缺氧的脂肪细胞与巨噬细胞条件培养基对骨骼肌细胞胰岛素作用的影响

发布时间：2019-05-29 22:30

【摘要】：目的：应用脂肪细胞和巨噬细胞条件培养基以及饱和脂肪酸孵育骨骼肌细胞,检测胰岛素刺激的骨骼肌葡萄糖转运子4(GLUT4)的转位和胰岛素信号分子的磷酸化水平,研究肥胖和缺氧对骨骼肌胰岛素作用的影响,探讨肥胖导致周身胰岛素抵抗的机制。方法： 1.脂肪细胞和巨噬细胞分别进行常氧和缺氧(1%02,5%CO2,94%N2)培养,制备条件培养基。 2.用脂肪细胞和巨噬细胞的条件培养基孵育骨骼肌细胞,采用偶联抗体的吸光度分析法测定骨骼肌GLUT4转位,采用(?)Vestern Blot法测定胰岛素信号分子的磷酸化水平。 3.用饱和脂肪酸棕榈酸孵育骨骼肌细胞,采用偶联抗体的吸光度分析法测定骨骼肌中GLUT4转位,采用(?)Vestern Blot法测定胰岛素信号分子的磷酸化水平。 4.采用ELISA法测定脂肪细胞和巨噬细胞条件培养基内TNF-α的水平。 5.采用Real-time PCR法,测定常氧和缺氧处理的脂肪细胞脂联素mRNA、 TNF-a mRNA和巨噬细胞TNF-a mRNA水平。 6.采用Transwell系统观察常氧和缺氧培养的脂肪细胞对巨噬细胞的趋化作用。 7.本研究的数据采用SPSS13.0软件进行统计学分析。结果： 1.缺氧处理脂肪细胞和巨噬细胞,GLUT1蛋白表达增加。 2.常氧处理的脂肪细胞条件培养基孵育骨骼肌细胞,在C2C12和L6两种骨骼肌细胞中,基础状态和胰岛素刺激状态下GLUT4转位与对照组相比无显著性差异；缺氧处理的脂肪细胞条件培养基孵育骨骼肌细胞骨骼肌细胞在基础状态下GLUT4转位与对照组相比有显著增加(p0.05,p0.01),而胰岛素刺激的GLUT4转位与对照组相比增加无显著性差异,C2C12骨骼肌细胞胰岛素刺激GLUT4转位增加的倍数与对照组相比呈下降趋势,但无显著性差异,而在L6细胞中则显著下降(p0.05)。 3.常氧处理的巨噬细胞条件培养基孵育C2C12骨骼肌细胞,与对照组相比,基础状态和胰岛素刺激状态下GLUT4转位有上升趋势,但无显著性差异。缺氧处理的巨噬细胞条件培养基孵育骨骼肌细胞,与对照组相比,基础状态下GLUT4转位有显著性升高(p0.05),胰岛素刺激状态下GLUT4转位有增加趋势,但无显著性差异。两组胰岛素刺激GLUT4转位增加的倍数与对照组相比均呈下降趋势,但无显著性差异。 4.常氧处理的脂肪细胞条件培养基孵育C2C12骨骼肌细胞,与对照组相比,基础状态下Akt磷酸化水平有升高的趋势,但无显著性差异,胰岛素刺激的Akt磷酸化水平与对照组相比无显著性差异。缺氧处理的脂肪细胞条件培养基孵育C2C12骨骼肌细胞,与对照组相比,基础状态下Akt磷酸化水平有升高的趋势,但无显著性差异,胰岛素刺激的Akt磷酸化水平与对照组相比无显著性差异。 5.常氧处理的巨噬细胞条件培养基孵育C2C12骨骼肌细胞,与对照组相比,基础状态下Akt磷酸化水平有升高的趋势,但无显著性差异,胰岛素剌激的Akt磷酸化水平与对照组相比无显著性差异。缺氧处理的巨噬细胞条件培养基孵育C2C12骨骼肌细胞,与对照组相比,基础状态下Akt磷酸化水平有升高趋势,但无显著性差异,而胰岛素刺激的Akt磷酸化水平与对照组相比无显著性差异。 6.棕榈酸孵育骨骼肌细胞,可使骨骼肌细胞在基础状态下GLUT4转位和对照组相比无明显改变,而在胰岛素刺激下的GLUT4转位和对照组相比显著降低(p0.05)。溶剂组BSA孵育的骨骼肌细胞,胰岛素增加骨骼肌细胞Akt的磷酸化水平,而棕榈酸孵育则使胰岛素刺激的Akt磷酸化水平显著性降低。胰岛素增加S6K的磷酸化水平,而棕榈酸孵育不影响胰岛素刺激的S6K的磷酸化以及IRS1$636/639磷酸化水平 7.缺氧处理的脂肪细胞和巨噬细胞条件培养基中TNF-a水平显著升高(p0.01)。 8.缺氧处理的脂肪细胞脂联素mRNA水平降低(p0.01),缺氧处理的脂肪细胞和巨噬细胞TNF-α mRNA水平显著升高(p0.01)。 9.缺氧处理的脂肪细胞吸引巨噬细胞迁移增加。结论： 1.缺氧培养箱培养细胞,可造成脂肪细胞和巨噬细胞缺氧,模拟肥胖者脂肪组织缺氧,制备条件培养基。 2.常氧处理的脂肪细胞条件培养基孵育骨骼肌细胞可造成骨骼肌细胞胰岛素刺激的GLUT4转位增加的倍数呈下降的趋势,但无显著性差异。而缺氧处理的脂肪细胞条件培养基孵育骨骼肌细胞可造成骨骼肌细胞胰岛素抵抗。 3.常氧处理和缺氧处理的巨噬细胞条件培养基孵育骨骼肌细胞使骨骼肌细胞胰岛素刺激的GLUT4转位增加的倍数呈下降的趋势,但无显著性差异。 4.饱和脂肪酸孵育可造成骨骼肌细胞胰岛素抵抗,其机制可能不涉及S6K。 5.脂肪细胞在缺氧状态下脂联素表达水平显著下降,TNF-α表达水平显著增加。巨噬细胞在缺氧状态下,TNF-α表达水平显著增加。这些因子可能参与条件培养基导致骨骼肌胰岛素抵抗的机制。 6.脂肪细胞缺氧可吸引更多的巨噬细胞,可能参与肥胖导致周身胰岛素抵抗的机制。
[Abstract]:Purpose: The translocation of the skeletal muscle glucose transporter 4 (GLUT4) and the phosphorylation of the insulin signal molecules of the skeletal muscle glucose transporter 4 (GLUT4) in the insulin-stimulated skeletal muscle were detected using the fatty cell and the macrophage conditioned medium and the saturated fatty acid. To study the effect of obesity and hypoxia on the insulin action of skeletal muscle, and to investigate the effect of obesity on the insulin resistance of skeletal muscle. System. Methods:1. The fat cells and macrophages were cultured by normal oxygen and hypoxia (1%02,5% CO2,94% N2), respectively. Conditioned medium.2. The skeletal muscle cells were incubated with the conditioned medium of adipocytes and macrophages, and the skeletal muscle GLUT was determined using the absorbance analysis method of the coupled antibody. 