本文關(guān)鍵詞：大氣顆粒污染物與臭氧對(duì)高果糖飲食大鼠心表面和腎周脂肪致炎和氧化應(yīng)激作用的研究　出處：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 大氣污染 大氣微粒子 臭氧 心表面脂肪組織 腎周脂肪組織 炎癥 氧化應(yīng)激

【摘要】：[背景]大氣污染介導(dǎo)的心血管疾病(cardiovascular diseases, CVD)是大氣污染相關(guān)人群死亡的首位病因,世界各地普遍存在大氣污染,其導(dǎo)致的心血管健康問(wèn)題已經(jīng)造成全球巨大的經(jīng)濟(jì)負(fù)擔(dān)。系統(tǒng)性炎癥和氧化應(yīng)激在大氣污染物觸發(fā)或加重CVD的發(fā)生、發(fā)展和預(yù)后中發(fā)揮著至關(guān)重要的作用。而大氣污染導(dǎo)致脂肪組織炎癥是環(huán)境與代謝心臟病學(xué)領(lǐng)域新近研究的熱點(diǎn)問(wèn)題,但對(duì)心表面和腎周脂肪的研究較少,尤其是吸入濃聚大氣微粒子(concentrated ambient particulates, CAPs)和(或)臭氧(ozone,O3)暴露對(duì)心表面和腎周脂肪的致炎和氧化應(yīng)激作用,目前國(guó)內(nèi)外均未見(jiàn)相關(guān)研究。 [目的]研究：心表面和腎周脂肪的特征；短期內(nèi)吸入性CAPs和(或)03暴露,對(duì)心表面和腎周脂肪組織中的白色和棕色脂肪特異性基因表達(dá)的調(diào)控作用,及其對(duì)心表面和腎周脂肪組織的致炎和氧化應(yīng)激作用。 [方法]8周齡大小的美國(guó)斯普拉格—道利(SD)雄性大鼠,隨機(jī)分為8組：4組正常飲食(ND),4組高果糖飲食(HF),8周后開(kāi)始吸入性CAPs和(或)03暴露,組別如下：ND-AIR, ND-CAPs, ND-O3, ND-CAPs/O3, HF-AIR, HF-CAPs, HF-O3和HF-CAPs/O3(n=8)。飲食和吸入性大氣污染暴露方案：8h/d,4-5d/w,共2周。所有大鼠均于暴露完成后24h內(nèi)解剖,采集心表面、腎周、肩胛間和大網(wǎng)膜脂等多部位脂肪標(biāo)本。上述大鼠的暴露和脂肪標(biāo)本的采集在美國(guó)密歇根州立大學(xué)(MSU)毒理學(xué)與病理生物學(xué)研究中心完成。各項(xiàng)實(shí)驗(yàn)在美國(guó)俄亥俄州立大學(xué)戴維斯心肺研究所(OSU-DHLI)完成：1.研究EAT和腎周脂肪的特征：病理學(xué)方法包括：(1)蘇木精—伊紅染色：光學(xué)顯微鏡20x放大,觀察脂肪細(xì)胞的形態(tài)學(xué)特征；(2)透射電子顯微鏡：觀察脂肪細(xì)胞線粒體的超微結(jié)構(gòu),30,000x放大；(3)白色和棕色脂肪組織的特異性基因表達(dá)：用Trizol試劑提取EAT、腎周、肩胛間和內(nèi)臟脂肪的RNA,用分光光度儀測(cè)定RNA濃度,后逆轉(zhuǎn)錄為cDNA,實(shí)時(shí)定量PCR檢測(cè)基因表達(dá)。2.評(píng)價(jià)CAPs和(或)03暴露后EAT和腎周脂肪的炎癥和氧化應(yīng)激：(1)酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)測(cè)定脂肪組織上清液中脂聯(lián)素的水平；(2)實(shí)時(shí)定量PCR：檢測(cè)大氣污染物暴露對(duì)脂肪組織中白色和棕色脂肪標(biāo)志性基因表達(dá)的調(diào)控,以及多種炎癥基因的表達(dá)變化：(3)免疫組織化學(xué)染色技術(shù)：半定量評(píng)價(jià)脂肪組織中巨噬細(xì)胞浸潤(rùn)程度；(4)免疫熒光染色技術(shù)：半定量評(píng)價(jià)誘導(dǎo)型一氧化氮合成酶(iNOS)熒光信號(hào)強(qiáng)度；(5)蛋白質(zhì)提取和免疫印跡法分離純化蛋白質(zhì)：定量評(píng)價(jià)iNOS蛋白質(zhì)的表達(dá)；(6)透射電子顯微鏡檢查：觀察脂肪細(xì)胞線粒體的改變,定量分析線粒體的數(shù)目和面積變化。 [結(jié)果]1.EAT和腎周脂肪均為白色脂肪：大體標(biāo)本呈白色；顯微鏡下見(jiàn)：?jiǎn)畏考?xì)胞、細(xì)胞核呈球形位于細(xì)胞邊緣、單個(gè)大脂滴、線粒體數(shù)目較棕色脂肪少,白色脂肪標(biāo)志性基因(Dpt和Hoxc9)表達(dá)與經(jīng)典的白色脂肪組織相近(p0.05),而其棕色脂肪特異性基因(UCP1, PGC-1α, Cidea, C/EBPβ和Dio2)表達(dá)低于棕色脂肪數(shù)百倍(p0.001),以上符合白色脂肪特征。但EAT與其他內(nèi)臟白色脂肪明顯不同,脂肪細(xì)胞直徑較其他白色脂肪小5～10倍,UCP-1, PGC-1α和Cidea表達(dá)較其他白色脂肪高約10倍(p0.05),提示EAT可能有棕色脂肪的某些特性。2.短期內(nèi)吸入性CAPs(?)口(或)03暴露,對(duì)心表面和腎周脂肪組織中的白色和棕色脂肪標(biāo)志性基因表達(dá)的調(diào)控,以及炎癥和氧化應(yīng)激作用：短期的CAPs(?)口(或)03暴露,導(dǎo)致高果糖組大鼠的心表面和腎周脂肪組織中白色(Dpt和Hoxc9)和棕色脂肪(UCP1,PGC-1α和Cidea)的標(biāo)志性基因下調(diào)(p0.001)。