發(fā)布時(shí)間：2018-08-19 06:15

【摘要】：目的:Vaspin(內(nèi)臟脂肪組織來(lái)源的絲氨酸蛋白酶抑制物)被證實(shí)能對(duì)抗動(dòng)脈粥樣硬化發(fā)生發(fā)展,其具體作用機(jī)制尚未被完全認(rèn)識(shí)。本研究主要探討Vaspin能否通過(guò)抑制NF-κB途徑上調(diào)THP-1巨噬細(xì)胞ATP結(jié)合盒轉(zhuǎn)運(yùn)體A1(ATP-binding cassette transporter A1,ABCA1)表達(dá)。方法:將人THP-1單核巨噬細(xì)胞誘導(dǎo)轉(zhuǎn)變成為泡沫細(xì)胞,經(jīng)不同濃度的Vaspin處理相同時(shí)間或同一濃度Vaspin(100ng/mL)處理不同時(shí)間(0,6,12,24,48h),用液體閃爍計(jì)數(shù)法檢測(cè)細(xì)胞內(nèi)膽固醇流出,油紅O染色觀察細(xì)胞內(nèi)脂滴情況,高效液相色譜法測(cè)定細(xì)胞內(nèi)總膽固醇、游離膽固醇及膽固醇酯含量;Western blot測(cè)定細(xì)胞內(nèi)ABCA1蛋白含量,實(shí)時(shí)定量PCR檢測(cè)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(xì)胞對(duì)LPS作用下的細(xì)胞處理24h,再加入vaspin處理24h,檢測(cè)細(xì)胞內(nèi)ABCA1、miR-33a和SREBP mRNA表達(dá)水平。用NF-κB抑制劑預(yù)處理LPS作用下的THP-1細(xì)胞24h,再加或不加vaspin處理,檢測(cè)細(xì)胞內(nèi)NF-κB p65蛋白、ABCA1蛋白和mRNA表達(dá)、miR-33a mRNA表達(dá)、細(xì)胞膽固醇的流出情況。結(jié)果:Vaspin上調(diào)THP-1巨噬細(xì)胞ABCA1蛋白及mRNA水平呈時(shí)間和劑量依賴(lài)性。進(jìn)一步實(shí)驗(yàn)證實(shí)vaspin可以抑制mi R-33am RNA及SREBP-2的表達(dá),上調(diào)ABCA1mRNA的表達(dá),促進(jìn)膽固醇流出;NF-κB的特異性抑制劑PDTC處理和荷脂的THP-1細(xì)胞,vaspin可削弱NF-κB/miR-33a信號(hào)途徑對(duì)ABCA1表達(dá)的抑制作用。結(jié)論:Vaspin可通過(guò)抑制NF-κB信號(hào)途徑,下調(diào)miR-33a表達(dá),進(jìn)而上調(diào)THP-1巨噬細(xì)胞ABCA1蛋白及mRNA表達(dá),并促進(jìn)細(xì)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(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R54

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 曾夢(mèng)雅;王燕;李伍夫;曾高峰;唐朝克;;Vaspin與動(dòng)脈粥樣硬化的研究進(jìn)展[J];生命的化學(xué);2016年01期

2 王海濤;孫雪;時(shí)立新;徐淑靜;;血清Vaspin水平與2型糖尿病代謝改變的相關(guān)性研究[J];齊齊哈爾醫(yī)學(xué)院學(xué)報(bào);2015年13期

3 高干;馬琦琳;劉偉琦;;原發(fā)性高血壓患者血清vaspin水平與血壓及UACR關(guān)系的研究[J];臨床心血管病雜志;2015年03期

4 蘇楊;明強(qiáng);李海玲;彭文輝;張毅;魏毅東;徐亞偉;;血清內(nèi)臟脂肪組織絲氨酸蛋白酶抑制因子水平與冠心病患者危險(xiǎn)因素及預(yù)后的關(guān)系[J];中國(guó)心臟起搏與心電生理雜志;2015年01期

5 邵冰;吳蔚;王曉萍;孫琳;宋平南;;血漿vaspin濃度與急性冠脈綜合征的相關(guān)性[J];中國(guó)老年學(xué)雜志;2015年02期

6 吳劍鋒;唐艷艷;曾高峰;唐朝克;;載脂蛋白AI對(duì)HDL生成影響的研究新進(jìn)展[J];生理科學(xué)進(jìn)展;2014年02期

7 徐玲玲;陳紅珊;杜敏聯(lián);馬華梅;李燕虹;蘇U，

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