發(fā)布時間：2018-01-04 21:29

  本文關(guān)鍵詞：篩選類風(fēng)濕關(guān)節(jié)炎中糖代謝關(guān)鍵基因的研究　出處：《山東大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 葡萄糖代謝 類風(fēng)濕關(guān)節(jié)炎 膠原誘導(dǎo)型關(guān)節(jié)炎 磷酸甘油酸酯激1(PGK1) 烯醇化酶α(ENO1)

【摘要】：類風(fēng)濕關(guān)節(jié)炎(Rheumatoid arthritis, RA)是一種自生免疫性疾病,主要出現(xiàn)滑膜炎癥、血管翳形成和隨后的關(guān)節(jié)損傷等病理改變。糖代謝是機(jī)體內(nèi)最主要的代謝途徑之一,主要是指葡萄糖在降解的過程,包括糖酵解、有氧氧化和磷酸戊糖代謝途徑。糖酵解參與是氨基酸、脂類、核苷酸類三大代謝,并能產(chǎn)生三磷酸腺苷(Adenosine Triphosphate, ATP)。糖代謝不僅為機(jī)體提供能量,還通過信號通路,信號網(wǎng)絡(luò)來調(diào)節(jié)多種生理過程。糖代謝在糖尿病、中風(fēng)、精神分裂癥和藥物濫用等疾病中發(fā)揮重要作用。有研究表明,糖代謝包括糖酵解這一過程,在RA的發(fā)病機(jī)制和發(fā)生發(fā)展中起著重要作用。有研究認(rèn)為,糖酵解活動增強(qiáng),在RA的發(fā)病機(jī)制中,可能發(fā)揮著重要作用。既往主要是從免疫學(xué)和遺傳學(xué)的角度進(jìn)行研究RA的相關(guān)致病機(jī)制,對于糖代謝如何參與影響RA發(fā)生發(fā)展尚無深入研究。而能否從糖代謝角度思考RA這一過程,我們的很多研究成果和最近的研究都提供了新的視角。PCR Array是一項敏感和可靠的基因表達(dá)分析技術(shù),不僅具有實時熒光定量PCR技術(shù)較好的準(zhǔn)確性和靈敏度,同時還具有微列陣的高通量優(yōu)勢,可以進(jìn)行信號轉(zhuǎn)導(dǎo)途徑、生理過程或與疾病相關(guān)的一組基因網(wǎng)絡(luò)分析。本研究利用PCRArray法檢測膠原誘導(dǎo)型關(guān)節(jié)炎(Collagen induced arthritis, CIA)大鼠的糖代謝相關(guān)基因,旨在檢測糖代謝的關(guān)鍵酶在RA中的表達(dá)水平改變,并闡明作用,進(jìn)一步探索糖代謝在類風(fēng)濕性關(guān)節(jié)炎的作用.進(jìn)一步探討糖代謝和類風(fēng)濕關(guān)節(jié)炎的相關(guān)性。方法：取牛II型膠原誘導(dǎo)大鼠(CIA組)和正常對照大鼠(NCR組)膝關(guān)節(jié)滑膜組織,提取總RNA,采用Rat Glucose Metabolism RT2 ProfilerTM PCR Array篩選表達(dá)不一致基因；采用RT-PCR法對CIA組和NCR組進(jìn)行mRNA水平的檢測,采用RT-PCR法和Western blot法檢測類風(fēng)濕關(guān)節(jié)炎(RA組)和骨關(guān)節(jié)炎(OA組)滑膜組織中基因mRNA水平和蛋白水平的表達(dá)。收集類風(fēng)濕關(guān)節(jié)炎血清(RAB組)和正常獻(xiàn)血者血清(NCB組),采用ELISA法檢測血清中的表達(dá)水平。取膝關(guān)節(jié)滑膜組織原代培養(yǎng)FLS；采用人工合成的Si-RNA-PGK1干擾劑特異性抑制PGK1在RAFLS中的表達(dá),陰性Si-RNA為陰性對照組,瞬時轉(zhuǎn)染24 h后,采用RT-PCR法和Western blot法檢測抑制效率,采用CCK-8、 Transwell、流式細(xì)胞術(shù)、ELISA等方法檢測干擾PGK1表達(dá)后對細(xì)胞增殖、侵襲、遷移、細(xì)胞周期和相關(guān)炎性因子分泌的影響。結(jié)果PCR Array檢測顯示,CIA組烯醇化酶(ENO1)、己糖激酶2(HK2)、磷酸甘油酸激酶-1(PGK1)較NCR組表達(dá)上調(diào)(P0.05),磷酸烯醇式丙酮酸羧激酶-1(PCK1)和丙酮酸脫氫酶激酶-4(PDK4)表達(dá)下調(diào)(P0.05); RT-PCR法和Western blot法檢測顯示,RA組和OA組相比, ENO1和PGK1表達(dá)上調(diào)(P0.05), ELISA法檢測結(jié)果顯示,RAB組和NCB組相比,PGK1表達(dá)升高(P0.05)。轉(zhuǎn)染特異性Si-RNA-PGK1后, 各干擾組與陰性對照組相比,Si-RNA-PGK1干擾劑轉(zhuǎn)染24 h時,PGK1 mRNA水平抑制效果顯著(P0.05),RAFLS的增殖受到抑制(P0.05), 細(xì)胞的侵襲能力下降(P0.05),IFN-γ和IL-1β分泌降低(P0.05)。結(jié)論：牛Ⅱ型膠原誘導(dǎo)大鼠和類風(fēng)濕關(guān)節(jié)炎滑膜組織中糖代謝關(guān)鍵酶ENO1和PGK1表達(dá)增高,類風(fēng)濕關(guān)節(jié)炎血液中PGK1表達(dá)增加,均提示類風(fēng)濕關(guān)節(jié)炎中糖代謝活動增強(qiáng),導(dǎo)致相關(guān)基因的表達(dá)異常。這說明糖代謝中的關(guān)鍵酶PGK1參與了細(xì)胞異常增殖、遷移以及IFN-γ和IL-1 p等炎癥因子的分泌表達(dá),說明PKK1對RA炎癥因子表達(dá)及RAFLS的生物學(xué)功能有影響。
[Abstract]:Rheumatoid arthritis (Rheumatoid arthritis RA) is a kind of spontaneous autoimmune disease, mainly inflammation of synovium, pannus formation change and subsequent joint injury pathology. Glucose metabolism is one of the most important metabolic pathways in the body, mainly refers to the process of glucose degradation, including glycolysis and aerobic oxidation and the pentose phosphate pathway is involved in glycolysis. Amino acids, lipids, nucleotides three metabolism, and can produce ATP (Adenosine Triphosphate, ATP). Glucose metabolism not only provides energy for the body, but also through the signal pathway, signal network to regulate a variety of physiological processes. Glucose metabolism in diabetes, stroke, schizophrenia play an important effect of disease and drug abuse and other diseases. Studies have shown that glucose metabolism including glycolysis in this process plays an important role in the pathogenesis of RA and development. Some studies suggested that the glycolytic activity The dynamic enhancement in the pathogenesis of RA, may play an important role. It is mainly related to the pathogenesis of RA from the perspective of immunology and genetics, the sugar metabolism