致炎基因：各組中IL-6的表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義；在HF組,TNF-α, MCP-1和leptin的表達(dá)呈明顯上調(diào),HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR (p0.0001);抗炎基因：IL-10和adiponectin表達(dá)呈明顯下調(diào),HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR (p0.05)。但在ND組內(nèi)比較,CAPs和03暴露對(duì)EAT和腎周脂肪組織的基因調(diào)控,與HF組呈現(xiàn)相同趨勢(shì),但ND-CAPs, ND-O3和ND-CAPs/O3vs. ND-AIR,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。免疫組織化學(xué)染色：大氣污染物暴露導(dǎo)致巨噬細(xì)胞在EAT和腎周脂肪組織中浸潤(rùn)明顯增加,HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR (p 0.05)。在ND組內(nèi),CAPs和03暴露后,巨噬細(xì)胞的浸潤(rùn)和炎癥基因的上調(diào)有增加趨勢(shì),但差異均無(wú)統(tǒng)計(jì)學(xué)意義,ND-CAPs, ND-O3和ND-CAPs/O3vs. ND-AIR (p0.05);而HF組和ND-AIR比較則呈現(xiàn)明顯差異(p0.05)。氧化應(yīng)激作用：大氣污染物暴露后,iNOS免疫熒光信號(hào)強(qiáng)度和蛋白質(zhì)表達(dá)均增加,HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。大氣污染物暴露導(dǎo)致線粒體的面積明顯減少,ND-AIR vs. HF-CAPs, HF-O3和HF-CAPs/O3(p0.05),線粒體的嵴減少、結(jié)構(gòu)模糊甚至破壞,但線粒體的數(shù)目無(wú)明顯減少,ND-AIR vs. HF-CAPs, HF-O3和HF-CAPs/O3,差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。此外,本研究中多數(shù)實(shí)驗(yàn)顯示：HF組中單獨(dú)CAPs暴露較CAPs/O3混合暴露的致炎和氧化應(yīng)激作用更強(qiáng)。 [結(jié)論]1.EAT和腎周脂肪均為白色脂肪,但EAT與其他內(nèi)臟白色脂肪不同,具有某些棕色脂肪的特征；2.短期吸入性CAPs和(或)O3暴露,下調(diào)心表面和腎周脂肪組織中白色和棕色脂肪特異性基因的表達(dá),可能參與調(diào)控脂肪代謝；3.高果糖飲食可導(dǎo)致大鼠心表面和腎周脂肪組織的炎癥和氧化應(yīng)激反應(yīng),而短期吸入性CAPs和(或)03暴露可促發(fā)炎癥和氧化應(yīng)激明顯惡化。大氣污染導(dǎo)致心表面和腎周脂肪組織的炎癥和氧化應(yīng)激,可能是其觸發(fā)和加重心血管病發(fā)生和發(fā)展的重要機(jī)制之一。
[Abstract]:[background] air pollution mediated cardiovascular disease (cardiovascular diseases CVD) is the first cause of death of air pollution related populations around the world, widespread air pollution and cardiovascular health problems which cause has caused huge economic burden in the world. The occurrence of systemic inflammation and oxidative stress triggered or exacerbation of CVD in atmospheric pollutants, play a crucial role in the development and prognosis. Air pollution caused adipose tissue inflammation is a hot issue in recent research on environmental and metabolic Cardiology, but research on the heart surface and perirenal fat less, especially the inhalation concentration of atmospheric particles (concentrated ambient, particulates, CAPs) and (or) ozone (ozone O3) exposure on the heart surface and perirenal fat inflammation and oxidative stress, there were no related research at home and abroad.
[Objective] to study the surface characteristics: heart and kidney fat; short term inhalation of CAPs and (or) 03 exposure regulation effect on the expression of white and brown fat specific genes of heart surface and perirenal adipose tissue in the heart, and on the surface and perirenal adipose tissue inflammation and oxidative stress.
