發(fā)布時(shí)間：2018-10-23 19:43

【摘要】：原發(fā)性膽汁性肝硬化（primary biliary cirrhosis，PBC）是一種慢性肝臟疾病，以肝內(nèi)中小膽管損傷、膽汁淤積以及部分患者出現(xiàn)肝硬化為特征，約90~95%的患者血清可出現(xiàn)高度特異的抗線粒體抗體（antimitochondrial autoantibodies，AMAs）[1]。PBC好發(fā)于中年女性，近年來(lái)其發(fā)病率逐年升高，但其發(fā)病機(jī)制仍不明確。在治療方面，熊去氧膽酸（ursodeoxycholic acid，UDCA）是目前唯一公認(rèn)的治療藥物，PBC患者對(duì)免疫抑制劑的治療反應(yīng)性差。 微小RNA（microRNA，miRNA）作為一類序列短、不具有編碼功能的小分子RNA，廣泛參與基因的轉(zhuǎn)錄后調(diào)控[2]。已經(jīng)有不少研究者證實(shí)了miRNA在類風(fēng)濕性關(guān)節(jié)炎（rheumatoid arthritis，RA）、系統(tǒng)性紅斑狼瘡（systemic lupus erythematosus，SLE）、多發(fā)性硬化癥（multiple sclerosis，MS）以及潰瘍性結(jié)腸炎（ulcerative colitis，UC）等一系列自身免疫性疾病的發(fā)生、發(fā)展以及治療過(guò)程中發(fā)揮著重要作用，近年來(lái)miRNA在PBC中的作用也逐漸得到關(guān)注。本實(shí)驗(yàn)小組之前已經(jīng)就PBC患者的外周血單個(gè)核細(xì)胞（peripheral blood mononuclear cells，PBMCs）進(jìn)行了差異性表達(dá)的miRNA芯片篩查，miR-17家族的miR-20a-5p、miR-106b和miR-93表達(dá)均顯著上調(diào)，且位于miRNA-gene-network位于中心位置。 白介素-8（interleukin-8，IL-8）是一種重要的炎癥介質(zhì)，Zimmermann H W等發(fā)現(xiàn)慢性肝臟疾�。╟hronic liver disease，CLD）患者血清IL-8水平升高，其中膽汁淤積性肝臟疾病和酒精性肝硬化患者IL-8水平升高最為顯著，且IL-8水平與CLD的疾病進(jìn)展以及非侵襲性肝硬化指標(biāo)呈正相關(guān)[3]。 本課題針對(duì)PBC患者miR-20a-5p和IL-8的表達(dá)水平進(jìn)行檢測(cè)，并對(duì)二者之間可能存在的調(diào)控關(guān)系進(jìn)行探討。 第一部分：PBC患者miR-20a-5p和IL-8表達(dá)水平的檢測(cè) 我們收集長(zhǎng)征醫(yī)院PBC、RA、SLE、病毒性肝炎肝硬化（Viral hepatitis cirrhosis，VHC）住院患者和正常對(duì)照各20例，利用RT-PCR檢測(cè)miR-20a-5p、IL-8在各類疾病PBMCs中的表達(dá)差異，血漿IL-8的表達(dá)水平采用ELISA方法檢測(cè)。結(jié)果顯示PBC患者的PBMCs中miR-20a-5p和IL-8的相對(duì)表達(dá)水平均較正常對(duì)照和其他疾病對(duì)照組顯著升高，差異均有統(tǒng)計(jì)學(xué)意義（P0.05）。miR-20a-5p的相對(duì)表達(dá)量為正常對(duì)照組的6.33倍，IL-8的相對(duì)表達(dá)量為正常對(duì)照的7.96倍，PBC患者血漿IL-8的平均水平為89.5±36.6ng/L。 第二部分：THP-1細(xì)胞中轉(zhuǎn)染miR-20a-5p對(duì)IL-8表達(dá)水平的影響 用100ng/ml的LPS對(duì)THP-1細(xì)胞進(jìn)行預(yù)處理，6小時(shí)后轉(zhuǎn)染miR-20a-5p的mimic和inhibitor，分別利用RT-PCR和ELISA檢測(cè)IL-8表達(dá)水平的變化。結(jié)果顯示轉(zhuǎn)染了miR-20a-5p的mimic后，THP-1細(xì)胞IL-8的相對(duì)表達(dá)量降低，為空白對(duì)照的0.25倍，而轉(zhuǎn)染了miR-20a-5p的inhibitor后THP-1細(xì)胞IL-8的相對(duì)表達(dá)量升高，為空白對(duì)照的4.02倍，差異均有統(tǒng)計(jì)學(xué)意義（P 0.05）。 第三部分：miR-20a-5p對(duì)IL-8調(diào)控機(jī)制的探究 為了進(jìn)一步探究miR-20a-5p對(duì)IL-8表達(dá)水平調(diào)控的機(jī)制，我們?cè)趍iRDB、TargetScan以及MicroRNA.org數(shù)據(jù)庫(kù)對(duì)miR-20a-5p的靶基因進(jìn)行預(yù)測(cè)，并利用雙熒光素酶報(bào)告實(shí)驗(yàn)進(jìn)行了miR-20a-5p與預(yù)測(cè)靶基因干擾素調(diào)節(jié)因子9（IFN regulatoryfactor9，IRF9）之間靶向作用關(guān)系的驗(yàn)證，最后針對(duì)IRF9設(shè)計(jì)了小干擾RNA（smallinterfering RNA，siRNA），利用RT-PCR和ELISA檢測(cè)轉(zhuǎn)染siRNA后IL-8表達(dá)的改變情況。MicroRNA.org數(shù)據(jù)庫(kù)顯示miR-20a-5p和IRF9之間有9個(gè)連續(xù)互補(bǔ)配對(duì)的堿基，存在緊密的靶向作用關(guān)系，，而將miR-20a-5p的mimic與構(gòu)建好的報(bào)告基因載體共轉(zhuǎn)后，報(bào)告基因相對(duì)熒光值下調(diào)了30.3%（P 0.05），也說(shuō)明miR-20a-5p對(duì)IRF9有靶向調(diào)節(jié)作用。在LPS預(yù)處理的THP-1單核細(xì)胞株內(nèi)轉(zhuǎn)染IRF9的siRNA以后，細(xì)胞和培養(yǎng)上清內(nèi)IL-8的表達(dá)均下調(diào)，其改變趨勢(shì)與轉(zhuǎn)染了miR-20a-5p的mimic之后改變一致。轉(zhuǎn)染miR-20a-5p以及siRNA后IRF9的蛋白表達(dá)水平均用Westernblot實(shí)驗(yàn)進(jìn)行檢測(cè)，結(jié)果與RT-PCR檢測(cè)結(jié)果一致。 結(jié)論P(yáng)BC患者外周血miR-20a-5p和IL-8的表達(dá)均升高；在THP-1細(xì)胞株內(nèi)miR-20a-5p對(duì)IL-8的表達(dá)有抑制作用，而這種抑制作用可能是通過(guò)靶作用于IRF9的表達(dá)而實(shí)現(xiàn)的。miR-20a-5p有可能作為一種保護(hù)性因素抑制IL-8的持續(xù)升高，而IRF9通過(guò)何種途徑影響IL-8的表達(dá)有待進(jìn)一步研究。
