RNA干扰铁蛋白轻链的表达对RAW264.7细胞炎症反应影响及其机制的研究

发布时间：2018-06-18 01:03

本文选题：铁蛋白轻链 + RNA干扰　；参考：《河北师范大学》2011年硕士论文

【摘要】：铁蛋白(ferritin)作为动植物细胞内的储铁蛋白,在维持铁代谢稳定平衡中发挥着重要的作用。近些年来,人们对于它的其他功能也有了越来越多的认识,铁蛋白亚基之一的铁蛋白轻链(ferritin light chain, FTL)除了在铁代谢过程中发挥储铁功能外,还很可能参与了炎症的发生。致炎因子刺激巨噬细胞可以产生多种炎症介质,包括活性氧物质(reactive oxidative species, ROS),前炎症因子如肿瘤坏死因子-α(tumor necrosis factor-alpha, TNF-α)、白介素-1β(interleukin-1 beta, IL-1β),诱导型一氧化氮合酶(inductible nitric oxide synthase, iNOS)、环氧合酶-2 (cyclooxygenase-2, COX-2),细胞毒素如一氧化氮(nitric oxide, NO)、前列腺素E2(prostaglandin E2,PGE2)等。炎症介质的大量产生可导致各种严重的炎性疾病的发生。如风湿性关节炎、脓毒症、心血管病、帕金森及肿瘤等。炎症介质需要通过信号通路的参与调控其产生及释放。丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和核因子-κB (nuclear factor-kappa B, NF-κB)是参与调控炎症介质的产生及释放的主要信号途径。在炎性疾病的发生过程中起到重要的调控作用。本课题研究了干扰FTL表达对炎症发生的作用。我们首先构建了针对FTL的短发夹RNA(shRNA)干扰载体即FTL-shRNA干扰载体,并转染入RAW264.7细胞,筛选出稳定表达FTL-shRNA的小鼠单核巨噬细胞RAW264.7作为实验模型,转染空载pRNA-U6.1质粒的RAW264.7作为实验对照。用脂多糖(lipopolysaccharide,LPS)(?)乍为炎症诱导剂,运用Western Blot、RT-PCR、和ELISA等方法技术研究了干扰FTL表达对LPS诱导的巨噬细胞炎症反应(即各种炎症介质的产生和释放)的影响以及分子机制。结果发现shRNA干扰FTL的表达增加了LPS诱导的RAW264.7细胞中前炎症因子TNF-a和IL-1β的mRNA水平和释放到细胞外的TNF-α、IL-1β的量；提高了LPS诱导的iNOS和COX-2的mRNA(?)口蛋白水平的增加,提高了LPS诱导的分泌至胞外的NO和PGE2的量。这些结果说明FTL参与了炎症反应并在其中发挥了重要的调节作用。MAPK和NF-κB信号通路在炎症反应中发挥重要作用,它们受致炎因子的激活,调控多种炎症介质的产生和表达。为了进一步阐明FTL的调节机制,我们研究了干扰FTL表达对LPS诱导的MAPK和NF-κB信号通路激活的影响。结果显示,干扰FTL表达能增强LPS诱导的MAPK家族中的ERK和JNK的磷酸化激活,并可通过增强LPS诱导的IKB磷酸化及其降解增强NF-κB信号通路的激活。说明FTL通过抑制LPS诱导的MAPK和NF-κB信号通路的激活从而抑制炎症介质的产生和释放,进而减弱LPS诱导的巨噬细胞炎症反应。本论文结果对于进一步深入研究FTL对炎症反应的调控,了解并掌握FTL在人体中的新功能有非常重要的意义,并为研究FTL在炎性疾病治疗中的作用提供新思路。
[Abstract]:Iron - protein plays an important role in maintaining iron metabolism and stability . In recent years , there has been a growing recognition of its other functions . The ferritin light chain ( FTL ) , one of the ferritin subunits , is likely to be involved in inflammation in addition to its iron - storing function during iron metabolism .

inflammatory mediators stimulate macrophages to produce various inflammatory mediators , including reactive oxygen species ( ros ) , pro - inflammatory factors such as tumor necrosis factor - alpha ( TNF - 伪 ) , interleukin - 1 beta ( IL - 1尾 ) , inducible nitric oxide synthase ( iNOS ) , cyclooxygenase - 2 ( COX - 2 ) , cellular toxins such as nitric oxide ( NO ) , prostaglandin E2 ( prostaglandin E2 , PGE2 ) , etc .

mitogen - activated protein kinase ( MAPK ) and nuclear factor - 魏B ( NF - 魏B ) play an important role in regulating the production and release of inflammatory mediators .

In this study , we studied the effect of interfering FTL expression on inflammation . We constructed a short hairpin RNA interference vector ( FTL - shRNA ) interference vector for FTL , transfected into RAW264.7 cells , screened a mouse mononuclear macrophage RAW264.7 stably expressing FTL - shRNA as an experimental model , and transfected with RAW264.7 of no - load pRNA - 6.1 plasmid as an experimental control . The effects of interfering FTL on LPS - induced inflammatory response of macrophages ( i.e . , the production and release of various inflammatory mediators ) and the molecular mechanism were studied by Western Blot , RT - PCR and ELISA .

It was found that the expression of shRNA interfering with FTL increased the mRNA levels of pro - inflammatory cytokines TNF - a and IL - 1尾 in RAW264.7 cells induced by LPS and the amount of TNF - 伪 and IL - 1尾 released outside the cells .
The expression of MAPK and NF - 魏B signaling pathway in LPS - induced MAPK and NF - 魏B signaling pathway was enhanced by inhibiting LPS - induced phosphorylation of MAPK and NF - 魏B signaling pathway .

The results of this paper are very important to further study the regulation , understanding and control of FTL in inflammatory response , and to provide a new idea for the study of the role of FTL in the treatment of inflammatory diseases .
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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