【摘要】：目的:以3T3-L1前脂肪細(xì)胞為研究對象,重點(diǎn)探討不同作用濃度Met對前脂肪細(xì)胞增殖分化的影響機(jī)制。觀察前脂肪細(xì)胞階段給予不同濃度二甲雙胍后,對前脂肪細(xì)胞增殖分化的影響,同時(shí)檢測成脂相關(guān)多種轉(zhuǎn)錄因子的變化情況。方法:購置3T3-L1前脂肪細(xì)胞進(jìn)行培養(yǎng),傳二代后,將脂肪細(xì)胞接種到孔板,培養(yǎng)24小時(shí)后,不同濃度的二甲雙胍0mM(control)、1mM、2mM、5mM、10mM作用48h,(1)用CCK-8檢測細(xì)胞增殖情況。(2)收集細(xì)胞用RT-PCR技術(shù)檢測脂肪細(xì)胞中GATA3、PPAR-γ、C/EBPα、β-catenin mRNA表達(dá)水平。(3)細(xì)胞過融合后,予成脂誘導(dǎo)液誘導(dǎo)成脂,A液3天,B液1天交替3次,共12天。誘導(dǎo)成脂后通過油紅O染色判定細(xì)胞成脂情況。實(shí)驗(yàn)數(shù)據(jù)采用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,以均數(shù)±標(biāo)準(zhǔn)差(x±s)的形式將研究所得結(jié)果進(jìn)行表示,組間差異比較采用單因素方差分析,組間多重比較用LSD檢驗(yàn),檢驗(yàn)水準(zhǔn)取α=0.05。結(jié)果:(1)CCK-8細(xì)胞增殖實(shí)驗(yàn):1mM、2mM、5mM具有促增殖作用,不同濃度間無明顯差別;與1mM相比,10mM促增殖作用減弱。(2)油紅O染色結(jié)果顯示:與對照組對比,不同濃度的二甲雙胍對脂肪細(xì)胞成脂均有一定的抑制作用,其中10mM抑制作用最強(qiáng)(圖2-1)。(3)10mM二甲雙胍較0、1、2mM相比,明顯促進(jìn)GATA3 mRNA的表達(dá);10mM二甲雙胍與5mM相比,明顯下調(diào)PPAR-γmRNA的表達(dá);與control相比,不同濃度二甲雙胍均可明顯抑制C/EBPα的表達(dá),與5mM相比,10mM有抑制增強(qiáng)的趨勢。(4)各濃度對β-catenin表達(dá)水平無顯著差別(表2-1)。結(jié)論:在前脂肪細(xì)胞階段給予二甲雙胍,10mM二甲雙胍可以明顯抑制脂肪細(xì)胞的成脂分化,其機(jī)制可能與促進(jìn)GATA3,抑制PPAR-γ和C/EBPα有關(guān)。近期研究表明,AICAR通過激活A(yù)MPK信號通路激活wnt經(jīng)典通路,促進(jìn)GATA3的表達(dá)從而抑制前脂肪細(xì)胞成脂。PPAR-γ和C/EBPα是脂肪細(xì)胞中調(diào)控脂肪細(xì)胞分化的關(guān)鍵轉(zhuǎn)錄分子。β-catenin是wnt經(jīng)典通路的關(guān)鍵分子。本項(xiàng)目推測高濃度的Met可能激活A(yù)MPK途徑,促進(jìn)GATA3表達(dá),抑制PPAR-γ和C/EBPα,但β-catenin無變化,可能與β-catenin非依賴性的非經(jīng)典途徑有關(guān),具體分子機(jī)制有待進(jìn)一步的蛋白水平及基因敲除等實(shí)驗(yàn)步驟的深入研究。
[Abstract]:Aim: to investigate the effects of 3T3-L1 preadipocytes on the proliferation and differentiation of preadipocytes with different concentrations of Met. The effects of different concentrations of metformin on the proliferation and differentiation of preadipocytes were observed. Methods: the adipocytes were cultured before 3T3-L1. After the second passage, the adipocytes were inoculated into the pore plate and cultured for 24 hours. (1) the proliferation of adipocytes was detected by CCK-8. (2) the expression of GATA3PPAR- 緯 PPAR- 緯 -PPAR- 緯 -EBP 偽, 尾 -catenin mRNA in adipocytes was detected by RT-PCR technique. (3) after the cells were fused, the cells were induced into lipopolysaccharide solution for 3 days. A total of 12 days. Oil red O staining was used to determine the adipogenic status of the cells. The experimental data were statistically analyzed by SPSS13.0 software, and the results were expressed in the form of mean 鹵standard deviation (x 鹵s). The differences between groups were compared by single factor analysis of variance, and the multiple comparisons between groups were tested by LSD test. The test level was 偽 -0.05. Results: (1) CCK-8 cell proliferation assay: 1. 1 mm M 2 mm M 5 mm M had the effect of promoting proliferation, but there was no significant difference between different concentrations, and the effect of 10 mm M on proliferation was weaker than that of 1mM. (2) the results of oil red O staining showed that: compared with the control group, the effect of oil red O staining was higher than that of the control group. Metformin at different concentrations could inhibit adipogenesis in adipocytes to a certain extent, and 10mM had the strongest inhibitory effect (fig. 2-1). (3) 10mM metformin significantly promoted the expression of GATA3 mRNA compared with 01mM. 10 mm metformin significantly down-regulated the expression of PPAR- 緯 mRNA compared with 5mM. Compared with control, metformin significantly inhibited the expression of C/EBP 偽 and increased the expression of 尾 -catenin in 10 mm compared with 5mM. (4) there was no significant difference in 尾 -catenin expression between different concentrations (Table 2-1). Conclusion: 10 mm metformin in preadipocytes can significantly inhibit adipogenic differentiation of adipocytes. The mechanism may be related to the promotion of GATA3 and the inhibition of PPAR- 緯 and C/EBP 偽. Recent studies have shown that AICAR activates the classical wnt pathway by activating the AMPK signaling pathway. Promoting the expression of GATA3 and inhibiting preadipocyte adipogenesis. PPAR- 緯 and C/EBP 偽 are the key transcription molecules in adipocytes to regulate adipocyte differentiation. 尾 -catenin is the key molecule of wnt classic pathway. Our study suggests that high concentration of Met may activate AMPK pathway, promote GATA3 expression and inhibit PPAR- 緯 and C/EBP 偽, but 尾 -catenin does not change, which may be related to 尾 -catenin independent non-classical pathway. The specific molecular mechanisms need to be further studied, such as protein level and gene knockout.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R589.2

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