【摘要】：目的：ABCA1在促進膽固醇逆向轉運和抗動脈粥樣硬化中發(fā)揮重要作用。EGCG是一種綠茶提取物，具有抗炎和調控脂代謝的藥理作用。以往的研究中發(fā)現(xiàn)SREBP-1c、apoB和LDLR等脂代謝相關蛋白參與了EGCG調控脂代謝及其抗炎作用。但是EGCG對ABCA1的作用尚不清楚。在我們的研究中，我們用TNF-α處理THP-1源性泡沫細胞，觀察EGCG預處理對ABCA1及其膽固醇流出影響及其機制。 方法：我們首先用PMA誘導THP-1細胞貼壁形成巨噬細胞，，加入OX-LDL形成泡沫細胞。在加入TNF-α (10ng/ml)前，分別用不同濃度的(0,10,20,40,80μg/mL)EGCG處理泡沫細胞，然后收集細胞進行實驗。膽固醇流出實驗和油紅O染色觀察細胞內膽固醇流出和脂質蓄積情況。實時定量PCR觀察ABCA1和LXRα/β mRNA水平。熒光素酶報告基因分析ABCA1啟動子活性。Western blot檢測ABCA1、LXRα/β、NF-κB p65, Nrf2, Keap1和IKKβ蛋白水平。EMSA檢測NF-κB和Nrf2的DNA結合活性。用siRNA轉染細胞沉默NF-κB和Nrf2. 結果：實驗結果表明，TNF-α處理細胞能夠導致ABCA1mRNA表達水平啟動子活性降低，以及膽固醇流出水平降低，而EGCG預處理能夠衰減TNF-α這一作用。EGCG通過抑制NF-κB途徑來衰減TNF-α對細胞毒性作用，而不是通過激活LXR途徑。在抑制NF-κB過程中，EGCG激活了Nrf2/Keap1途徑，一方面，解離的Nrf2進入細胞核內激活啟動子區(qū)域含有ARE的相關基因，抑制NF-κB活化；另一方面，Keap1從復合體上解離后，直接與IKKβ結合，阻滯其下游對NF-κB的激活。 結論：在THP-1源性泡沫細胞中，EGCG通過激活Nrf2/Keap1途徑抑制TNF-α誘導的NF-κB激活，上調ABCA1的表達促進細胞內膽固醇流出。
[Abstract]:Aim: ABCA1 plays an important role in promoting cholesterol reverse transport and anti-atherosclerosis. EGCG is a green tea extract with anti-inflammation and regulation of lipid metabolism. Previous studies have found that lipid metabolism-related proteins such as SREBP-1c,apoB and LDLR are involved in the regulation of lipid metabolism and its anti-inflammatory effects by EGCG. But the effect of EGCG on ABCA1 is unclear. In our study, we treated THP- 1-derived foam cells with TNF- 偽, and observed the effect and mechanism of EGCG pretreatment on ABCA1 and cholesterol efflux. Methods: we first induced THP-1 cells to adhere to the wall to form macrophages with PMA, and then added OX-LDL to form foam cells. Foam cells were treated with (0,10,20,40,80 渭 g / mL) EGCG) of (0,10,20,40,80 渭 g / mL) EGCG) respectively before TNF- 偽 (10ng/ml) was added, and then the cells were collected for experiment. Cholesterol efflux test and oil red O staining were used to observe intracellular cholesterol efflux and lipid accumulation. The levels of ABCA1 and LXR 偽 / 尾 mRNA were measured by real-time quantitative PCR. The promoter activity of ABCA1 was analyzed by luciferase reporter gene analysis. The levels of ABCA1,LXR 偽 / 尾, NF- 魏 B p65, Nrf2, Keap1 and IKK 尾 proteins were detected by Western blot, and the DNA binding activities of NF- 魏 B and Nrf2 were detected by EMSA. Cell silencing of NF- 魏 B and Nrf2. by siRNA transfection Results: the results showed that TNF- 偽 could decrease the promoter activity of ABCA1mRNA expression level and decrease the cholesterol efflux level. EGCG pretreatment can attenuate the effect of TNF- 偽. EGCG attenuates the cytotoxicity of TNF- 偽 by inhibiting the NF- 魏 B pathway, rather than activating the LXR pathway. In the process of inhibiting NF- 魏 B, EGCG activates the Nrf2/Keap1 pathway. On the one hand, the dissociated Nrf2 enters the activation promoter region of nucleus and contains the related gene of ARE, which inhibits the activation of NF- 魏 B; On the other hand, when Keap1 is dissociated from the complex, it binds directly to IKK 尾 and blocks its downstream activation of NF- 魏 B. Conclusion: in THP- 1-derived foam cells, EGCG inhibits TNF- 偽-induced NF- 魏 B activation by activating Nrf2/Keap1 pathway, and up-regulates the expression of ABCA1 to promote intracellular cholesterol efflux.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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