缺氧脂肪细胞条件培养基造成骨骼肌细胞胰岛素抵抗

发布时间：2018-10-10 18:52

【摘要】：研究目的：脂肪组织缺氧是肥胖的早期表现,与巨噬细胞浸润和局部炎症相关。我们研究在缺氧环境中培养的脂肪细胞,释放促炎因子对巨噬细胞迁移以及骨骼肌胰岛素作用的影响。方法： 1.在21%O2,5%CO2(常氧)或1%O2,94%N2,5%CO2(缺氧)培箱中培养脂肪细胞,并提取条件培养基。 2. Real-time PCR测定缺氧脂肪细胞的脂肪因子的mRNA水平。 3. ELISA测定脂肪细胞条件培养基CM-H中TNFa、IL-6、MCP-1和脂联素的水平。 4. Transwell系统观察缺氧培养的脂肪细胞对巨噬细胞的趋化作用。 5.偶联抗体的吸光度分析法测定骨骼肌中GLUT4转位。 6. Western Blot法测定胰岛素信号分子的磷酸化水平。 7.热灭活CM-H后检测对骨骼肌细胞GLUT4转位的影响。 8.中和抗体中和CM-H中MCP-1、IL-6后检测对骨骼肌细胞作用的影响。 9.加入AMPK抑制剂Compound C后检测其对骨骼肌细胞作用的影响。结果： 1.3T3-L1脂肪细胞缺氧后缺氧诱导因子HIFla和葡萄糖转运子GLUT1的蛋白表达升高。 2.缺氧脂肪细胞的TNFa, IL-6和MCP-1的mRNA表达增加,而脂联素的mRNA表达降低。 3.CM-H中的TNFa, IL-6和MCP-1浓度增加,而脂联素浓度降低。 4.在Transwell的共培养系统中,缺氧脂肪细胞比常氧脂肪细胞募集更多巨噬细胞。用中和抗体中和共培养系统中的MCP-1抑制巨噬细胞的迁移。 5.CM-H导致C2C12肌管基础状态葡萄糖转运子GLUT4转位增加而胰岛素刺激的GLUT4转位减少。 6.CM-H导致C2C12肌管中介导胰岛素调节的GLUT4外排的两个关键信号分子Akt和AS160磷酸化水平明显降低。CM-H磷酸化JNK和S6K,并增加C2C12肌管细胞的IRS1丝氨酸磷酸化水平。 7.热灭活CM-H中的细胞因子逆转其对肌细胞GLUT4转位的影响。 8.在CM-H中加入IL-6中和抗体,部分逆转条件培养基造成的骨骼肌细胞基础状态GLUT4转位及胰岛素刺激的GLUT4转位增加。 9.在CM-H中加入AMPK抑制剂Compound C,部分逆转条件培养基对骨骼肌细胞基础状态GLUT4转位及胰岛素刺激的GLUT4转位增加。 10.在CM-H中加入MCP-1中和抗体,部分逆转条件培养基造成的骨骼肌细胞胰岛素刺激的GLUT4转位减少。结论： 1.缺氧可以使脂肪细胞TNFα, IL-6和MCP-1表达与分泌增加,脂联素的表达与分泌降低。 2.升高的趋化因子MCP-1可以促进巨噬细胞向脂肪细胞迁移。 3.CM-H造成骨骼肌细胞膜上基础状态GLUT4转位增加而胰岛素刺激的转位减少,及骨骼肌细胞胰岛素信号通路受损。 4.热灭活可以逆转CM对肌细胞GLUT4的转位影响。. 5.IL-6可能通过AMPK磷酸化既参与CM-H造成的骨骼肌细胞基础状态GLUT4转位增加和胰岛素刺激的转位增加。 6.MCP-1参与CM-H造成的骨骼肌细胞胰岛素刺激的转位减少。
[Abstract]:Objective: adipose tissue hypoxia is an early manifestation of obesity and is associated with macrophage infiltration and local inflammation. We investigated the effects of pro-inflammatory factor release on macrophage migration and skeletal muscle insulin in cultured adipocytes in anoxic environment. Methods: 1. The adipocytes were cultured in 21O _ 2 ~ (5) CO _ 2 (normoxic) or 1O _ (2) C _ (94) N _ (2) O _ (2) culture box, and the conditioned medium was 2. 2. Real-time PCR was used to measure the mRNA level of adipocyte adipocytes. The levels of TNFa,IL-6,MCP-1 and adiponectin in adipocyte conditioned medium CM-H were determined by ELISA. 4. 4. Transwell system was used to observe the chemotaxis of hypoxia-cultured adipocytes to macrophages. Spectrophotometric determination of GLUT4 translocation in skeletal muscle by coupling antibody. 6. 6. The phosphorylation level of insulin signaling molecules was determined by Western Blot method. Effects of heat inactivated CM-H on GLUT4 translocation of skeletal muscle cells. 8. 8. Effect of neutralizing antibody on skeletal muscle cells after neutralizing MCP-1,IL-6 in CM-H. 9. 9. The effect of AMPK inhibitor Compound C on skeletal muscle cells was detected. Results: the protein expression of hypoxia inducible factor HIFla and glucose transporter GLUT1 increased after hypoxia in 1.3T3-L1 adipocytes. 2. The mRNA expression of TNFa, IL-6 and MCP-1 in anoxic adipocytes was increased, while the mRNA expression of adiponectin was decreased, TNFa, IL-6 and MCP-1 in 3.CM-H were increased, and adiponectin was decreased. In the co-culture system of Transwell, anoxic adipocytes recruit more macrophages than normoxic adipocytes. MCP-1 in neutralizing co-culture system with neutralizing antibody inhibited the migration of macrophages. 5.CM-H increased the translocation of glucose transporter GLUT4 in the basic state of the C2C12 myotube and decreased the GLUT4 translocation stimulated by insulin. 6.CM-H induced the translocation of macrophages. The phosphorylation levels of Akt and AS160, two key signaling molecules of insulin-mediated GLUT4 efflux in C2C12 myotubes, were significantly decreased, while CM-H phosphorylation of JNK and S6K increased IRS1 serine phosphorylation level of C2C12 myotube cells. The effect of cytokines in heat inactivated CM-H on GLUT4 transposition of myocytes was reversed. 8. 8. When IL-6 neutralizing antibody was added to CM-H, partial reversal of GLUT4 translocation induced by conditioned medium and insulin stimulated GLUT4 translocation increased. When AMPK inhibitor Compound C was added to CM-H, partial reverse conditioned medium increased GLUT4 translocation and insulin stimulated GLUT4 translocation in skeletal muscle cells. When MCP-1 neutralizing antibody was added to CM-H, the GLUT4 translocation induced by insulin stimulation in skeletal muscle cells was partly reversed by conditioned medium. Conclusion: 1. Hypoxia can increase the expression and secretion of TNF 伪, IL-6 and MCP-1 in adipocytes, and decrease adiponectin expression and secretion. 2. Increased chemokine MCP-1 could promote macrophage migration to adipocytes. 3.CM-H increased basal GLUT4 translocation and decreased insulin-stimulated translocation in skeletal muscle cell membrane. Damage of insulin signaling pathway in skeletal muscle cells. 4. 4. Heat inactivation can reverse the effect of CM on GLUT4 translocation of myocytes. 5.IL-6 may be involved in the increase of GLUT4 translocation and insulin stimulation translocation of skeletal muscle cells induced by CM-H through AMPK phosphorylation. The translocation of insulin stimulation in skeletal muscle cells caused by CM-H was reduced.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329.2

【共引文献】