[methods]8 weeks of age the size of the United States Sprague Dawley (SD) male rats were randomly divided into 8 groups: 4 normal diet group (ND), 4 groups of high fructose diet (HF), 8 weeks after the start of inhalation of CAPs and (or) 03 exposure groups as follows: ND-AIR, ND-CAPs, ND-O3. ND-CAPs/O3, HF-AIR, HF-CAPs, HF-O3 and HF-CAPs/O3 (n=8). The diet and inhalation of air pollution exposure scheme: 8h/d, 4-5d/w, a total of 2 weeks. All rats were exposed after the completion of 24h in heart anatomy, collecting surface, perirenal, scapular and omental fat and other parts. The fat samples of rats the exposure and fat were collected at the Michigan State University (MSU) Center for toxicological pathology and biology research. The experiments at the Ohio State University Davies heart and Lung Institute (OSU-DHLI) completed: 1. the characteristics of EAT and perirenal fat: including pathological method: (1) hematoxylin - eosin staining Color: optical microscopy 20x amplification, observe the morphological characteristics of fat cells; (2) transmission electron microscopy: ultrastructure observation, adipose mitochondrial 30000x amplification; (3) the expression of specific genes in white and brown adipose tissue: EAT was extracted with Trizol reagent perirenal, scapular and visceral fat with RNA. Concentration of RNA was determined by spectrophotometer after reverse transcription of cDNA, real-time quantitative PCR detection of.2. gene expression and evaluation of CAPs (or 03) inflammation after exposure to EAT and perirenal fat and oxidative stress: (1) enzyme-linked immunosorbent assay (ELISA) was measured in the supernatant of adipose tissue adiponectin level; (2) real time quantitative PCR detection: air pollutant exposure on adipose tissue in white and brown fat labeling regulation of gene expression, and the expression of inflammatory gene changes: (3) immunohistochemistry: semi quantitative evaluation of fatty tissue The degree of macrophage infiltration; (4) immunofluorescence staining: semi quantitative evaluation of inducible nitric oxide synthase (iNOS) fluorescence signal intensity; (5) the separation and purification of protein extraction and Western blot: the expression of iNOS protein quantitative evaluation; (6) transmission electron microscopy observation: fat cell mitochondria, quantitative analysis the number and area change of mitochondria.
