本文選題：蛻皮甾酮 + 3T3-L1脂肪細胞��； 參考：《瀘州醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：通過培養(yǎng)、誘導(dǎo)、分化小鼠的3T3-L1前脂肪細胞，使用游離脂肪酸（FFA）建立胰島素抵抗模型，在此基礎(chǔ)上使用蛻皮甾酮（ECR）對其增殖進行干預(yù)，探究蛻皮甾酮在體外對細胞胰島素抵抗的影響及可能的分子作用機制。重點觀察蛻皮甾酮能否通過抑制核因子κB(NF-κB)炎性路徑上IKKβ蛋白與NF-κB蛋白的表達改善的胰島素抵抗，能否通過下調(diào)跨膜受體TLR4的基因表達來減輕脂肪炎癥。方法：將小鼠3T3-L1前脂肪細胞常規(guī)培養(yǎng)于高糖DMEM培養(yǎng)液中，2天換液一次，當(dāng)細胞融合至75%左右時，用含10μg/ml胰島素、1μmol/L地塞米松、0.5mmol/L1-甲基-3異丁基-黃嘌呤（IBMX）的高糖DMEM培養(yǎng)液培養(yǎng)2天，然后換上含10μg/L胰島素的完全培養(yǎng)液再培養(yǎng)6-8天，每2天換液一次；最后以完全培養(yǎng)液繼續(xù)培養(yǎng)，共誘導(dǎo)分化12-14天，成3T3-L1脂肪細胞；將誘導(dǎo)分化成熟的3T3-L1脂肪細胞換上含2g/L牛血清白蛋白(BSA)的高糖DMEM培養(yǎng)液培養(yǎng)12h后，換上含0.5mmol/L軟脂酸（PA）、10g/L無脂酸牛血清白蛋白（FAFBSA）的DMEM培養(yǎng)24h，建成胰島素抵抗模型。在培養(yǎng)液中加入不同濃度蛻皮甾酮及濃度為1×10~(-5)mol/L的吡格列酮同時孵育24小時。用MTT法檢測蛻皮甾酮對3T3-L1脂肪細胞增殖的影響，用葡萄糖檢測試劑盒（氧化酶法）檢測各組細胞24小時培養(yǎng)液中葡萄糖的消耗量，用Western印跡法檢測各組IKKβ蛋白與NF-κB蛋白表達水平，用RT-PCR檢測TLR4受體m RNA基因表達水平。實驗共分為六組：（1）空白對照組：3T3-L1脂肪細胞；（2）模型組：肥胖細胞，即具有胰島素抵抗的脂肪細胞；（3）ECR組：分組為ECR1×10~(-7)組、ECR1×10~(-6)組、ECR1×10~(-5)組；（4）吡格列酮組，濃度為1×10~(-5)mol/L。結(jié)果：蛻皮甾酮濃度1×10-4mol/L時，細胞OD值顯著降低（P0.01），細胞生長受到抑制；蛻皮甾酮濃度在1×10~(-7)～1×10~(-5)mol/L時，細胞生長率為92.70%～100%，生長良好；與模型組相比較，蛻皮甾酮在1×10~(-7)～1×10~(-5)mol/L濃度范圍的各組，可使肥胖細胞的葡萄糖消耗量增加。模型組胰島素抵抗細胞中IKKβ蛋白與NF-κB蛋白的表達顯著增加，TLR4受體m RNA基因的表達亦顯著增加，，經(jīng)1×10~(-7)～1×10~(-5)mol/L濃度的蛻皮甾酮干預(yù)后，各組IKKβ蛋白與NF-κB蛋白表達及TLR4受體m RNA基因表達有不同程度的下降。結(jié)論：（1）游離脂肪酸使脂肪細胞處于炎癥狀態(tài)，激活TLR4信號轉(zhuǎn)導(dǎo)通路，使脂肪細胞產(chǎn)生胰島素抵抗；（2）蛻皮甾酮在濃度1×10-4mol/L時對脂肪細胞生長有明顯抑制作用，故本實驗選取蛻皮甾酮濃度為1×10~(-7)mol/L、1×10~(-6)mol/L、1×10~(-5)mol/L；（3）蛻皮甾酮可以增加肥胖細胞葡萄糖消耗量，可以抑制肥胖細胞炎癥反應(yīng)通路TLR4信號轉(zhuǎn)導(dǎo)途徑中IKKβ蛋白與NF-κB蛋白的表達，可以使肥胖細胞細胞膜TLR4受體m RNA的表達減少，提示蛻皮甾酮可以改善游離脂肪酸誘導(dǎo)的胰島素抵抗，其分子機制可能與蛻皮甾酮抑制TLR4信號轉(zhuǎn)導(dǎo)途徑有關(guān)。
[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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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