本文選題：鐵蛋白輕鏈 + RNA干擾��； 參考：《河北師范大學(xué)》2011年碩士論文

【摘要】：鐵蛋白(ferritin)作為動植物細(xì)胞內(nèi)的儲鐵蛋白,在維持鐵代謝穩(wěn)定平衡中發(fā)揮著重要的作用。近些年來,人們對于它的其他功能也有了越來越多的認(rèn)識,鐵蛋白亞基之一的鐵蛋白輕鏈(ferritin light chain, FTL)除了在鐵代謝過程中發(fā)揮儲鐵功能外,還很可能參與了炎癥的發(fā)生。 致炎因子刺激巨噬細(xì)胞可以產(chǎn)生多種炎癥介質(zhì),包括活性氧物質(zhì)(reactive oxidative species, ROS),前炎癥因子如腫瘤壞死因子-α(tumor necrosis factor-alpha, TNF-α)、白介素-1β(interleukin-1 beta, IL-1β),誘導(dǎo)型一氧化氮合酶(inductible nitric oxide synthase, iNOS)、環(huán)氧合酶-2 (cyclooxygenase-2, COX-2),細(xì)胞毒素如一氧化氮(nitric oxide, NO)、前列腺素E2(prostaglandin E2,PGE2)等。炎癥介質(zhì)的大量產(chǎn)生可導(dǎo)致各種嚴(yán)重的炎性疾病的發(fā)生。如風(fēng)濕性關(guān)節(jié)炎、膿毒癥、心血管病、帕金森及腫瘤等。炎癥介質(zhì)需要通過信號通路的參與調(diào)控其產(chǎn)生及釋放。 絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和核因子-κB (nuclear factor-kappa B, NF-κB)是參與調(diào)控炎癥介質(zhì)的產(chǎn)生及釋放的主要信號途徑。在炎性疾病的發(fā)生過程中起到重要的調(diào)控作用。 本課題研究了干擾FTL表達(dá)對炎癥發(fā)生的作用。我們首先構(gòu)建了針對FTL的短發(fā)夾RNA(shRNA)干擾載體即FTL-shRNA干擾載體,并轉(zhuǎn)染入RAW264.7細(xì)胞,篩選出穩(wěn)定表達(dá)FTL-shRNA的小鼠單核巨噬細(xì)胞RAW264.7作為實(shí)驗(yàn)?zāi)Ｐ?轉(zhuǎn)染空載pRNA-U6.1質(zhì)粒的RAW264.7作為實(shí)驗(yàn)對照。用脂多糖(lipopolysaccharide,LPS)(?)乍為炎癥誘導(dǎo)劑,運(yùn)用Western Blot、RT-PCR、和ELISA等方法技術(shù)研究了干擾FTL表達(dá)對LPS誘導(dǎo)的巨噬細(xì)胞炎癥反應(yīng)(即各種炎癥介質(zhì)的產(chǎn)生和釋放)的影響以及分子機(jī)制。 結(jié)果發(fā)現(xiàn)shRNA干擾FTL的表達(dá)增加了LPS誘導(dǎo)的RAW264.7細(xì)胞中前炎癥因子TNF-a和IL-1β的mRNA水平和釋放到細(xì)胞外的TNF-α、IL-1β的量；提高了LPS誘導(dǎo)的iNOS和COX-2的mRNA(?)口蛋白水平的增加,提高了LPS誘導(dǎo)的分泌至胞外的NO和PGE2的量。這些結(jié)果說明FTL參與了炎癥反應(yīng)并在其中發(fā)揮了重要的調(diào)節(jié)作用。MAPK和NF-κB信號通路在炎癥反應(yīng)中發(fā)揮重要作用,它們受致炎因子的激活,調(diào)控多種炎癥介質(zhì)的產(chǎn)生和表達(dá)。為了進(jìn)一步闡明FTL的調(diào)節(jié)機(jī)制,我們研究了干擾FTL表達(dá)對LPS誘導(dǎo)的MAPK和NF-κB信號通路激活的影響。結(jié)果顯示,干擾FTL表達(dá)能增強(qiáng)LPS誘導(dǎo)的MAPK家族中的ERK和JNK的磷酸化激活,并可通過增強(qiáng)LPS誘導(dǎo)的IKB磷酸化及其降解增強(qiáng)NF-κB信號通路的激活。說明FTL通過抑制LPS誘導(dǎo)的MAPK和NF-κB信號通路的激活從而抑制炎癥介質(zhì)的產(chǎn)生和釋放,進(jìn)而減弱LPS誘導(dǎo)的巨噬細(xì)胞炎癥反應(yīng)。 本論文結(jié)果對于進(jìn)一步深入研究FTL對炎癥反應(yīng)的調(diào)控,了解并掌握FTL在人體中的新功能有非常重要的意義,并為研究FTL在炎性疾病治療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 - 偽 ) , 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 - 偽 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 .
【學(xué)位授予單位】：河北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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相關(guān)期刊論文 前2條

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