本文選題：miR-322 + NF-κB1��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：炎癥反應(yīng)是機(jī)體抵抗病原入侵,消滅病原體的正常自我保護(hù)機(jī)制,然而過度的炎癥反應(yīng)會(huì)導(dǎo)致機(jī)體損傷,導(dǎo)致相關(guān)免疫性疾病的發(fā)生,對(duì)炎癥反應(yīng)的精確調(diào)控顯得尤為重要。微小RNA(mi RNA)是一段長(zhǎng)為19~24nt的非編碼小RNA分子,在進(jìn)化過程中高度保守。mi RNA通過與靶基因m RNA 3'UTR完全或部分結(jié)合,對(duì)靶m RNA剪切降解或抑制其翻譯,mi RNA介導(dǎo)的轉(zhuǎn)錄后水平在炎癥反應(yīng)和免疫調(diào)節(jié)中發(fā)揮重要作用。mmu-mi R-322與人mi R-424同源,是mi R-322家族的重要成員,mi R-322在多種疾病情況下差異表達(dá),且在不同組織細(xì)胞中具有高度的保守性。研究表明,mi R-322在細(xì)胞增殖、細(xì)胞分化等生物學(xué)過程中發(fā)揮重要作用,但mi R-322在免疫調(diào)節(jié)中的作用及生物學(xué)功能尚待進(jìn)一步研究。因此,本研究通過分析炎癥反應(yīng)與mi R-322的關(guān)系;研究mi R-322在巨噬細(xì)胞增殖分化中的作用;預(yù)測(cè)mi R-322靶基因并揭示其相互作用,闡明mi R-322在炎性反應(yīng)中的調(diào)控作用,為揭示其免疫調(diào)控功能奠定基礎(chǔ)。具體研究方法如下:q PCR檢測(cè)mi R-322的表達(dá)變化:LPS刺激RAW264.7細(xì)胞,q PCR檢測(cè)mi R-322的表達(dá),結(jié)果顯示,LPS刺激后mi R-322表達(dá)下調(diào),且具有時(shí)間依賴性,提示mi R-322可能參與了炎癥反應(yīng)的調(diào)控。mi R-322對(duì)炎癥反應(yīng)的影響:將mi R-322 mimics轉(zhuǎn)染RAW264.7細(xì)胞,轉(zhuǎn)染24h后LPS刺激,q PCR檢測(cè)IL-1β、IL-6、TNF-α的表達(dá),結(jié)果顯示,LPS刺激后IL-1β、IL-6、TNF-α大量表達(dá),而在轉(zhuǎn)染mi R-322后IL-1β、IL-6、TNF-α的表達(dá)被顯著抑制。由此表明,mi R-322負(fù)調(diào)控LPS誘導(dǎo)的炎癥反應(yīng)。mi R-322對(duì)LPS介導(dǎo)的巨噬細(xì)胞增殖的影響:將mi R-322 mimics轉(zhuǎn)染RAW264.7細(xì)胞,轉(zhuǎn)染24h后LPS刺激,q PCR檢測(cè)細(xì)胞周期蛋白cyclin D、cyclin E、P21、P27的表達(dá),結(jié)果顯示,LPS刺激后cyclin D、cyclin E顯著下調(diào),P21、P27顯著上調(diào),說明LPS刺激導(dǎo)致細(xì)胞周期紊亂,抑制了細(xì)胞增殖,而在轉(zhuǎn)染mi R-322后,LPS誘導(dǎo)的cyclin D、cyclin E下調(diào)以及P21、P27上調(diào)被明顯抑制,表明mi R-322能夠影響細(xì)胞周期進(jìn)程,隨后利用流式細(xì)胞技術(shù)進(jìn)一步驗(yàn)證發(fā)現(xiàn)mi R-322能夠促進(jìn)巨噬細(xì)胞周期進(jìn)程,進(jìn)而促進(jìn)細(xì)胞增殖,緩解細(xì)胞損傷。利用MTT法檢測(cè)活細(xì)胞數(shù),同樣證明了該結(jié)果,即LPS刺激后,活細(xì)胞數(shù)顯著減少,細(xì)胞損傷嚴(yán)重,而在轉(zhuǎn)染mi R-322后細(xì)胞損傷得到緩解,活細(xì)胞數(shù)明顯增加。以上結(jié)果共同表明,mi R-322能夠促進(jìn)巨噬細(xì)胞增殖,緩解炎性損傷。靶基因的預(yù)測(cè):利用mi RNA靶基因預(yù)測(cè)軟件(mi Randa、targetscan)預(yù)測(cè)mi R-322靶基因,發(fā)現(xiàn)mi R-322能夠與NF-κB1互補(bǔ)結(jié)合,提示NF-κB1可能是mi R-322的靶基因。mi R-322靶基因的驗(yàn)證:將NF-κB1 3'UTR克隆至雙熒光素酶質(zhì)粒上,構(gòu)建雙熒光素酶報(bào)告載體以及突變體,將其與mi R-322 mimics(模擬物)以及mi R-322 inhibitors(抑制劑)共轉(zhuǎn)染至293T細(xì)胞,檢測(cè)熒光素酶活性,結(jié)果顯示,mi R-322轉(zhuǎn)染組熒光素酶活性明顯下調(diào),且差異顯著(P0.0001),mi R-322 inhibitors組熒光素酶活性顯著上調(diào),差異顯著(P0.05),表明mi R-322能夠與NF-κB1相互作用。隨后利用q PCR以及western blot技術(shù)檢測(cè)mi R-322對(duì)NF-κB1 m RNA及蛋白水平表達(dá)的影響,結(jié)果顯示轉(zhuǎn)染mi R-322 mimics后NF-κB1 m RNA及蛋白水平均明顯下調(diào),轉(zhuǎn)染mi R-322 inhibitors后結(jié)果相反。表明NF-κB1是mi R-322的靶基因,并且通過mi R-322通過降解NF-κB1 m RNA來實(shí)現(xiàn)對(duì)NF-κB1的表達(dá)抑制。結(jié)論:mi R-322通過靶向抑制NF-κB1表達(dá)負(fù)調(diào)控LPS介導(dǎo)的炎癥反應(yīng),并且mi R-322能促進(jìn)細(xì)胞增殖,緩解炎性損傷。
