LASER通过HNF-1α-PCSK9调控胆固醇代谢及zonulin易化肠道细菌移位致动脉硬化的研究

发布时间：2018-05-31 11:40

本文选题：长链非编码RNA + 胆固醇　；参考：《第三军医大学》2017年博士论文

【摘要】：第一部分LASER通过HNF-1α-PCSK9途径调控肝脏胆固醇代谢研究背景高脂血症是动脉硬化性心血管疾病最重要的危险因素之一。血脂水平与许多因素有关,其中遗传变异是研究热点。全基因组关联分析发现许多基因的单核苷酸多态性与血脂水平相关,这些变异可以解释约10-12%的变异幅度。然而约43%的变异位于基因间的非编码区域,其功能尚不清楚。最近的研究发现基因的非编码区域可以转录出众多的非编码RNA,而长链非编码RNA更易于分布在疾病相关的全基因组关联区域。如9p21编码ANRIL并与PRC2蛋白结合而调控下游基因的表达。因此,我们提出脂蛋白相关的关联分析区域同样可以编码lnc RNA并调控脂代谢。研究目标研究肝脏中是否存在调控脂代谢的lnc RNA。研究方法以q RT-PCR的方法测定外周白细胞中LASER的表达。以RNA干扰的方法降低Hep G2细胞内LASER的表达后,以酶法或Filipin染色测定细胞内胆固醇水平,以基因芯片分析干扰LASER后有哪些基因发生了改变。RNA-蛋白质间的相互作用通过RNA免疫共沉淀证实,HNF-1α基因启动子区域的组蛋白甲基化通过染色质免疫共沉淀检测。研究结果我们在血脂相关多态性热点区域鉴定了一个lnc RNA(LASER)。临床研究发现LASER的表达与含胆固醇的脂蛋白及PCSK9含量呈正相关。降低LASER的表达后可以降低细胞内胆固醇水平并影响胆固醇代谢的相关基因,其中PCSK9及其转录因子HNF-1α的表达均下降。同时,在给予HNF-1α的天然的拮抗剂黄连素后可以阻断LASER对PCSK9的作用。LASER通过在核内与Co REST/REST蛋白复合体的LSD1结合干扰了LSD1的活性,从而导致HNF-1α基因启动子抑制性的H3K4me2增加。与此同时,降低LSD1的表达则可以阻断LASER对HNF-1α-PCSK9的作用。最后,我们发现他汀类可以升高LASER的表达,同时导致PCSK9增加,胆固醇可以反向激活LXR受体调控LASER的表达。我们的研究可能部分解释了“他汀逃逸”的现象。研究结论我们发现一个新的调控肝脏胆固醇代谢的lnc RNA-LASER。而抑制LASER可与他汀协同发挥联合调脂的效应。第二部分Zonulin易化肠道细菌移位参与动脉硬化的机制研究研究背景动脉硬化性心血管疾病导致的死亡仍高居不下,其中炎症激活在动脉硬化的进展中发挥了重要的作用。大量的流行病学研究发现冠心病的发生与多种病原微生物相关(肺炎衣原体、幽门螺杆菌及巨细胞病毒等)。肠道中存在着种类众多的共生菌。肠道共生菌可以通过磷脂酰胆碱和左卡尼汀的代谢而间接致动脉硬化,但目前仍缺乏肠道微生物直接致动脉硬化的直接证据。约95%的动脉硬化斑块中能检测到细菌的16S r RNA基因成分,其中以肠杆菌科为主。同时,我们前期发现冠心病人全血中肠道菌属的荧光假单胞菌及沙雷氏菌含量增加,提示除了间接作用外,肠道细菌还可能直接参与了动脉硬化的进展。生理状况下肠道间的紧密连接将肠道细菌与循环隔绝开来,在动脉硬化等病理情况下细菌能否穿过肠道屏障并进入动脉硬化斑块内目前尚不清楚。肠道细胞间的紧密连接在决定肠道通透性及控制肠道细菌移位方面发挥了重要的作用,其中zonulin(连蛋白)是肠道细胞合成的分泌蛋白,在调节紧密连接开放和决定肠道细胞通透性方面发挥了重要的作用。既往研究表明循环中zonulin的水平在糖尿病、肥胖等动脉硬化的危险因素中升高。研究目标动脉硬化的发生和发展中,是否也有zonulin的改变,而zonulin调控的肠道通透性改变是否参与了肠道细菌的移位呢?研究方法以ELISA的方法测定血浆中的zonulin含量。体外培养Caco2细胞层构建肠上皮屏障模型,培养荧光假单胞菌并以菌液干预肠上皮屏障模型。于不同时间点取Transwell小室的上层及下层的培养基进行细菌培养,并照相记录菌落生长情况。以扫描电镜观察荧光假单胞菌暴露对肠上皮屏障结构的影响,以激光共聚焦显微镜观察对zonulin分泌的影响。研究结果冠心病人中血浆中zonulin含量增加,而荧光假单胞菌的暴露促进了肠道上皮细胞内zonulin的分泌。分泌的zonulin改变了肠上皮屏障的细胞结构,导致肠道紧密连接的开放而促进荧光假单胞菌在肠道上皮的移位。研究结论荧光假单胞菌可以促进zonulin的分泌并易化肠道细菌移位。
[Abstract]:Part one LASER regulates liver cholesterol metabolism through the HNF-1 alpha -PCSK9 pathway. Hyperlipidemia is one of the most important risk factors for atherosclerotic cardiovascular disease. Blood lipid levels are related to many factors. Genetic variation is the focus of research. Lipid levels are related, and these variations can explain the variation of about 10-12%. However, about 43% of the variation is located in the non coding region of the gene, and its function is not clear. Recent studies have found that the non coding region of the gene can be transcribed more than the uncoded RNA, while the long chain non coded RNA is more easily distributed in the whole genome associated area related to the disease. Domain. Such as 9p21 encoding ANRIL and binding with PRC2 protein to regulate the expression of downstream genes. Therefore, we suggest that the association analysis area of lipoprotein related can also encode LNC RNA and regulate lipid metabolism. Study the objective of the study of whether there is a LNC RNA. study in the liver to regulate lipid metabolism in the liver to determine LASE in peripheral leukocytes by Q RT-PCR R expression. After the expression of LASER in Hep G2 cells was reduced by RNA interference, the intracellular cholesterol levels were measured by enzyme or Filipin staining, and the gene chips were used to analyze the genes that were interfered with LASER after the interference of the.RNA- protein interaction through the RNA immunoprecipitation syndrome, and the histone a in the promoter region of the HNF-1 alpha gene. We have identified a LNC RNA (LASER) in the blood lipid related polymorphism hot spots. The clinical study found that the expression of LASER is positively correlated with the cholesterol containing lipoprotein and PCSK9 content. Reducing the expression of LASER can reduce the level of intracellular cholesterol and affect the metabolism of cholesterol. Related genes, in which the expression of PCSK9 and its transcriptional factor HNF-1 alpha decreased. At the same time, a natural antagonist of berberine, a natural antagonist of HNF-1 alpha, could block the effect of LASER on PCSK9 by interfering with the activity of LSD1 through the LSD1 