【摘要】：目的類風(fēng)濕關(guān)節(jié)炎(RA)屬于風(fēng)濕性疾病的一種,也是全世界范圍內(nèi)最為嚴(yán)重和普遍的自身免疫性疾病。RA通常起病隱匿,發(fā)病痛苦,病程漫長;且病患個體差異性大,缺乏徹底治愈的治療藥品和手段;病情的惡化常引發(fā)關(guān)節(jié)畸形,嚴(yán)重者甚至致殘。目前,關(guān)于RA的發(fā)病機制所公認的說法是免疫系統(tǒng)的紊亂,該過程中涉及到多個細胞因子在滑膜中的非正常作用,與許多復(fù)雜免疫反應(yīng)有較大關(guān)聯(lián)。對RA的表觀遺傳研究陸續(xù)發(fā)現(xiàn)有部分miRNA,不僅受到某些炎性細胞因子的調(diào)節(jié),也可通過調(diào)控某些細胞因子或靶向某些免疫相關(guān)的基因的表達來影響RA的發(fā)生和發(fā)展。目前,RA領(lǐng)域已有數(shù)個miRNA的作用被一步步揭示出來,而miR-145在RA中的作用卻未曾有人關(guān)注。癌癥中miR-145的研究成果的較突出,可調(diào)節(jié)多種惡性表型相關(guān)的基因,抑制其轉(zhuǎn)錄或蛋白功能的正常行使,發(fā)揮抑制腫瘤的作用。因此,本課題把miR-145-5p作為研究對象,以期發(fā)現(xiàn)miR-145-5p在RA中的調(diào)控作用。方法本實驗首先采用熒光定量PCR技術(shù)檢測了20例RA患者PBMC細胞中miR-145-5p的相對表達量,并以20例普通人的PBMC中的含量作為對比,得到miR-145-5p的差異表達的結(jié)果。隨后在RA患者的RA-FLS中轉(zhuǎn)染miR-145-5p的mimic或inhibitor實現(xiàn)對miR-145-5p的過表達和敲減之后,進行RA-FLS細胞的增殖、周期、凋亡實驗,得出miR-145-5p對細胞表型的影響。并通過熒光定量PCR實驗檢測過表達miR-145-5p后RA相關(guān)炎性因子基因的表達。接著運用生物信息學(xué)軟件和通路分析來尋找與細胞增殖、周期和凋亡有關(guān)或與骨代謝通路相關(guān)的基因作為候選研究的靶基因,接著采用Q-PCR實驗在轉(zhuǎn)錄水平上進行初步檢測,最后經(jīng)由western blot對待驗靶在翻譯水平上進行驗證,最終明確miR-145-5p在RA中的功能及作用機制。結(jié)果1.對20例RA患者和20例正常人PBMC細胞中miR-145-5p的表達檢測發(fā)現(xiàn),RA病人PBMC中miR-145-5p的表達量比正常人PBMC中顯著升高。2.RA-FLS中,過表達miR-145-5p后,細胞的增殖能力的降低比較明顯;敲減miR-145-5p后,細胞的增殖能力的上調(diào)比較明顯。該結(jié)果提示,miR-145-5p可能通過調(diào)節(jié)RA-FLS細胞的增值能力發(fā)揮對RA的作用。3.RA-FLS中,miR-145-5p含量升高后,可顯著發(fā)現(xiàn)G2期的細胞數(shù)目減少;敲減miR-145-5p后,可顯著發(fā)現(xiàn)G2期的細胞數(shù)目增多。分析上述可得出,miR-145-5p可能通過影響RA-FLS細胞的周期進程來影響細胞的增殖能力。4.RA-FLS中,過表達miR-145-5p的樣本,凋亡的細胞數(shù)目比對照組的數(shù)目明顯要高;敲減miR-145-5p的樣本,凋亡的細胞數(shù)目比對照組顯著減少。分析上述可得出,miR-145-5p可以影響RA-FLS的細胞凋亡過程。5.RA-FLS中,miR-145-5p水平的升高,可造成MMP9表達的下調(diào)。6.生物信息學(xué)預(yù)測出ADAM17為miR-145-5p的靶基因,其mRNA和蛋白含量在過表miR-145-5p后下調(diào),敲減miR-145-5p后上調(diào)。結(jié)論miR-145-5p在RA患者PBMC中表達升高。在RA-FLS中,miR-145-5p可能通過靶基因ADAM17的表達來調(diào)節(jié)TNF-α、細胞增殖相關(guān)通路以及MMP9的表達,從而影響細胞的增殖、凋亡和細胞周期進程,對滑膜的炎癥程度造成影響。
[Abstract]:Objective Rheumatoid arthritis (RA) is one of the most serious and widespread autoimmune diseases in the world. RA usually has a hidden onset, painful onset, and a long course of disease. The individual variability of RA patients is large, and the lack of thorough treatment drugs and means. The deterioration of RA often leads to joint deformities, and even serious cases. Disability. At present, it is generally accepted that the pathogenesis of RA is the disorder of the immune system, which involves the abnormal action of many cytokines in the synovium, and is closely related to many complex immune responses. At present, the role of several microRNAs in RA has been revealed step by step. However, the role of microRNAs in RA has not been paid attention to. In cancer, microRNAs-145 can regulate many malignant phenotypes. In order to discover the regulatory role of microRNAs-145-5p in RA, we first detected the relative expression of