4 Determination of insulin signal by using (?) western Blot method Phosphorylation level of the child.3. The skeletal muscle cells were incubated with saturated fatty acid palmitic acid, and the GL of the skeletal muscle was determined using the absorbance analysis method of the coupled antibody. U Transposition of T4, using (?) Western Blot to measure the insulin letter Phosphorylation level of the number of molecules.4. The method of ELISA was used to determine the conditions of the adipocytes and macrophages. The levels of TNF-a and TNF-a mRNA, TNF-a mRNA and macrophagocytosis in the fat cells treated with normal oxygen and hypoxia were determined by the Real-time PCR. Cell TNF-a mRNA level.6. Using the Transwell system to observe the culture of normal oxygen and hypoxia 7. The chemotaxis of fat cells to macrophages. SPS S13.1.0 Statistical analysis of the software. Results:1. Anoxia treatment Adipocytes and macrophages, GLUT1 protein expression increased.2. Normal oxygen treated fat cell conditioned medium for skeletal muscle cells, in both C2C12 and L6 skeletal muscle cells, basal and insulin Compared with the control group, the expression of GLUT4 was significantly higher than that in the control group (p0.05, p0.01) compared with the control group (p0.05, p0.01). There was no significant difference between the shock-induced GLUT4 index and the control group, and the increase of the insulin-stimulated GLUT4 index in the C2C12 skeletal muscle cells decreased in comparison with the control group, but no significant difference was observed. The difference in sex and the significant decrease in L6 cells (p0.05).3. The C2C12 skeletal muscle cells were incubated with normal oxygen-treated macrophage conditioned medium and the basal and insulin levels were compared to the control group. There was no significant difference in GLUT4 transposition in the condition of stimulation, but there was no significant difference. In the conditioned medium of the macrophages treated with hypoxia, the skeletal muscle cells were incubated, and compared with the control group, there was a significant increase in GLUT4 transposition in the basal state (p0.05), and insulin. There was an increased tendency of GLUT4 translocation in the stimulated state, but there was no significant difference. The two groups of insulin-stimulated GLUT4 translocation The increased fold decreased with the control group, but there was no significant difference.4. The cultured C2C12 skeletal muscle cells were incubated with normal oxygen treated fat cell conditioned medium, but there was no significant difference in the level of Akt phosphorylation in the basal state compared with the control group. There was no significant difference in the level of Akt phosphorylation of insulin stimulation compared with the control group. The cultured C2C12 skeletal muscle cells were incubated with hypoxic-treated fat cell conditioned medium, but there was no significant difference in Akt phosphorylation in the basal state compared with the control group. There was no significant difference in the level of Akt phosphorylation of insulin stimulation compared with that in the