binding in the nucleus with the Co REST/REST protein complex, resulting in the inhibition of the HNF-1 alpha gene promoter. At the same time, reducing the expression of LSD1 could block the effect of LASER on HNF-1 alpha -PCSK9. Finally, we found that statins can increase the expression of LASER and increase PCSK9, and the cholesterol can activate the LXR receptor to regulate the expression of LASER. Our study may decompose the phenomenon of "statin escape". We found a new LNC RNA-LASER. that regulates the metabolism of cholesterol in the liver and inhibits the synergistic effect of LASER with statins. Second part Zonulin facilitates the mechanism of intestinal bacterial translocation in arteriosclerosis; background the death of arteriosclerotic cardiovascular disease is still high, and inflammation is activated A large number of epidemiological studies have found that the occurrence of coronary heart disease is associated with a variety of pathogenic microbes (Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus). There are many kinds of symbiotic bacteria in the intestine. Intestinal symbiotic bacteria can be metabolized by phosphatidylcholine and L-carnitine. Indirect arteriosclerosis, but there is still a lack of direct evidence of direct arteriosclerosis by intestinal microbes. About 95% of the atherosclerotic plaques can detect the 16S R RNA gene in bacteria, mainly Enterobacteriaceae. At the same time, we have found that the content of Pseudomonas fluorescens and salibacillae in the whole blood of coronary heart disease patients increased in the early stage. It is suggested that in addition to indirect effects, intestinal bacteria may also be directly involved in the progress of arteriosclerosis. Under physiological conditions, close connections between the intestinal tract isolate intestinal bacteria from circulation. It is not clear whether bacteria can pass through the intestinal barrier and enter the atherosclerotic plaque under the pathological conditions of arteriosclerosis. Connection has played an important role in determining intestinal permeability and controlling intestinal bacterial translocation, in which zonulin (protein) is a secretory protein synthesized by intestinal cells. It plays an important role in regulating close connection and opening and determining the permeability of intestinal cells. Previous studies have shown that the level of zonulin in the circulation is in diabetes and obesity. The risk factors of arteriosclerosis are elevated. Is there any change in zonulin in the occurrence and development of target arteriosclerosis, and does the intestinal permeability change in the zonulin regulate intestinal bacterial translocation? The method of ELISA is used to determine the zonulin content in the plasma. In vitro culture, the Caco2 cell layer is used to construct the intestinal epithelium. A barrier model was used to cultivate Pseudomonas fluorescens and to interfere with the intestinal epithelial barrier model with bacterial fluid. Bacteria culture was cultured at the upper and lower layers of the Transwell chamber at different time points, and the growth of the colonies was recorded. The effects of Pseudomonas fluorescens exposure on the upper intestinal barrier structure were observed by scanning electron microscopy, and laser confocal microscopy was used to observe the effect of the Pseudomonas fluorescens on the structure of the upper intestinal barrier. The effect of microscopic observation on the secretion of zonulin. The results of the study showed that the content of zonulin in the plasma increased in crowns and the exposure of Pseudomonas fluorescens promoted the secretion of zonulin in the intestinal epithelial cells. The secreted zonulin changed the cell structure of the intestinal epithelial barrier, leading to the opening of the intestinal close connection and the promotion of Pseudomonas fluorescens in the intestinal epithelium. Conclusion: Pseudomonas fluorescens can promote zonulin secretion and facilitate intestinal bacterial translocation.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R589.2

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