二甲双胍对前脂肪细胞增殖分化的影响
发布时间：2018-07-24 20:36
【摘要】：目的:以3T3-L1前脂肪细胞为研究对象,重点探讨不同作用浓度Met对前脂肪细胞增殖分化的影响机制。观察前脂肪细胞阶段给予不同浓度二甲双胍后,对前脂肪细胞增殖分化的影响,同时检测成脂相关多种转录因子的变化情况。方法:购置3T3-L1前脂肪细胞进行培养,传二代后,将脂肪细胞接种到孔板,培养24小时后,不同浓度的二甲双胍0mM(control)、1mM、2mM、5mM、10mM作用48h,(1)用CCK-8检测细胞增殖情况。(2)收集细胞用RT-PCR技术检测脂肪细胞中GATA3、PPAR-γ、C/EBPα、β-catenin mRNA表达水平。(3)细胞过融合后,予成脂诱导液诱导成脂,A液3天,B液1天交替3次,共12天。诱导成脂后通过油红O染色判定细胞成脂情况。实验数据采用SPSS13.0软件进行统计学分析,以均数±标准差(x±s)的形式将研究所得结果进行表示,组间差异比较采用单因素方差分析,组间多重比较用LSD检验,检验水准取α=0.05。结果:(1)CCK-8细胞增殖实验:1mM、2mM、5mM具有促增殖作用,不同浓度间无明显差别;与1mM相比,10mM促增殖作用减弱。(2)油红O染色结果显示:与对照组对比,不同浓度的二甲双胍对脂肪细胞成脂均有一定的抑制作用,其中10mM抑制作用最强(图2-1)。(3)10mM二甲双胍较0、1、2mM相比,明显促进GATA3 mRNA的表达;10mM二甲双胍与5mM相比,明显下调PPAR-γmRNA的表达;与control相比,不同浓度二甲双胍均可明显抑制C/EBPα的表达,与5mM相比,10mM有抑制增强的趋势。(4)各浓度对β-catenin表达水平无显著差别(表2-1)。结论:在前脂肪细胞阶段给予二甲双胍,10mM二甲双胍可以明显抑制脂肪细胞的成脂分化,其机制可能与促进GATA3,抑制PPAR-γ和C/EBPα有关。近期研究表明,AICAR通过激活AMPK信号通路激活wnt经典通路,促进GATA3的表达从而抑制前脂肪细胞成脂。PPAR-γ和C/EBPα是脂肪细胞中调控脂肪细胞分化的关键转录分子。β-catenin是wnt经典通路的关键分子。本项目推测高浓度的Met可能激活AMPK途径,促进GATA3表达,抑制PPAR-γ和C/EBPα,但β-catenin无变化,可能与β-catenin非依赖性的非经典途径有关,具体分子机制有待进一步的蛋白水平及基因敲除等实验步骤的深入研究。
[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- 纬 PPAR- 纬 -PPAR- 纬 -EBP 伪, 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 卤standard deviation (x 卤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 伪 -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- 纬 mRNA compared with 5mM. Compared with control, metformin significantly inhibited the expression of C/EBP 伪 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- 纬 and C/EBP 伪. 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- 纬 and C/EBP 伪 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- 纬 and C/EBP 伪, 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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2
本文编号：2142582
[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- 纬 PPAR- 纬 -PPAR- 纬 -EBP 伪, 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 卤standard deviation (x 卤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 伪 -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- 纬 mRNA compared with 5mM. Compared with control, metformin significantly inhibited the expression of C/EBP 伪 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- 纬 and C/EBP 伪. 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- 纬 and C/EBP 伪 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- 纬 and C/EBP 伪, 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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2
【参考文献】
相关期刊论文 前2条
1 王慧;刘四红;翟元梅;朱晓宇;章菁;万云;路伟;石军;;二甲双胍通过调节成脂影响脂肪细胞对白血病细胞作用[J];中国实验血液学杂志;2015年02期
2 宋文华;王贵;王哲;;脂肪细胞培养研究现状[J];动物医学进展;2006年04期
相关硕士学位论文 前2条
1 杨霞;二甲双胍在3T3-L1前脂肪细胞诱导过程中对v-SNAREs蛋白家族mRNA表达的影响[D];山西医科大学;2016年
2 车选强;二甲双胍对3T3-L1前脂肪细胞增殖和分化的影响[D];山东大学;2015年
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