[Abstract]:Primary biliary cirrhosis (PBC) is a kind of chronic liver disease, characterized by small and medium-sized bile duct injury, bile siltation and partial cirrhosis. About 90-95% of patients have high specific anti-mitochondrial antibody (AMAs)[1]. PBC is a middle-aged woman, whose incidence has increased year by year, but its pathogenesis is still unclear. In the treatment aspect, ursodic acid (UDCA) is the only recognized therapeutic drug, and PBC patients have poor therapeutic response to immunosuppressive agents. MicroRNA (miRNA) is a kind of short-molecule RNA with short sequence and no coding function, and is widely involved in the post-transcriptional regulation of gene. 2] Many researchers have demonstrated that miRNA is a series of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and chronic colitis (UC). In recent years, the role of miRNA in PBC has also been gradually obtained. The results showed that the expression of miR-20a-5p, miR-106b and miR-93 in the peripheral blood mononuclear cells (PBMNC) of PBC patients was significantly increased, and the expression of miR-20a-5p, miR-106b and miR-93 was significantly increased, and the miRNA-gene-network was located at the center. Interleukin-8 (IL-8) is an important inflammatory mediator, and the levels of IL-8 in serum of patients with chronic liver disease (CLD) are elevated in patients with chronic liver disease (CLD). The highest and most significant, and the level of IL-8 was positive with the disease progression of CLD and the non-invasive liver cirrhosis index.[3]. The objective of this study was to detect the expression levels of miR-20a-5p and IL-8 in patients with PBC. Regulatory relationships. Part 1: PBC patient miR-20a-5p The levels of IL-8 expression in patients with PBC, RA, SLE, viral hepatitis cirrhosis (VHC) and 20 normal controls were collected by RT-PCR, and miR-20a-5p and IL-8 were detected by RT-PCR. Differences in expression in PBRER and Table of plasma IL-8 The results showed that the relative expression levels of miR-20a-5p and IL-8 in PBMNC of PBC patients were significantly higher than those of normal control group and other control group (P <0.05). The relative expression of miR-20a-5p was 6.33 times that of normal control group. The expression level was 7. 96 times the normal control, and the level of plasma IL-8 in PBC patients was higher than that of normal control. miR-1 cells were transfected with miR-The effect of 20a-5p on the expression level of IL-8 was pretreated with 100ng/ ml LPS and then transfected into mimeic and intreitor of miR-20a-5p after 6 hours, respectively using RT-PC. The expression level of IL-8 was detected by R and ELISA. The results showed that the relative expression of IL-8 of IL-8 decreased in the transfected cells of miR-20a-5p, the relative expression of IL-8 was 0. 25 times of that of blank control, but the relative expression of IL-8 was increased after transfection of inactivator of miR-20a-5p. 02-fold, difference was statistically significant (P 0.05). In order to further explore the mechanism of miR-20a-5p on the regulation of IL-8 expression levels, we used miRDB, TargetScan and

