E2F2在类风湿关节炎滑膜成纤维细胞中的作用及其机制研究

发布时间：2018-05-20 02:01

  本文选题：E2F2 + 类风湿关节炎滑膜成纤维细胞　； 参考：《济南大学》2017年硕士论文

【摘要】：目的:1、探讨E2F2在RASFs中发挥的生物学功能;2、研究E2F2在RASFs中异常表达的调控机制;3、探讨E2F2调控RASFs活性的致病途径。方法:1、取置换关节手术的RA患者膝关节滑膜组织,体外进行原代培养,获取RA滑膜成纤维细胞RASFs;RT-qPCR和Western Blot法分别检测E2F2在RASFs和OASFs中的表达情况;设计并合成E2F2的small interfering RNA(siRNA),转染RASFs以沉默E2F2的表达,以转染negative siRNA(NC)为阴性对照组;检测干扰效率并筛选出干扰效率最佳的siE2F2,用于后续实验;MTS法检测E2F2沉默后RASFs的增殖能力变化;细胞划痕和Transwell法检测E2F2沉默后RASFs的迁移能力变化;Transwell法检测E2F2沉默后RASFs的侵袭能力变化。2、选择重组白细胞介素-6(interleukin-6,IL-6),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),脂多糖(lipopolysaccharides,LPS)三种促炎因子刺激RASFs,RT-qPCR和Western Blot法检测E2F2的变化及其与浓度和时间的关系;PDTC、static、PD98059分别阻断NF-κB、STAT3、ERK信号通路的同时,用IL-6、TNF-α、LPS刺激RASFs,RT-qPCR和Western Blot法分别检测E2F2的表达变化;染色质免疫共沉淀法(Chromatin Immunoprecipitation,Ch IP)验证信号通路与E2F2启动子区的结合情况。3、IL-6、TNF-α、LPS分别刺激RASFs,提取分离核蛋白及浆蛋白,Western Blot检测E2F2在细胞核及细胞浆中的表达变化,探讨促炎因子对转录因子E2F2的核定位的影响;RT-qPCR及ELISA检测E2F2沉默后在RASFs中相关基因的表达;RT-qPCR检测RA血浆中E2F2与促炎因子水平之间的关系;用Ch IP验证E2F2与促炎因子启动子区的结合;E2F2沉默后,促炎因子刺激RASFs,RTqPCR法和Western Blot检测与E2F2相关的促炎因子的表达变化。结果:1、E2F2在RASFs的表达高于在OASFs中的表达(*p0.05)。E2F2沉默后,在mRNA以及蛋白质水平显著降低RASFs中E2F2的表达(*p0.05),干扰效率为78%;E2F2沉默后,RASFs的增殖速率降低(*p0.05),迁移能力显著降低(*p0.05),同时其侵袭能力也显著下降(**p0.01)。2、IL-6、TNF-α、LPS分别刺激RASFs,E2F2在mRNA以及蛋白水平的表达均显著增加,且对三种促炎因子均有时间和浓度依赖性(*p0.05,**p0.01);ERK信号通路抑制剂PD98059能够显著逆转LPS对E2F2的诱导(*p0.05,**p0.01),而NF-κB信号通路抑制剂PDTC能够显著逆转IL-6和TNF-α对E2F2的诱导(*p0.05,**p0.01)。以上结果说明,促炎因子刺激能够诱导RASFs中E2F2的表达,其中LPS通过ERK信号通路、IL-6和TNF-α通过NF-κB信号通路诱导E2F2的表达。进一步的ChIP结果显示NF-κB能够与E2F2的启动子区直接结合,验证了以上结果。3、TNF-α,IL-6,LPS均能够促进E2F2入核,其中IL-6作用最显著。E2F2沉默后RASFs中一些与炎症、侵袭密切相关的基因包括TNF-α、IL-1α、IL-1β、IL-6、前列腺素E2(prostaglandin E2,PGE2)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)、MMP9和MMP13的表达均降低(*p0.05,**p0.01)。ELISA结果证实炎症因子TNF-α,IL-1α,IL-1β,IL-6分泌显著降低(*p0.05,**p0.01)。沉默E2F2可以逆转TNF-α对RASFs中IL-6的诱导;ELISA结果显示RA血浆中E2F2与TNF-α(r2=0.6142,P=0.0005)、IL-6(r2=0.5940,P=0.0008)均呈较强的线性关系。此外,ChIP结果显示E2F2能够与IL-6的启动子区直接结合。结论:E2F2在RA滑膜组织中高表达,并参与RA滑膜成纤维细胞的异常增殖、迁移、侵袭,促进细胞因子的产生。在炎症条件下,炎性因子TNF-α、LPS、IL-6均可诱导RASFs中E2F2的表达;我们还发现,在RASFs中存在NF-κB/E2F2/IL-6之间的正反馈循环。说明E2F2参与介导RA中的炎症。总之,本研究证实E2F2在RA的炎症、滑膜增生等病理过程中起着重要的促进作用,是RA临床治疗的潜在靶标。
[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.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22

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