二甲双胍对3T3-L1前脂肪细胞增殖和分化的影响

发布时间：2018-04-28 05:35

本文选题：二甲双胍 + 3T3-L1脂肪前体细胞　；参考：《山东大学》2015年硕士论文

【摘要】：目的：(1)研究二甲双胍对3T3-L1脂肪前体细胞细胞增殖过程的影响；(2)研究二甲双胍对3T3-L1脂肪前体细胞细胞分化过程的影响；通过上述研究,探讨二甲双胍对脂肪细胞的作用,以期明确二甲双胍对脂肪前体细胞增殖和分化过程的干预是否为二甲双胍减轻体重的机制之一,进一步为肥胖的治疗寻找新的理论依据。方法：(1)3T3-L1脂肪前体细胞细胞增殖的测定：①用不同浓度的二甲双胍(0.05mmol/L, 0.5mmol/L,5mmol/L,10 mmol/L,20 mmol/L,50mmol/L)对体外培养的3T3-L1脂肪前体细胞细胞增殖过程进行干预。②应用倒置显微镜对细胞形态进行观察,应用MTT法测定二甲双胍细胞增殖的影响并与对照组(二甲双胍浓度为Ommol/L)进行对照。③应用酶标仪在OD570nm处测量各组光密度值,以此来判断不同浓度二甲双胍对细胞增殖的影响。(2)3T3-L1脂肪前体细胞细胞分化的测定：①用不同浓度的二甲双胍(0.05mmol/L, 0.5mmol/L,5mmol/L)对体外培养的3T3-L1脂肪前体细胞细胞分化过程进行干预。②应用倒置显微镜对细胞形态进行观察,油红O染色法鉴定脂肪细胞的分化程度,并与阴性对照组(未加成脂诱导液)和阳性对照组(加成脂诱导液,二甲双胍浓度为Ommol/L)进行对照。③应用酶标仪在OD510nm处测量各组的光密度值,以此来判断不同浓度二甲双胍对细胞分化的影响。结果：(1)二甲双胍对细胞增殖的影响：显微镜下观察细胞形态：在显微镜下观察贴壁后的3T3-L1脂肪前体细胞,细胞成长条梭形,类似成纤维细胞。体外培养24小时,0.05mmol/L和0.5 mmol/L的二甲双胍组和对照组相比,镜下细胞形态和密度无明显差异,而5 mmol/L,10 mmol/L,20 mmol/L和50mmol/L的二甲双胍组和对照组相比,细胞形态发生改变(变形、破坏、凋亡小体形成),数量明显减少。且为浓度依赖性,浓度越高,细胞破坏越严重,细胞数量。继续培养至72h,0.05 mmol/L和0.5 mmol/L的二甲双胍组和对照组相比,镜下细胞形态和密度亦无明显差异,而5 mmol/L,10 mmol/L,20 mmol/L和50mmol/L的二甲双胍组和对照组相比,随着浓度的增加细胞破坏越严重,细胞数量越少。测量各组光密度值：(D0.05mmol/L(OD值：2.0±0.09)和0.5 mmol/L(OD值：1.99±0.09)二甲双胍和对照组(OD值：2.14±0.14)比较,均无统计学意义(P0.05)。②5mmol/L(OD值：1.53±0.20)、10mmol/L(OD值：1.29±0.14)、20mmol/L (OD值：1.03±0.12)和50 mmol/L二甲双胍(OD值：0.70±0.13)和对照组(OD值：2.14±0.14)比较,均有明显统计学意义(P0.001)。③5mmol/L(OD值：1.53±0.20)、1 Ommol/L(OD值：1.29±0.14)、20mmol/L(OD值：1.03±0.12)和50 mmol/L二甲双胍(OD值：0.70±0.13)各组之间比较,均有统计学意义(P0.05),且浓度越高,光密度值越小。(2)二甲双胍对细胞分化的影响显微镜下观察细胞形态(油红0染色)：①阴性对照组细胞仍呈成梭形或椭圆形,油红0染色无明显着色。②阳性对照组可见80-90%的细胞细胞内有脂滴聚积,油红0染色着色明显。③0.05 mmol/L和0.5 mmol/L的二甲双胍组亦可见80-90%的呈脂肪细胞表型,细胞内有脂滴聚积,油红0染色着色明显,与阳性对照组相似。④5 mmol/L的二甲双胍组脂肪细胞表型数量较对照组明显减少,油红0染色着色较对照组明显减少。测量各组光密度值：①阳性对照组(OD值：0.61±0.060)、0.05mmol/L(OD值：0.59±0.055)、0.5 mmol/L(OD值：0.59±0.047)和5 mmol/L(OD值：0.42±0.027)和阴性对照组比较(OD值：0.34±0.017)均有明显统计学意义(P0.001)。②0.05mmol/L(OD值：0.59±0.055)、0.5 mmol/L(OD值：0.589±0.047)二甲双胍和阳性对照组(OD值：0.61±0.060)比较均无统计学意义(P0.05)。③5 mmol/L (OD值：0.42±0.027)二甲双胍和阳性对照组(OD值：0.61±0.060)比较,有明显的统计学意义(P0.001)。结论：(1) 0.05,0.5 mmol/L的二甲双胍对3T3-L1脂肪前体细胞细胞增殖无影响；大于5mmol/L的二甲双胍明显抑制3T3-L1脂肪前体细胞细胞增殖,且随着药物浓度的增加抑制作用增强；大于5 mmol/L的二甲双胍可能有促进3T3-L1脂肪前体细胞细胞凋亡的作用。(2) 0.05、0.5 mmol/L的二甲双胍对3T3-L1脂肪前体细胞细胞分化过程无影响；5mmol/L的二甲双胍抑制3T3-L1脂肪前体细胞细胞分化。
[Abstract]:Objective: (1) to study the effect of metformin on the proliferation of 3T3-L1 fat precursor cells; (2) to study the effect of metformin on the differentiation of 3T3-L1 adipose progenitor cells, and to explore the effect of metformin on the adipocytes in order to clarify the effect of two metformin on the proliferation and differentiation of fat precursor cells. Whether it is one of the mechanisms of metformin reducing weight, and finding a new theoretical basis for the treatment of obesity. Methods: (1) the proliferation of 3T3-L1 fat precursor cells: (1) different concentrations of metformin (0.05mmol/L, 0.5mmol/L, 5mmol/L, 10 mmol/L, 20 mmol/L, 50mmol/L) were used for the in vitro culture of 3T3-L1 fat precursor cells The cell morphology was observed by inverted microscope. The effect of metformin cell proliferation was measured by MTT method and compared with the control group (metformin concentration of Ommol/L). (3) the light density values of each group were measured at OD570nm by the enzyme labeling instrument, so as to determine the increase of metformin at different concentrations to cells. Colonization. (2) determination of cell differentiation in 3T3-L1 fat precursor cells: (1) the differentiation process of 3T3-L1 adipose progenitor cells cultured in vitro was intervened with different concentrations of metformin (0.05mmol/L, 0.5mmol/L, 5mmol/L). Second, the cell morphology was observed by inverted microscope, and the differentiation of adipocytes was identified by oil red O staining. Degree, and compared with negative control group (without adipogenic inducer) and positive control group (addition of lipid inducer, metformin concentration of Ommol/L). (3) the effects of different concentrations of metformin on cell differentiation were measured at OD510nm