本文選題：E2F2 + 類風(fēng)濕關(guān)節(jié)炎滑膜成纖維細(xì)胞　； 參考：《濟(jì)南大學(xué)》2017年碩士論文

【摘要】：目的:1、探討E2F2在RASFs中發(fā)揮的生物學(xué)功能;2、研究E2F2在RASFs中異常表達(dá)的調(diào)控機(jī)制;3、探討E2F2調(diào)控RASFs活性的致病途徑。方法:1、取置換關(guān)節(jié)手術(shù)的RA患者膝關(guān)節(jié)滑膜組織,體外進(jìn)行原代培養(yǎng),獲取RA滑膜成纖維細(xì)胞RASFs;RT-qPCR和Western Blot法分別檢測E2F2在RASFs和OASFs中的表達(dá)情況;設(shè)計并合成E2F2的small interfering RNA(siRNA),轉(zhuǎn)染RASFs以沉默E2F2的表達(dá),以轉(zhuǎn)染negative siRNA(NC)為陰性對照組;檢測干擾效率并篩選出干擾效率最佳的siE2F2,用于后續(xù)實驗;MTS法檢測E2F2沉默后RASFs的增殖能力變化;細(xì)胞劃痕和Transwell法檢測E2F2沉默后RASFs的遷移能力變化;Transwell法檢測E2F2沉默后RASFs的侵襲能力變化。2、選擇重組白細(xì)胞介素-6(interleukin-6,IL-6),腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α),脂多糖(lipopolysaccharides,LPS)三種促炎因子刺激RASFs,RT-qPCR和Western Blot法檢測E2F2的變化及其與濃度和時間的關(guān)系;PDTC、static、PD98059分別阻斷NF-κB、STAT3、ERK信號通路的同時,用IL-6、TNF-α、LPS刺激RASFs,RT-qPCR和Western Blot法分別檢測E2F2的表達(dá)變化;染色質(zhì)免疫共沉淀法(Chromatin Immunoprecipitation,Ch IP)驗證信號通路與E2F2啟動子區(qū)的結(jié)合情況。3、IL-6、TNF-α、LPS分別刺激RASFs,提取分離核蛋白及漿蛋白,Western Blot檢測E2F2在細(xì)胞核及細(xì)胞漿中的表達(dá)變化,探討促炎因子對轉(zhuǎn)錄因子E2F2的核定位的影響;RT-qPCR及ELISA檢測E2F2沉默后在RASFs中相關(guān)基因的表達(dá);RT-qPCR檢測RA血漿中E2F2與促炎因子水平之間的關(guān)系;用Ch IP驗證E2F2與促炎因子啟動子區(qū)的結(jié)合;E2F2沉默后,促炎因子刺激RASFs,RTqPCR法和Western Blot檢測與E2F2相關(guān)的促炎因子的表達(dá)變化。結(jié)果:1、E2F2在RASFs的表達(dá)高于在OASFs中的表達(dá)(*p0.05)。E2F2沉默后,在mRNA以及蛋白質(zhì)水平顯著降低RASFs中E2F2的表達(dá)(*p0.05),干擾效率為78%;E2F2沉默后,RASFs的增殖速率降低(*p0.05),遷移能力顯著降低(*p0.05),同時其侵襲能力也顯著下降(**p0.01)。2、IL-6、TNF-α、LPS分別刺激RASFs,E2F2在mRNA以及蛋白水平的表達(dá)均顯著增加,且對三種促炎因子均有時間和濃度依賴性(*p0.05,**p0.01);ERK信號通路抑制劑PD98059能夠顯著逆轉(zhuǎn)LPS對E2F2的誘導(dǎo)(*p0.05,**p0.01),而NF-κB信號通路抑制劑PDTC能夠顯著逆轉(zhuǎn)IL-6和TNF-α對E2F2的誘導(dǎo)(*p0.05,**p0.01)。以上結(jié)果說明,促炎因子刺激能夠誘導(dǎo)RASFs中E2F2的表達(dá),其中LPS通過ERK信號通路、IL-6和TNF-α通過NF-κB信號通路誘導(dǎo)E2F2的表達(dá)。進(jìn)一步的ChIP結(jié)果顯示NF-κB能夠與E2F2的啟動子區(qū)直接結(jié)合,驗證了以上結(jié)果。3、TNF-α,IL-6,LPS均能夠促進(jìn)E2F2入核,其中IL-6作用最顯著。E2F2沉默后RASFs中一些與炎癥、侵襲密切相關(guān)的基因包括TNF-α、IL-1α、IL-1β、IL-6、前列腺素E2(prostaglandin E2,PGE2)、基質(zhì)金屬蛋白酶2(matrix metalloproteinase 2,MMP2)、MMP9和MMP13的表達(dá)均降低(*p0.05,**p0.01)。ELISA結(jié)果證實炎癥因子TNF-α,IL-1α,IL-1β,IL-6分泌顯著降低(*p0.05,**p0.01)。沉默E2F2可以逆轉(zhuǎn)TNF-α對RASFs中IL-6的誘導(dǎo);ELISA結(jié)果顯示RA血漿中E2F2與TNF-α(r2=0.6142,P=0.0005)、IL-6(r2=0.5940,P=0.0008)均呈較強(qiáng)的線性關(guān)系。此外,ChIP結(jié)果顯示E2F2能夠與IL-6的啟動子區(qū)直接結(jié)合。結(jié)論:E2F2在RA滑膜組織中高表達(dá),并參與RA滑膜成纖維細(xì)胞的異常增殖、遷移、侵襲,促進(jìn)細(xì)胞因子的產(chǎn)生。在炎癥條件下,炎性因子TNF-α、LPS、IL-6均可誘導(dǎo)RASFs中E2F2的表達(dá);我們還發(fā)現(xiàn),在RASFs中存在NF-κB/E2F2/IL-6之間的正反饋循環(huán)。說明E2F2參與介導(dǎo)RA中的炎癥�？傊�,本研究證實E2F2在RA的炎癥、滑膜增生等病理過程中起著重要的促進(jìn)作用,是RA臨床治療的潛在靶標(biāo)。
[Abstract]:Objective: 1, to explore the biological functions of E2F2 in RASFs; 2, to study the regulation mechanism of abnormal expression of E2F2 in RASFs; 3, to explore the pathogeny way of E2F2 regulation of RASFs activity. Method: 1, take the knee joint synovial tissue of the RA patients undergoing replacement arthroplasty for primary culture in vitro, and obtain RA synovial fibroblasts RASFs; RT-qPCR and Western Blot method. To detect the expression of E2F2 in RASFs and OASFs, and to design and synthesize small interfering RNA (siRNA) of E2F2, transfect RASFs to silence E2F2 expression, to transfect negative siRNA (negative siRNA) as negative control group, to detect interference efficiency and to screen out the best interference efficiency. Ability change, cell scratch and Transwell method to detect the change of migration ability of RASFs after E2F2 silence; Transwell assay was used to detect RASFs invasion ability of.2 after E2F2 silencing, and three kinds of proinflammatory cells were selected as recombinant interleukin -6 (interleukin-6, IL-6), tumor necrosis factor