大气颗粒污染物与臭氧对高果糖饮食大鼠心表面和肾周脂肪致炎和氧化应激作用的研究

发布时间：2018-01-10 04:17

本文关键词：大气颗粒污染物与臭氧对高果糖饮食大鼠心表面和肾周脂肪致炎和氧化应激作用的研究　出处：《北京协和医学院》2012年博士论文　论文类型：学位论文

【摘要】：[背景]大气污染介导的心血管疾病(cardiovascular diseases, CVD)是大气污染相关人群死亡的首位病因,世界各地普遍存在大气污染,其导致的心血管健康问题已经造成全球巨大的经济负担。系统性炎症和氧化应激在大气污染物触发或加重CVD的发生、发展和预后中发挥着至关重要的作用。而大气污染导致脂肪组织炎症是环境与代谢心脏病学领域新近研究的热点问题,但对心表面和肾周脂肪的研究较少,尤其是吸入浓聚大气微粒子(concentrated ambient particulates, CAPs)和(或)臭氧(ozone,O3)暴露对心表面和肾周脂肪的致炎和氧化应激作用,目前国内外均未见相关研究。 [目的]研究：心表面和肾周脂肪的特征；短期内吸入性CAPs和(或)03暴露,对心表面和肾周脂肪组织中的白色和棕色脂肪特异性基因表达的调控作用,及其对心表面和肾周脂肪组织的致炎和氧化应激作用。 [方法]8周龄大小的美国斯普拉格—道利(SD)雄性大鼠,随机分为8组：4组正常饮食(ND),4组高果糖饮食(HF),8周后开始吸入性CAPs和(或)03暴露,组别如下：ND-AIR, ND-CAPs, ND-O3, ND-CAPs/O3, HF-AIR, HF-CAPs, HF-O3和HF-CAPs/O3(n=8)。饮食和吸入性大气污染暴露方案：8h/d,4-5d/w,共2周。所有大鼠均于暴露完成后24h内解剖,采集心表面、肾周、肩胛间和大网膜脂等多部位脂肪标本。上述大鼠的暴露和脂肪标本的采集在美国密歇根州立大学(MSU)毒理学与病理生物学研究中心完成。各项实验在美国俄亥俄州立大学戴维斯心肺研究所(OSU-DHLI)完成：1.研究EAT和肾周脂肪的特征：病理学方法包括：(1)苏木精—伊红染色：光学显微镜20x放大,观察脂肪细胞的形态学特征；(2)透射电子显微镜：观察脂肪细胞线粒体的超微结构,30,000x放大；(3)白色和棕色脂肪组织的特异性基因表达：用Trizol试剂提取EAT、肾周、肩胛间和内脏脂肪的RNA,用分光光度仪测定RNA浓度,后逆转录为cDNA,实时定量PCR检测基因表达。2.评价CAPs和(或)03暴露后EAT和肾周脂肪的炎症和氧化应激：(1)酶联免疫吸附实验(ELISA)测定脂肪组织上清液中脂联素的水平；(2)实时定量PCR：检测大气污染物暴露对脂肪组织中白色和棕色脂肪标志性基因表达的调控,以及多种炎症基因的表达变化：(3)免疫组织化学染色技术：半定量评价脂肪组织中巨噬细胞浸润程度；(4)免疫荧光染色技术：半定量评价诱导型一氧化氮合成酶(iNOS)荧光信号强度；(5)蛋白质提取和免疫印迹法分离纯化蛋白质：定量评价iNOS蛋白质的表达；(6)透射电子显微镜检查：观察脂肪细胞线粒体的改变,定量分析线粒体的数目和面积变化。 [结果]1.EAT和肾周脂肪均为白色脂肪：大体标本呈白色；显微镜下见：单房细胞、细胞核呈球形位于细胞边缘、单个大脂滴、线粒体数目较棕色脂肪少,白色脂肪标志性基因(Dpt和Hoxc9)表达与经典的白色脂肪组织相近(p0.05),而其棕色脂肪特异性基因(UCP1, PGC-1α, Cidea, C/EBPβ和Dio2)表达低于棕色脂肪数百倍(p0.001),以上符合白色脂肪特征。但EAT与其他内脏白色脂肪明显不同,脂肪细胞直径较其他白色脂肪小5～10倍,UCP-1, PGC-1α和Cidea表达较其他白色脂肪高约10倍(p0.05),提示EAT可能有棕色脂肪的某些特性。2.短期内吸入性CAPs(?)口(或)03暴露,对心表面和肾周脂肪组织中的白色和棕色脂肪标志性基因表达的调控,以及炎症和氧化应激作用：短期的CAPs(?)口(或)03暴露,导致高果糖组大鼠的心表面和肾周脂肪组织中白色(Dpt和Hoxc9)和棕色脂肪(UCP1,PGC-1α和Cidea)的标志性基因下调(p0.001)。致炎基因：各组中IL-6的表达差异无统计学意义；在HF组,TNF-α, MCP-1和leptin的表达呈明显上调,HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR (p0.0001);抗炎基因：IL-10和adiponectin表达呈明显下调,HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR (p0.05)。但在ND组内比较,CAPs和03暴露对EAT和肾周脂肪组织的基因调控,与HF组呈现相同趋势,但ND-CAPs, ND-O3和ND-CAPs/O3vs. ND-AIR,差异无统计学意义(p0.05)。免疫组织化学染色：大气污染物暴露导致巨噬细胞在EAT和肾周脂肪组织中浸润明显增加,HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR (p 0.05)。在ND组内,CAPs和03暴露后,巨噬细胞的浸润和炎症基因的上调有增加趋势,但差异均无统计学意义,ND-CAPs, ND-O3和ND-CAPs/O3vs. ND-AIR (p0.05);而HF组和ND-AIR比较则呈现明显差异(p0.05)。氧化应激作用：大气污染物暴露后,iNOS免疫荧光信号强度和蛋白质表达均增加,HF-CAPs, HF-O3和HF-CAPs/O3vs. ND-AIR,差异有统计学意义(p0.05)。大气污染物暴露导致线粒体的面积明显减少,ND-AIR vs. HF-CAPs, HF-O3和HF-CAPs/O3(p0.05),线粒体的嵴减少、结构模糊甚至破坏,但线粒体的数目无明显减少,ND-AIR vs. HF-CAPs, HF-O3和HF-CAPs/O3,差异无统计学意义(p0.05)。此外,本研究中多数实验显示：HF组中单独CAPs暴露较CAPs/O3混合暴露的致炎和氧化应激作用更强。 [结论]1.EAT和肾周脂肪均为白色脂肪,但EAT与其他内脏白色脂肪不同,具有某些棕色脂肪的特征；2.短期吸入性CAPs和(或)O3暴露,下调心表面和肾周脂肪组织中白色和棕色脂肪特异性基因的表达,可能参与调控脂肪代谢；3.高果糖饮食可导致大鼠心表面和肾周脂肪组织的炎症和氧化应激反应,而短期吸入性CAPs和(或)03暴露可促发炎症和氧化应激明显恶化。大气污染导致心表面和肾周脂肪组织的炎症和氧化应激,可能是其触发和加重心血管病发生和发展的重要机制之一。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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