本文選題：長(zhǎng)鏈非編碼RNA + 膽固醇��； 參考：《第三軍醫(yī)大學(xué)》2017年博士論文

【摘要】：第一部分LASER通過(guò)HNF-1α-PCSK9途徑調(diào)控肝臟膽固醇代謝研究背景高脂血癥是動(dòng)脈硬化性心血管疾病最重要的危險(xiǎn)因素之一。血脂水平與許多因素有關(guān),其中遺傳變異是研究熱點(diǎn)。全基因組關(guān)聯(lián)分析發(fā)現(xiàn)許多基因的單核苷酸多態(tài)性與血脂水平相關(guān),這些變異可以解釋約10-12%的變異幅度。然而約43%的變異位于基因間的非編碼區(qū)域,其功能尚不清楚。最近的研究發(fā)現(xiàn)基因的非編碼區(qū)域可以轉(zhuǎn)錄出眾多的非編碼RNA,而長(zhǎng)鏈非編碼RNA更易于分布在疾病相關(guān)的全基因組關(guān)聯(lián)區(qū)域。如9p21編碼ANRIL并與PRC2蛋白結(jié)合而調(diào)控下游基因的表達(dá)。因此,我們提出脂蛋白相關(guān)的關(guān)聯(lián)分析區(qū)域同樣可以編碼lnc RNA并調(diào)控脂代謝。研究目標(biāo)研究肝臟中是否存在調(diào)控脂代謝的lnc RNA。研究方法以q RT-PCR的方法測(cè)定外周白細(xì)胞中LASER的表達(dá)。以RNA干擾的方法降低Hep G2細(xì)胞內(nèi)LASER的表達(dá)后,以酶法或Filipin染色測(cè)定細(xì)胞內(nèi)膽固醇水平,以基因芯片分析干擾LASER后有哪些基因發(fā)生了改變。RNA-蛋白質(zhì)間的相互作用通過(guò)RNA免疫共沉淀證實(shí),HNF-1α基因啟動(dòng)子區(qū)域的組蛋白甲基化通過(guò)染色質(zhì)免疫共沉淀檢測(cè)。研究結(jié)果我們?cè)谘嚓P(guān)多態(tài)性熱點(diǎn)區(qū)域鑒定了一個(gè)lnc RNA(LASER)。臨床研究發(fā)現(xiàn)LASER的表達(dá)與含膽固醇的脂蛋白及PCSK9含量呈正相關(guān)。降低LASER的表達(dá)后可以降低細(xì)胞內(nèi)膽固醇水平并影響膽固醇代謝的相關(guān)基因,其中PCSK9及其轉(zhuǎn)錄因子HNF-1α的表達(dá)均下降。同時(shí),在給予HNF-1α的天然的拮抗劑黃連素后可以阻斷LASER對(duì)PCSK9的作用。LASER通過(guò)在核內(nèi)與Co REST/REST蛋白復(fù)合體的LSD1結(jié)合干擾了LSD1的活性,從而導(dǎo)致HNF-1α基因啟動(dòng)子抑制性的H3K4me2增加。與此同時(shí),降低LSD1的表達(dá)則可以阻斷LASER對(duì)HNF-1α-PCSK9的作用。最后,我們發(fā)現(xiàn)他汀類(lèi)可以升高LASER的表達(dá),同時(shí)導(dǎo)致PCSK9增加,膽固醇可以反向激活LXR受體調(diào)控LASER的表達(dá)。我們的研究可能部分解釋了“他汀逃逸”的現(xiàn)象。研究結(jié)論我們發(fā)現(xiàn)一個(gè)新的調(diào)控肝臟膽固醇代謝的lnc RNA-LASER。而抑制LASER可與他汀協(xié)同發(fā)揮聯(lián)合調(diào)脂的效應(yīng)。第二部分Zonulin易化腸道細(xì)菌移位參與動(dòng)脈硬化的機(jī)制研究研究背景動(dòng)脈硬化性心血管疾病導(dǎo)致的死亡仍高居不下,其中炎癥激活在動(dòng)脈硬化的進(jìn)展中發(fā)揮了重要的作用。大量的流行病學(xué)研究發(fā)現(xiàn)冠心病的發(fā)生與多種病原微生物相關(guān)(肺炎衣原體、幽門(mén)螺桿菌及巨細(xì)胞病毒等)。腸道中存在著種類(lèi)眾多的共生菌。腸道共生菌可以通過(guò)磷脂酰膽堿和左卡尼汀的代謝而間接致動(dòng)脈硬化,但目前仍缺乏腸道微生物直接致動(dòng)脈硬化的直接證據(jù)。約95%的動(dòng)脈硬化斑塊中能檢測(cè)到細(xì)菌的16S r RNA基因成分,其中以腸桿菌科為主。同時(shí),我們前期發(fā)現(xiàn)冠心病人全血中腸道菌屬的熒光假單胞菌及沙雷氏菌含量增加,提示除了間接作用外,腸道細(xì)菌還可能直接參與了動(dòng)脈硬化的進(jìn)展。生理狀況下腸道間的緊密連接將腸道細(xì)菌與循環(huán)隔絕開(kāi)來(lái),在動(dòng)脈硬化等病理情況下細(xì)菌能否穿過(guò)腸道屏障并進(jìn)入動(dòng)脈硬化斑塊內(nèi)目前尚不清楚。腸道細(xì)胞間的緊密連接在決定腸道通透性及控制腸道細(xì)菌移位方面發(fā)揮了重要的作用,其中zonulin(連蛋白)是腸道細(xì)胞合成的分泌蛋白,在調(diào)節(jié)緊密連接開(kāi)放和決定腸道細(xì)胞通透性方面發(fā)揮了重要的作用。既往研究表明循環(huán)中zonulin的水平在糖尿病、肥胖等動(dòng)脈硬化的危險(xiǎn)因素中升高。研究目標(biāo)動(dòng)脈硬化的發(fā)生和發(fā)展中,是否也有zonulin的改變,而zonulin調(diào)控的腸道通透性改變是否參與了腸道細(xì)菌的移位呢?研究方法以ELISA的方法測(cè)定血漿中的zonulin含量。體外培養(yǎng)Caco2細(xì)胞層構(gòu)建腸上皮屏障模型,培養(yǎng)熒光假單胞菌并以菌液干預(yù)腸上皮屏障模型。于不同時(shí)間點(diǎn)取Transwell小室的上層及下層的培養(yǎng)基進(jìn)行細(xì)菌培養(yǎng),并照相記錄菌落生長(zhǎng)情況。以?huà)呙桦婄R觀察熒光假單胞菌暴露對(duì)腸上皮屏障結(jié)構(gòu)的影響,以激光共聚焦顯微鏡觀察對(duì)zonulin分泌的影響。研究結(jié)果冠心病人中血漿中zonulin含量增加,而熒光假單胞菌的暴露促進(jìn)了腸道上皮細(xì)胞內(nèi)zonulin的分泌。分泌的zonulin改變了腸上皮屏障的細(xì)胞結(jié)構(gòu),導(dǎo)致腸道緊密連接的開(kāi)放而促進(jìn)熒光假單胞菌在腸道上皮的移位。研究結(jié)論熒光假單胞菌可以促進(jìn)zonulin的分泌并易化腸道細(xì)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R589.2

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