蜕皮甾酮对游离脂肪酸诱导的3T3-L1肥胖细胞TLR4信号转导途径的影响

发布时间：2018-04-27 20:51

本文选题：蜕皮甾酮 + 3T3-L1脂肪细胞　；参考：《泸州医学院》2012年硕士论文

【摘要】：目的：通过培养、诱导、分化小鼠的3T3-L1前脂肪细胞，使用游离脂肪酸（FFA）建立胰岛素抵抗模型，在此基础上使用蜕皮甾酮（ECR）对其增殖进行干预，探究蜕皮甾酮在体外对细胞胰岛素抵抗的影响及可能的分子作用机制。重点观察蜕皮甾酮能否通过抑制核因子κB(NF-κB)炎性路径上IKKβ蛋白与NF-κB蛋白的表达改善的胰岛素抵抗，能否通过下调跨膜受体TLR4的基因表达来减轻脂肪炎症。方法：将小鼠3T3-L1前脂肪细胞常规培养于高糖DMEM培养液中，2天换液一次，当细胞融合至75%左右时，用含10μg/ml胰岛素、1μmol/L地塞米松、0.5mmol/L1-甲基-3异丁基-黄嘌呤（IBMX）的高糖DMEM培养液培养2天，然后换上含10μg/L胰岛素的完全培养液再培养6-8天，每2天换液一次；最后以完全培养液继续培养，共诱导分化12-14天，成3T3-L1脂肪细胞；将诱导分化成熟的3T3-L1脂肪细胞换上含2g/L牛血清白蛋白(BSA)的高糖DMEM培养液培养12h后，换上含0.5mmol/L软脂酸（PA）、10g/L无脂酸牛血清白蛋白（FAFBSA）的DMEM培养24h，建成胰岛素抵抗模型。在培养液中加入不同浓度蜕皮甾酮及浓度为1×10~(-5)mol/L的吡格列酮同时孵育24小时。用MTT法检测蜕皮甾酮对3T3-L1脂肪细胞增殖的影响，用葡萄糖检测试剂盒（氧化酶法）检测各组细胞24小时培养液中葡萄糖的消耗量，用Western印迹法检测各组IKKβ蛋白与NF-κB蛋白表达水平，用RT-PCR检测TLR4受体m RNA基因表达水平。实验共分为六组：（1）空白对照组：3T3-L1脂肪细胞；（2）模型组：肥胖细胞，即具有胰岛素抵抗的脂肪细胞；（3）ECR组：分组为ECR1×10~(-7)组、ECR1×10~(-6)组、ECR1×10~(-5)组；（4）吡格列酮组，浓度为1×10~(-5)mol/L。结果：蜕皮甾酮浓度1×10-4mol/L时，细胞OD值显著降低（P0.01），细胞生长受到抑制；蜕皮甾酮浓度在1×10~(-7)～1×10~(-5)mol/L时，细胞生长率为92.70%～100%，生长良好；与模型组相比较，蜕皮甾酮在1×10~(-7)～1×10~(-5)mol/L浓度范围的各组，可使肥胖细胞的葡萄糖消耗量增加。模型组胰岛素抵抗细胞中IKKβ蛋白与NF-κB蛋白的表达显著增加，TLR4受体m RNA基因的表达亦显著增加，，经1×10~(-7)～1×10~(-5)mol/L浓度的蜕皮甾酮干预后，各组IKKβ蛋白与NF-κB蛋白表达及TLR4受体m RNA基因表达有不同程度的下降。结论：（1）游离脂肪酸使脂肪细胞处于炎症状态，激活TLR4信号转导通路，使脂肪细胞产生胰岛素抵抗；（2）蜕皮甾酮在浓度1×10-4mol/L时对脂肪细胞生长有明显抑制作用，故本实验选取蜕皮甾酮浓度为1×10~(-7)mol/L、1×10~(-6)mol/L、1×10~(-5)mol/L；（3）蜕皮甾酮可以增加肥胖细胞葡萄糖消耗量，可以抑制肥胖细胞炎症反应通路TLR4信号转导途径中IKKβ蛋白与NF-κB蛋白的表达，可以使肥胖细胞细胞膜TLR4受体m RNA的表达减少，提示蜕皮甾酮可以改善游离脂肪酸诱导的胰岛素抵抗，其分子机制可能与蜕皮甾酮抑制TLR4信号转导途径有关。
[Abstract]:Aim: to establish an insulin resistance model by culture, induction and differentiation of mouse preadipocytes from 3T3-L1, and to establish insulin resistance model by using free fatty acid (FFA), and on the basis of which, ecdysterone was used to interfere with the proliferation of preadipocytes. To investigate the effect of ecdysterone on insulin resistance in vitro and its possible molecular mechanism. Whether ecdysterone can ameliorate insulin resistance by inhibiting the expression of IKK 尾 and NF- 魏 B on the inflammatory pathway of nuclear factor- 魏 B, or by down-regulating the gene expression of transmembrane receptor TLR4 to alleviate fat inflammation. Methods: the preadipocytes of mouse 3T3-L1 were routinely cultured in high glucose DMEM medium for 2 days. When the cells were fused to 75%, the cells were cultured in high glucose DMEM medium containing 10 渭 g/ml insulin 1 渭 mol/L dexamethasone 0.5 mmol / L 1-methyl-3 isobutyl-xanthine for 2 days. Then the whole culture medium containing 10 渭 g / L insulin was recultured for 6-8 days and changed every 2 days, and then cultured in the complete culture medium for 12-14 days to form 3T3-L1 adipocytes. 