MicroRNA. org database, and the relationship between miR-20a-5p and the target gene interferon regulatory factor 9 (IF9) was verified by double luciferase reporter assay. Finally, small interfering RNA was designed for IRF9. The changes of IL-8 expression after transfection of siRNA by RT-PCR and ELISA showed that there were 9 consecutive complementary pairs of bases between miR-20a-5p and IRF9 by RT-PCR and ELISA. The relative fluorescence value of reporter gene decreased by 30.3% (P 0.05). It is also indicated that miR-20a-5p has targeted regulation effect on IRF9. After transfected with IRF9 siRNA, the expression of IL-8 in cells and cultured supernatant is down regulated. After transfection with mimeic transfected with miR-20a-5p, the expression level of IRF9 after transfection of miR-20a-5p and siRNA was expressed by Western blot. The results showed that the expression of miR-20a-5p and IL-8 in peripheral blood of PBC was increased, and the expression of miR-20a-5p in human IL-1 cell line was inhibited by RT-PCR. This inhibition may be achieved by the expression of the target acting on IRF9. miR-20a-5p is likely to inhibit the continuous rise of IL-8 as a protective facto
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.22

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相關(guān)期刊論文 前2條

1 張振武;安洋;滕春波;;miR-17-92基因簇microRNAs對(duì)哺乳動(dòng)物器官發(fā)育及腫瘤發(fā)生的調(diào)控[J];遺傳;2009年11期

2 ;Interleukin-8, a promising predictor for prognosis of pancreatic cancer[J];World Journal of Gastroenterology;2012年10期

本文編號(hào)：2290282