microRNAs-145-5p in PBMC cells of 20 patients with RA by fluorescence quantitative PCR. The results of differentially expressed microRNAs-145-5p were obtained by comparing the contents of PBMC in a normal person.Mimic or inhibitor transfected with microRNAs-145-5p in RA-FLS of RA patients overexpressed and knocked down microRNAs-145-5p.The proliferation, cycle and apoptosis experiments of RA-FLS cells were carried out to determine the effect of microRNAs-145-5p on cell phenotype. The expression of RA-related inflammatory factor gene was detected by photoquantitative polymerase chain reaction (PCR) assay after the expression of microRNAs-145-5p. Bioinformatics software and pathway analysis were used to identify genes related to cell proliferation, cycle and apoptosis or bone metabolism pathway as candidate genes. Q-PCR assay was used to detect these genes at transcriptional level. Results 1. The expression of microRNA-145-5p in PBMC cells of 20 RA patients and 20 normal subjects was detected. The expression of microRNA-145-5p in PBMC cells of RA patients was significantly higher than that in normal PBMC. Mi-145-5p significantly decreased the proliferation of RA cells, and the up-regulation of cell proliferation was more obvious after the knockdown of Mi-145-5p. The results suggested that Mi-145-5p might play a role in RA by regulating the proliferation of RA-FLS cells. 3. In RA-FLS, when the content of Mi-145-5p increased, the number of G2 cells decreased significantly. Mi-145-5p could significantly increase the number of G2 cells after reducing the expression of Mi-145-5p. These results suggest that Mi-145-5p may affect the proliferation of RA-FLS cells by influencing the cell cycle progression. 4. In RA-FLS, the number of apoptotic cells was significantly higher than that in control group when the expression of Mi-145-5p was over-expressed; Mi-145-5p knocked down, apoptotic cells were observed. The number of cells was significantly lower than that of the control group. The results showed that the expression of MMP9 in RA-FLS was down-regulated by increasing the level of microRNAs-145-5p. Bioinformatics predicted that ADAM17 was the target gene of microRNAs-145-5p, and its mRNA and protein contents were down-regulated after overexpression of microRNAs-145-5p, knocking down microRNAs-145-5p. Conclusion The expression of microRNAs-145-5p in PBMC of RA patients is elevated. In RA-FLS, microRNAs-145-5p may regulate the expression of TNF-alpha, cell proliferation-related pathways and MMP9 through the expression of target gene ADAM17, thereby affecting cell proliferation, apoptosis and cell cycle progression, and affecting the degree of synovial inflammation.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R593.22

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