3T3-L1 adipocytes were cultured in high-glucose DMEM medium containing 2g/L bovine serum albumin (BSA) for 12 h, then DMEM containing 10 g / L 0.5mmol/L palmitate was added to DMEM for 24 h to establish insulin resistance model. Different concentrations of ecdysterone and 1 脳 10~(-5)mol/L pioglitazone were added to culture medium for 24 hours. The effects of ecdysterone on the proliferation of 3T3-L1 adipocytes were detected by MTT assay, and glucose consumption in 24 hours culture medium of each group was detected by glucose detection kit (oxidase method). The expression of IKK 尾 protein and NF- 魏 B protein was detected by Western blot, and the expression of TLR4 receptor m RNA gene was detected by RT-PCR. The experiment was divided into six groups: control group (control group: 1: 3T3-L1 adipocyte) model group: obese cells, adipocytes with insulin resistance: ECR1 脳 10 ~ (-7) group (ECR1 脳 10 ~ (1 脳 10 ~ (-6) group (n = 4) pioglitazone group with a concentration of 1 脳 10 ~ (-5) mol 路L ~ (-1). Results: when ecdysterone concentration was 1 脳 10-4mol/L, cell OD value decreased significantly and cell growth was inhibited. When ecdysterone concentration was 1 脳 10 ~ (-1) -7 脳 10~(-5)mol/L, cell growth rate was 92.70% and grew well. Compared with model group, ecdysone was in 1 脳 10 ~ (-7) 10~(-5)mol/L concentration group, and ecdysone concentration was 1 脳 10 ~ (-7) 10~(-5)mol/L concentration in each group. Increase glucose consumption in obese cells. In the model group, the expression of IKK 尾 protein and NF- 魏 B protein increased significantly in insulin resistant cells, and the expression of m RNA gene of TLR4 receptor was also significantly increased. After the intervention of 1 脳 10 ~ (-7) 10~(-5)mol/L concentration, the expression of IKK 尾 protein and NF- 魏 B protein was increased. The expression of IKK 尾 protein, NF- 魏 B protein and TLR4 receptor m RNA gene decreased in different degree. Conclusion: free fatty acids can cause adipocytes to be inflamed, activate TLR4 signal transduction pathway, and induce insulin resistance in adipocytes. Ecdysterone can inhibit adipocyte growth at a concentration of 1 脳 10-4mol/L. Therefore, ecdysterone concentration was 1 脳 10 ~ (-7) mol / L ~ (-1) 脳 10 ~ (-6) mol / L ~ (1 脳 10 ~ (-5) mol / L ~ (3) ecdysterone could increase glucose consumption of obese cells and inhibit the expression of IKK 尾 and NF- 魏 B protein in TLR4 signal transduction pathway of inflammatory response pathway in obese cells. The results suggest that ecdysterone can improve insulin resistance induced by free fatty acids, and its molecular mechanism may be related to the inhibition of TLR4 signal transduction pathway by ecdysterone.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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