本文關(guān)鍵詞： miR-101 ABCA1 炎癥因子　出處：《重慶醫(yī)科大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目的：探討在炎癥或非炎癥條件下microRNA-101(miR-101)對人單核巨噬細(xì)胞(THP-1)和人肝癌細(xì)胞(HepG2)膽固醇外排的影響,為揭示miR-101在動脈粥樣硬化發(fā)生中的作用機(jī)制提供實(shí)驗(yàn)依據(jù)。材料與方法：構(gòu)建miR-101或Anti-miR-101慢病毒載體并建立高表達(dá)miR-101或Anti-miR-101細(xì)胞模型。使用雙熒光素酶報告基因檢測驗(yàn)證miR-101與三磷酸腺苷結(jié)合盒轉(zhuǎn)運(yùn)體Al(ABCAl) mRNA的3’非編碼區(qū)(3’UTR)互補(bǔ)結(jié)合,分組為野生型(WT)+Con-miR組、定點(diǎn)突變(SDM)+miR-101組與WT+miR-101組。非炎癥條件下THP-1與HepG2各分為對照組,miR-101組和Anti-miR-101組；炎癥條件下各分為對照組,miR-101組,Anti-miR-101組,IL-6組(0.2%BSA+20ng/ml IL-6), TNF-a組(0.2%BSA+25ng/ml TNF-a), IL-6+miR-101/ Anti-miR-101組及TNF-a+Anti-miR-101組。各組再根據(jù)是否用LDL (0.2%BSA+25μg/ml LDL)處理,分為LDL負(fù)荷和無LDL負(fù)荷。采用oil red O染色檢測細(xì)胞內(nèi)脂質(zhì)蓄積,酶法測定胞內(nèi)膽固醇含量,熒光標(biāo)記膽固醇法(BODIPY-cholesterol)檢測載脂蛋白A-I(apoA-I)介導(dǎo)的膽固醇外排。運(yùn)用實(shí)時熒光定量RT-PCR檢測miR-101基因表達(dá),Western Blotting檢測ABCA1蛋白表達(dá)。結(jié)果：在結(jié)合位點(diǎn)2,與WT+Con-miR組和SDM+miR-101組比較,WT+miR-101組ABCA1 3'UTR活性顯著降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。非炎癥狀態(tài),THP-1和HepG2在有無LDL負(fù)荷條件下,與對照組相比,miR-101組的ABCA1蛋白表達(dá)顯著降低,apoA-I介導(dǎo)的細(xì)胞膽固醇外排明顯減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。Anti-miR-101組ABCA1蛋白表達(dá)明顯增加,apoA-I介導(dǎo)的細(xì)胞膽固醇外排顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。miR-101組或Anti-miR-101組分別可以促進(jìn)或減少THP-1和HepG2胞內(nèi)膽固醇聚集,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。在炎癥狀態(tài)下,與對照組比較,IL-6組與TNF-a組HepG2miR-101表達(dá)顯著增加,IL-6組THP-1 miR-101表達(dá)明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。與IL-6組和TNF-α組比較,IL-6+Anti-miR-101組與TNF-a+Anti-miR-101組THP-1和HepG2 ABCA1蛋白表達(dá)明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：miR-101通過與ABCA1基因3’UTR端互補(bǔ)結(jié)合下調(diào)細(xì)胞ABCA1蛋白表達(dá)抑制膽固醇外排；炎癥可以使細(xì)胞miR-101表達(dá)增加并增強(qiáng)miR-101促進(jìn)膽固醇聚集的作用；下調(diào)細(xì)胞miR-101表達(dá)可以減輕炎癥對ABCA1蛋白抑制作用。miR-101在動脈粥樣硬化的發(fā)生發(fā)展過程中起到重要作用。
[Abstract]:Objective: to investigate the effects of microRNA-101 miR-101) on cholesterol efflux in human mononuclear macrophages (THP-1) and hepatocellular carcinoma cells (HepG2) under inflammatory or non-inflammatory conditions. Materials and methods: construction of miR-101 or Anti-miR-101 lentivirus vector and establishment of high expression miR-101 or Anti-miR-101 cell model. Double luciferase reporter gene detection test. The results show that miR-101 is complementary to the 3'noncoding region of the adenosine triphosphate binding cassette transporter (Alo ABCAl) mRNA. They were divided into two groups: wild type WTM Con-miR group, site-directed mutagenesis (SDM) miR-101 group and WT miR-101 group. Under non-inflammatory condition, THP-1 and HepG2 were divided into two groups: control group (n = 10) and control group (n = 10) and Anti-miR-101 group (n = 10). Under the inflammatory condition, each group was divided into two groups: control group, control group, anti-miR-101 group, anti-miR-101 group, IL-6 group, BSA 20 ng / ml IL-6, TNF-a group, 0.2 BSA25 ng / ml TNF-aI, IL-6 miR-101 / Anti-miR-101 group and TNF-a Anti-miR-101 group. Each group was divided into LDL load and no LDL load according to LDL 0.2BSA 25 渭 g / ml LDL. Oil red O staining was used to detect intracellular lipid accumulation. Enzymatic determination of intracellular cholesterol, The cholesterol efflux mediated by apolipoprotein A-Iapo A-I was detected by fluorescent cholesterol labeling method. The expression of ABCA1 protein was detected by real-time fluorescence quantitative RT-PCR. Results: at binding site 2, it was compared with WT Con-miR group and SDM miR-101 group. The activity of ABCA1 3 UTR was significantly decreased in WT miR-101 group. The difference was statistically significant (P 0.05). The expression of ABCA1 protein in THP 1 and HepG2 group was significantly lower than that in control group under the condition of LDL loading or not, and the cholesterol efflux mediated by apoA-I was significantly decreased in the control group. The expression of ABCA1 protein in P0.05 + Anti-miR-101 group was significantly increased, and the cholesterol efflux mediated by apoA-I was significantly increased. The difference was statistically significant (P 0.05). MiR-101 group or Anti-miR-101 group could promote or decrease the accumulation of intracellular cholesterol in THP-1 and HepG2, respectively. Under the condition of inflammation, the expression of HepG2miR-101 in IL-6 group and TNF-a group was significantly increased compared with that in control group. The expression of THP-1 miR-101 in IL-6 group was significantly higher than that in control group, and the expression of THP-1 miR-101 in IL-6 group was significantly higher than that in control group. Compared with IL-6 group and TNF- 偽 group, the expression of THP-1 and HepG2 ABCA1 in IL-6 Anti-miR-101 group and TNF-a Anti-miR-101 group were significantly increased. The difference was statistically significant (P 0.05). Conclusion: miR-101 can inhibit cholesterol efflux by complementarily binding with ABCA1 gene 3UTR, inflammation can increase the expression of miR-101 and enhance the effect of miR-101 on the accumulation of cholesterol. Down-regulation of miR-101 expression can reduce the inhibitory effect of inflammation on ABCA1 protein. MiR-101 plays an important role in the development of atherosclerosis.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R543.5

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