at different concentrations. Results: (1) the effect of metformin on cell proliferation The morphology of the cells was observed under the microscope: the 3T3-L1 fat precursor cells after the wall were observed under the microscope, the cells grew spindle shaped and similar to fibroblasts. The cells were cultured for 24 hours in vitro. Compared with the control group, the morphology and density of the 0.05mmol/L and 0.5 mmol/L metformin groups were not significantly different, and 5 mmol/L, 10 mmol/L, 20 mmol/L and 50m. In the metformin group of mol/L, the cell morphology changes (deformation, destruction, and the formation of apoptotic bodies), and the number of cells decreased significantly. And the concentration dependent, the higher the concentration, the more serious the cell destruction, the number of cells. The cell morphology and density of the cells under the microscope were compared with the control group compared with the 0.05 mmol/L and 0.5 mmol/L metformin groups. There was no significant difference. Compared with the 5 mmol/L, 10 mmol/L, 20 mmol/L and 50mmol/L metformin groups, the more serious the cell destruction was, the less the cell number. The values of the light density were measured (D0.05mmol/L (OD: 2 + 0.09) and 0.5 mmol/L (OD: 1.99 + 0.09) and the control group (o value: 2.14 + 0.14) ratio. There was no statistical significance (P0.05). 2 5mmol/L (OD: 1.53 + 0.20), 10mmol/L (OD: 1.29 + 0.14), 20mmol/L (o value: 1.03 + 0.12) and 50 mmol/L metformin (o value: 0.70 + 0.13) and comparison group (o value: 2.14 + 0.14), there were significant statistical significance (P0.001), 5mmol/L (OD: 1.53 + 0.20) and 1 Ommol/L (OD value: 0.12 Ommol/L) 4), 20mmol/L (OD: 1.03 + 0.12) and 50 mmol/L metformin (OD: 0.70 + 0.13) had statistical significance (P0.05), and the higher the concentration, the smaller the light density. (2) the effect of metformin on cell differentiation was observed under microscope (oil red 0 staining): (1) the cells in the negative control group were still spindle shaped or oval, There was no obvious coloring in the oil red 0 staining. (2) the cells in the positive control group showed the accumulation of lipid droplets in the cell cells of 80-90% and the coloring of oil red 0. (3) the phenotype of 80-90% was also found in the group of 0.05 mmol/L and 0.5 mmol/L in metformin, and the lipid droplets were accumulated in the cells, and the oil red 0 staining was obvious, similar to the positive control group. (4) two of 5 mmol/L. The number of adipocyte phenotypes in metformin group was significantly lower than that of the control group, and the color of oil red 0 coloring was significantly lower than that of the control group. The values of light density were measured in each group: (1) positive control group (o value: 0.61 + 0.060), 0.05mmol/L (OD value: 0.59 + 0.055), 0.5 mmol/L (OD value: 0.59 + 0.047) and 5 mmol/L (o value: 0.42 + 0.027) and negative control group (o value) 0.34 + 0.017) had significant statistical significance (P0.001). 0.05mmol/L (OD: 0.59 + 0.055), 0.5 mmol/L (OD: 0.589 + 0.047) metformin and positive control group (o value: 0.61 + 0.060) had no statistical significance (P0.05). (3) 5 mmol/L (o value: 0.42 + 0.027) metformin and positive control group (OD: 0.61 + 0.060), there were Significant statistical significance (P0.001). Conclusion: (1) metformin in 0.05,0.5 mmol/L has no effect on the proliferation of 3T3-L1 adipose progenitor cells, and metformin, which is greater than 5mmol/L, obviously inhibits the proliferation of 3T3-L1 fat precursor cells and increases with the increase of drug concentration; metformin, which is greater than 5 mmol/L, may be promoted. The effect of 3T3-L1 fat precursor cell apoptosis. (2) metformin, 0.05,0.5 mmol/L, has no effect on the differentiation of 3T3-L1 adipose progenitor cells; 5mmol/L metformin inhibits the differentiation of 3T3-L1 fat precursor cells.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1;R589.2

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