alpha (tumor necrosis) alpha, and lipopolysaccharide. RASFs, RT-qPCR and Western Blot were used to detect the changes in E2F2 and their relationship with the concentration and time. PDTC, static, PD98059 blocked the NF- kappa B, STAT3, and ERK signal pathways, respectively. Cipitation, Ch IP) verify the combination of signal pathway and E2F2 promoter region.3, IL-6, TNF- a, LPS stimulate RASFs, extract separate nucleoprotein and plasma protein, Western Blot detect the expression of E2F2 in the nucleus and cytoplasm, and explore the effect of pro-inflammatory factors on the nuclear location of the transcription factor. The expression of related genes in RASFs; RT-qPCR detection of the relationship between E2F2 and the level of pro-inflammatory factors in RA plasma; using Ch IP to verify the combination of the promoter region of E2F2 and proinflammatory factors; after E2F2 is silent, the proinflammatory factor stimulates the expression of proinflammatory factors associated with RASFs, RTqPCR and Western Blot. Results: 1 After the expression of (*p0.05).E2F2 in OASFs, the expression of E2F2 in RASFs was significantly reduced at the level of mRNA and protein (*p0.05), and the interference efficiency was 78%. After E2F2 was silent, the proliferation rate of RASFs decreased (*p0.05), and the ability to migrate significantly decreased (*p0.05). The expression of 2F2 at mRNA and protein levels increased significantly, and had time and concentration dependence on three proinflammatory factors (*p0.05, **p0.01). ERK signaling pathway inhibitor PD98059 could significantly reverse the induction of E2F2 (*p0.05, **p0.01), while NF- kappa B signaling pathway inhibitor could significantly reverse the induction of E2F2. *p0.01). The above results show that proinflammatory factor stimulation can induce the expression of E2F2 in RASFs, in which LPS can induce the expression of E2F2 through the ERK signaling pathway, IL-6 and TNF- alpha through the NF- kappa B signaling pathway. 2 of the most significant.E2F2 silencing of IL-6 after silencing, some genes associated with inflammation, which are closely related to inflammation, include TNF- alpha, IL-1 alpha, IL-1 beta, IL-6, prostaglandin E2 (prostaglandin E2, PGE2), matrix metalloproteinase 2 (matrix 2). The secretion of TNF- alpha, IL-1 a, IL-1 beta, and IL-6 decreased significantly (*p0.05, **p0.01). Silent E2F2 can reverse the induction of TNF- alpha to IL-6 in RASFs; ELISA results show that RA plasma is strongly linear. Conclusion: E2F2 is highly expressed in RA synovial tissue and participates in the abnormal proliferation, migration and invasion of RA synovial fibroblasts, promoting the production of cytokines. In inflammatory conditions, inflammatory factors TNF- a, LPS, IL-6 can induce the expression of E2F2 in RASFs; we also found that there is a positive feedback loop between NF- kappa B/E2F2/IL-6 in RASFs. In addition to mediating inflammation in RA, this study confirms that E2F2 plays an important role in the pathogenesis of RA inflammation, synovial hyperplasia and other pathological processes, and is a potential target for the clinical treatment of RA.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R593.22

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 陳玉婷;曹永芬;;淺談NF-kB信號在類風(fēng)濕關(guān)節(jié)炎中的作用及臨床意義[J];現(xiàn)代養(yǎng)生;2015年12期

2 王碩;李時榮;;VEGF、IL-6在類風(fēng)濕關(guān)節(jié)炎發(fā)病中的作用研究進(jìn)展[J];山東醫(yī)藥;2014年07期

3 李香斌;連金饒;林娜;孔祥英;;類風(fēng)濕關(guān)節(jié)炎滑膜血管生成和血管翳[J];醫(yī)學(xué)綜述;2010年01期

4 侯亞楠;郭禮和;;滑膜成纖維細(xì)胞在類風(fēng)濕性關(guān)節(jié)炎中的作用[J];細(xì)胞生物學(xué)雜志;2009年02期

，

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