[results]1.EAT and perirenal fat were white fat: white specimen; microscope: single cell nuclei are spherical, located on the cell edge, a single large lipid droplet, the number of mitochondria is less brown fat, white fat marker genes (Dpt and Hoxc9) and the expression of classic white adipose tissue (similar to P0.05), and the brown fat specific genes (UCP1, Cidea, PGC-1 alpha, C/EBP beta and Dio2) was lower than that of brown adipose tissue (p0.001), hundreds of times more than meets white fat. But EAT and other visceral white adipose tissue were significantly different, fat cell diameter than the other small white fat 5 ~ 10 times, UCP-1 PGC-1, alpha and Cidea expression than the other white fat is about 10 times higher (P0.05), suggesting that EAT may have some characteristics of brown fat in the short term inhalation of CAPs (.2.?) and (or) 03 exposure of fat tissues, heart surface and perirenal in brown fat and white flag The regulation of gene expression, as well as inflammation and oxidative stress: short term CAPs (?) and (or) 03 lead exposure, white heart surface and perirenal adipose tissue of high fructose group in rats (Dpt and Hoxc9) and brown fat (UCP1, PGC-1 alpha and Cidea) marker gene transfer (p0.001). The inflammatory gene expression: there was no significant difference in each group IL-6; in group HF, TNF- alpha, the expression of MCP-1 and leptin was obviously up-regulated, HF-CAPs, HF-O3 and HF-CAPs/O3vs. ND-AIR (P0.0001); anti inflammatory genes: IL-10 and adiponectin expression was down regulated, HF-CAPs, HF-O3 and HF-CAPs/O3vs. ND-AIR (P0.05). But in ND group, and 03 CAPs exposure to control gene EAT and perirenal adipose tissue, and the HF group showed the same trend, but ND-CAPs, ND-O3 and ND-CAPs/O3vs. ND-AIR, the difference was not statistically significant (P0.05). Immunohistochemical staining: the air pollutant exposure to giant Macrophage infiltration in EAT and perirenal adipose tissue increased significantly, HF-CAPs, HF-O3 and HF-CAPs/O3vs. ND-AIR (P 0.05). In group ND, and 03 CAPs after exposure, macrophage infiltration and inflammatory gene upregulation increased, but there were no significant differences in ND-CAPs, ND-O3, and ND-CAPs/O3vs. ND-AIR (P0.05) group HF and ND-AIR; and the comparison showed significant differences (P0.05). The role of oxidative stress: air pollutants after exposure, the expression of iNOS protein and immune fluorescence signal intensity was increased, HF-CAPs, HF-O3 and HF-CAPs/O3vs. ND-AIR, the difference was statistically significant (P0.05). Air pollutant exposure results in mitochondrial ND-AIR vs. area decreased significantly, HF-CAPs. HF-O3 and HF-CAPs/O3 (P0.05), mitochondrial cristae decreased, fuzzy or even destroy the structure, but no significant decrease in the number of mitochondria, ND-AIR vs. HF-CAPs, HF-O3 and HF-CAPs/O3, the difference was not statistically significant (P0.05). In addition, most of the experiments in this study showed that CAPs exposure in the HF group was more exposed to inflammation and oxidative stress.
[conclusion]1.EAT and perirenal fat were white fat, but the EAT and other visceral white fat, brown fat has certain characteristics; 2. short term inhalation of CAPs and (or) O3 exposure, expression of white and brown fat specific genes of heart surface and perirenal adipose tissue may be involved in the regulation of fat metabolism. 3.; high fructose diet can lead to rat heart surface and perirenal adipose tissue inflammation and oxidative stress, while the short-term inhalation of CAPs and (or) 03 exposure can promote inflammation and oxidative stress significantly deteriorated. Air pollution caused inflammation in adipose tissue and heart surface perirenal and oxidative stress may be one of the the trigger and aggravate the occurrence of cardiovascular disease and the development of an important mechanism.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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