influence how to participate in the occurrence and development of RA. There is no in-depth study and whether this process on RA from the angle of glucose metabolism, many of our research results and recent studies have provided a new perspective.PCR Array expression analysis technique is a sensitive and reliable gene, not only has the accuracy and sensitivity of real-time fluorescence quantitative PCR technology is good, but also has the advantages of high-throughput microarray, can signal transduction pathways, analysis of physiological processes associated with disease or a group of gene network. The detection of collagen induced arthritis by PCRArray (Collagen induced arthritis, CIA) glucose metabolism related genes in rats, to detect the key enzyme in sugar metabolism The expression level of RA in the change, and to elucidate the role, to further explore the effect of glucose metabolism in rheumatoid arthritis. To further explore the correlation between glucose metabolism and rheumatoid arthritis. Methods: bovine type II collagen induced rats (CIA group) and normal control rats (group NCR) of knee joint synovial tissue, extraction of total RNA, Rat Glucose Metabolism RT2 ProfilerTM PCR by Array gene expression screening is not consistent; detection of mRNA level of CIA group and NCR group by RT-PCR method, rheumatoid arthritis was detected by RT-PCR method and Western blot method (group RA) and osteoarthritis (OA) gene expression level of mRNA and protein in synovial tissue the serum was collected. Rheumatoid arthritis (RAB group) and normal blood donors serum (NCB group), the expression levels in serum were detected by ELISA. The knee joint synovial tissue cultured FLS; using Si-RNA-PGK1 as artificial interference The expression of agent specific inhibition of PGK1 in RAFLS, negative Si-RNA as negative control group, 24 h after transient transfection, using RT-PCR method and Western blot method to detect the inhibition efficiency, using CCK-8, Transwell, flow cytometry, the expression of ELISA interference detection method PGK1 on cell proliferation, invasion, migration, secretion of cells cycle and related inflammatory factors. PCR Array results showed that group CIA enolase (ENO1), hexokinase 2 (HK2), phosphoglycerate kinase -1 (PGK1) than group NCR (P0.05), expression of phosphoenolpyruvate carboxykinase kinase -1 (PCK1) and pyruvate dehydrogenase kinase -4 (PDK4) expression of RT-PCR (P0.05); display detection method and Western blot method, RA group compared with OA group, expression of ENO1 and PGK1 (P0.05), ELISA assay showed that RAB group compared with NCB group, the expression of PGK1 increased (P0.05). After transfection of specific Si-RNA-PGK1, the interference group and the negative Compared with the control group, Si-RNA-PGK1 interference was transfected 24 h, significant inhibitory effect of PGK1 mRNA (P0.05), RAFLS (P0.05) inhibited proliferation, decreased cell invasion ability (P0.05), IFN- IL-1 and gamma beta secretion (P0.05). Conclusion: the increased expression of rat synovial tissue in rheumatoid arthritis and the key enzyme ENO1 and PGK1 induced by bovine type II collagen, rheumatoid arthritis and blood PGK1 expression increased, suggesting metabolic activity in rheumatoid arthritis related gene expression leads to enhanced anomaly. This shows that PGK1 is the key enzyme in sugar metabolism is involved in cell proliferation, migration and secretion expression of IFN- gamma and IL-1 P inflammation the influence factor of the biological function of PKK1 RA on the expression of inflammatory factors and RAFLS.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R593.22

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