【摘要】：目的:Vaspin(內(nèi)臟脂肪組織來源的絲氨酸蛋白酶抑制物)被證實能對抗動脈粥樣硬化發(fā)生發(fā)展,其具體作用機制尚未被完全認識。本研究主要探討Vaspin能否通過抑制NF-κB途徑上調(diào)THP-1巨噬細胞ATP結(jié)合盒轉(zhuǎn)運體A1(ATP-binding cassette transporter A1,ABCA1)表達。方法:將人THP-1單核巨噬細胞誘導(dǎo)轉(zhuǎn)變成為泡沫細胞,經(jīng)不同濃度的Vaspin處理相同時間或同一濃度Vaspin(100ng/mL)處理不同時間(0,6,12,24,48h),用液體閃爍計數(shù)法檢測細胞內(nèi)膽固醇流出,油紅O染色觀察細胞內(nèi)脂滴情況,高效液相色譜法測定細胞內(nèi)總膽固醇、游離膽固醇及膽固醇酯含量;Western blot測定細胞內(nèi)ABCA1蛋白含量,實時定量PCR檢測ABCA1 mRNA水平。分別用miR-33a模擬肽(miR-33a mimic)和anti-miR-33a轉(zhuǎn)染THP-1細胞對LPS作用下的細胞處理24h,再加入vaspin處理24h,檢測細胞內(nèi)ABCA1、miR-33a和SREBP mRNA表達水平。用NF-κB抑制劑預(yù)處理LPS作用下的THP-1細胞24h,再加或不加vaspin處理,檢測細胞內(nèi)NF-κB p65蛋白、ABCA1蛋白和mRNA表達、miR-33a mRNA表達、細胞膽固醇的流出情況。結(jié)果:Vaspin上調(diào)THP-1巨噬細胞ABCA1蛋白及mRNA水平呈時間和劑量依賴性。進一步實驗證實vaspin可以抑制mi R-33am RNA及SREBP-2的表達,上調(diào)ABCA1mRNA的表達,促進膽固醇流出;NF-κB的特異性抑制劑PDTC處理和荷脂的THP-1細胞,vaspin可削弱NF-κB/miR-33a信號途徑對ABCA1表達的抑制作用。結(jié)論:Vaspin可通過抑制NF-κB信號途徑,下調(diào)miR-33a表達,進而上調(diào)THP-1巨噬細胞ABCA1蛋白及mRNA表達,并促進細胞內(nèi)膽固醇流出。
[Abstract]:Objective: Vaspin (visceral adipose tissue derived serine protease inhibitor) has been proved to be able to prevent the development of atherosclerosis, and its mechanism has not been fully understood. The aim of this study was to investigate whether Vaspin can up-regulate the expression of ATP binding cassette transporter A1 (ATP-binding cassette transporter A1) in THP-1 macrophages by inhibiting NF- 魏 B pathway. Methods: human THP-1 mononuclear macrophages were induced to be foam cells and treated with different concentrations of Vaspin for the same time or at the same concentration of Vaspin (100ng/mL) for 48 h. Cholesterol efflux was detected by liquid scintillation counting. Lipid droplets were observed by oil red O staining, total cholesterol, free cholesterol and cholesterol ester were determined by HPLC, ABCA1 protein was detected by Western blot and ABCA1 mRNA was detected by real-time quantitative PCR. THP-1 cells were transfected with miR-33a mimic peptide (miR-33a mimic) and anti-miR-33a for 24 h, then treated with vaspin for 24 h. The expression levels of ABCA1 miR-33a and SREBP mRNA were detected. LPS cells were pretreated with NF- 魏 B inhibitor for 24 h, then treated with or without vaspin for 24 h. The expression of ABCA1 protein and mRNA protein of NF- 魏 B p65 was detected. The expression of miR-33a mRNA and cholesterol efflux were detected. Results Vaspin upregulated the levels of ABCA1 protein and mRNA in THP-1 macrophages in a time and dose-dependent manner. It was further confirmed that vaspin could inhibit the expression of mi R-33am RNA and SREBP-2, up-regulate the expression of ABCA1mRNA, promote the treatment of PDTC, a specific inhibitor of cholesterol efflux of NF- 魏 B, and inhibit the expression of ABCA1 by NF- 魏 B/miR-33a signaling pathway. Conclusion Vaspin can down-regulate the expression of miR-33a and up-regulate the expression of ABCA1 and mRNA in THP-1 macrophages by inhibiting NF- 魏 B signaling pathway, and promote intracellular cholesterol efflux.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R54

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