2的表達(dá)。2.Akt信號通路可能參與GLP-1的上述作用過程。3.100nM GLP-1能促進(jìn)更多小體積脂肪細(xì)胞的形成,該細(xì)胞中CPT-1A的mRNA表達(dá)顯著增加。

    Background:Nowadays, obesity and type2diabetes had been the serious public health problems in China. They are both metabolic diseases that have the similar basic reasons. Abnormal lipid deposition and insulin resistance are closely related to them [1]. Although a lot of genetic reasons contribute to the outcomes of obesity and insulin resistance, the most important factor in the environment is that the imbalance between energy intake and expenditure, thus causing too much fat accumulation in the body and causing obesity and lipid metabolism disorders[2.3]. The cellular mechanisms for obesity include the expansion of white adipose tissue via the hypertrophy of preexisting adipocytes and hyperplasia resulting from the adipogenesis of preadipocytes. Recent studies have shown that both the hypertrophy and hyperplasia have played the role in the pathology of obesity in human adults. However, there are significant differences in lipid and glucose metabolism between adipocyte hypertrophy and hyperplasia. Adipocyte hypertrophy is negatively correlated with dyslipidema and insulin resistance, independent of body composition. Interestingly, hyperplasia, which is characterized by an increased number of small subcutaneous adipocytes, may have a positive effect on lipid metabolism and insulin sensitivity through preadipocyte differentiation[4-6]. Glucagon-like peptide1(GLP-1) is the most effective incretin until now, which is secreted from intestinal L-cells and exerts multiple biological effects. Not only can it regulate the blood glucose, appetite, blood pressure and blood lipid; but also improve the cardiac and vascular endothelial function[7] Recent studies have revealed that GLP-1can significantly reduce fat mass and adipocyte hypertrophy, improve insulin sensitivity in adipocyte by up-regulating the expression of insulin receptor, insulin receptor substrate and Glut-4, reducing macrophage infiltration and inhibiting inflammatory adipocytes [8-10]. However, the effect of GLP-1on adipogenesis is less clear; and if GLP-1can improve the information of small adipocyte is also don’t have enough research. In this study, using3T3-L1preadipocyte, we examined the effect of GLP-1on preadipocyte differentiation and the mechanisms involved.Objective:1. To observe the effect of GLP-1on the adipocyte-specific proteins LPL, aP2and the transcription factors PPAR-y, CEBP/a during the process of3T3-L1preadipocyte differentiation.2. To observe the Effect of GLP-1on lipid accumulation and the cytomorphology of adipocyte.3. To investigate the possible mechanism involved in the GLP-1induced effect.Methods:1.3T3-L1preadipocytes culture and differentiation:3T3-L1preadipocytes were cultured in aseptic condition and the traditional "Cocktail method" was adopted to induce the3T3-L1preadipocyte differentiation.2. GLP-1was added to the medium of3T3-L1cells at different concentrations, MTT assay was used to test the cell viability.3. RT-PCR and Western-blot were used to exam the express levels of the adipocyte-specific markers.4. Oil Red O staining to examine the effect of GLP-1on lipid droplet accumulation at the8th day of differentiation, and the Image Pro plus5.02was used to analyse the size and number of lipid droplet.5. RT-PCR was used to exam the express level of Uncoupling protein-1(UCP-1) and Carnitine palmitoyltransferase1(CPT-1A) at the8th day of differentiation.6. Under the treatment of GLP-1, we investigated the levels of Akt, P38and ERK1/2 signaling pathway.Results:1. At varying times during differentiation, GLP-1enhanced the mRNA levels of the transcription factors PPAR-γ,CEBP/α and the adipocyte-specific markers LPL, aP2significantly in a dose-and time-dependent way, as compared to the control group(P<0.05).2. GLP-1enhanced the protein levels of the PPAR-γ and aP2significantly in a dose-and time-dependent way, as compared to the control group(P<0.05).3. During the first24h of differentiation, Akt, P38and ERK1/2signaling way were activated in different degrees. Compared to the control group, the protein level of p Akt in GLP-1group was increased markedly (P<0.05), while the protein levels of pP38and pERK1/2were not observed any significantly changes.4. Oil Red O staining results were shown that the lipid accumulation in experimental group were not increased as the enhanced concentrations of GLP-1. As compared to the control and other concentration groups, the cytomorphology of adipocyte in100nM GLP-1group was shown there were increased numbers of small adipocyte (P <0.05)5. Under the treatment of100nM GLP-1, the mRNA level of CPT-1A was increased significantly, while the mRNA level of UCP-1was not changed as compared to the control and other concentration groups (P<0.05).Conclusion:1. GLP-1enhanced the express of PPAR-γ, CEBP/α, LPL and aP2significantly in a dose-and time-dependent way during the period of3T3-L1preadipocyte differentiation,2. Akt signaling way may be involved in the above GLP-1-induced effects.3.100nM GLP-1can promote the formation of small adipocytes, which have more mRNA level of CPT-1A.

GLP-1對3T3-L1前脂肪細(xì)胞分化的影響及其調(diào)控機(jī)制研究

中文摘要6-9ABSTRACT9-11符號說明12-15前言15-17材料與方法17-33結(jié)果33-36討論36-42創(chuàng)新點(diǎn)42局限性42-43結(jié)論43-44附圖44-53參考文獻(xiàn)53-59致謝59-60攻讀碩士學(xué)位期間發(fā)表的學(xué)術(shù)論文60-61附件61

  本文關(guān)鍵詞：GLP-1對3T3-L1前脂肪細(xì)胞分化的影響及其調(diào)控機(jī)制研究，由筆耕文化傳播整理發(fā)布。