control group.5. The level of Akt phosphorylation in normal oxygen treated macrophage conditioned medium was higher than that of the control group, but there was no significant difference in the level of Akt phosphorylation in the basal state. There was no significant difference between the level of Akt phosphorylation and the control group, but there was no significant difference in the level of Akt phosphorylation in the basal state compared with the control group. There was no significant difference in the level of Akt phosphorylation of the insulin-stimulated cells compared with the control group.6. The skeletal muscle cells were incubated with palmitic acid, which could make the skeletal muscle cells not change significantly compared with the control group in the basal state. The GLUT4 index in the insulin-stimulated group was significantly lower than that in the control group (p0.05). The skeletal muscle cells incubated with the solvent group BSA, insulin, and the phosphoric acid of the skeletal muscle cell Akt were increased. The level of phosphorylation of the Akt phosphorylation in insulin-stimulated insulin was significantly reduced by the incubation of palmitic acid, while insulin increased the level of phosphorylation of S6K, while the incubation of palmitic acid did not affect Phosphorylation of S6K stimulated by insulin and phosphorylation of IRS1 $636/639 phosphorylation 7. Hypoxic treatment The level of TNF-a in the conditioned medium of adipocytes and macrophages increased significantly (p0.01).8. The level of adiponectin mRNA in the cultured adipocytes (p0.01) decreased (p0.01). Oxygen-treated adipocytes and macrophages, TNF-1 mRNA horizontal display Conclusion:1. Anoxia tissue culture The culture cells of the culture box can cause hypoxia of the fat cells and the macrophages, and can simulate the hypoxia of the fat tissues of the obesity patients and prepare the conditioned medium. the myoblasts can cause a downward trend in the increase in the number of GLUT4 transposition of the skeletal muscle cell insulin stimulation, However, there was no significant difference. The incubation of skeletal muscle cells with hypoxic-treated fatty cell conditioned medium could cause insulin resistance in skeletal muscle cells.3. The conditioned medium of macrophage conditioned by normal oxygen treatment and hypoxia The incubation of skeletal muscle cells resulted in a decrease in the number of GLUT4 transposition of the skeletal muscle cell insulin-stimulated 4. The incubation of saturated fatty acids can lead to insulin resistance of skeletal muscle cells, which The system may not involve S6K.5. The fat cells are fat in the oxygen-deficient state. The level of expression of adiponectin decreased significantly, and the level of TNF-VEGF was significantly increased. The levels of TNF-VEGF in the cells were significantly increased in the condition of hypoxia. These factors may be involved in the conditioned medium
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R587.1;R329

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