[Abstract]:Inflammation is the body's resistance to pathogen invasion, destroy pathogens normal self protection mechanism, but the excessive inflammatory reaction will cause body damage, lead to autoimmune diseases, precise regulation of inflammatory responses is particularly important. The tiny RNA (MI RNA) is a 19~24nt encoding RNA in non small molecules. The evolutionary process is highly conserved.Mi RNA binding with target gene m RNA 3'UTR completely or partially, translation of target m RNA shear degradation or inhibit the transcription of MI, RNA mediated level in inflammation and immune regulation play an important role in.Mmu-mi R-322 and MI R-424 homology, is an important member of MI R-322 family mi R-322, in a variety of diseases under the condition of differential expression, and is highly conserved in different tissues. The results show that MI R-322 play an important role in cell proliferation, cell differentiation and other biological processes, but Mi R-322 still needs further research on the effects of immune regulation and biological function. Therefore, this research through the analysis on the relationship between inflammation and MI R-322; study the role of MI R-322 in the proliferation and differentiation of macrophages; MI prediction of R-322 target genes and reveal the interaction, clarify the regulatory role of MI R-322 in inflammatory reaction. Lay a foundation for revealing its immune regulation function. Specific research methods are as follows: to examine the expression of MI R-322 Q PCR: LPS stimulation of RAW264.7 cells, the expression of MI, R-322 Q PCR detection showed that LPS stimulated mi R-322 expression, which is time-dependent effect of R-322 Mi regulation of.Mi R-322 may be involved in inflammation in response to inflammatory reaction: Mi R-322 mimics was transfected into RAW264.7 cells. After transfection of 24h LPS stimulation, Q PCR detection of IL-1 beta, IL-6, expression of TNF- alpha showed that after LPS stimulation of IL-1 beta, IL-6, TNF- a large number of tables Da, IL-1 in transfected mi R-322 beta, IL-6, TNF- expression was significantly inhibited. The effect of.Mi R-322 mi R-322 negatively regulates the inflammatory response induced by LPS on LPS mediated macrophage proliferation: Mi R-322 mimics was transfected into RAW264.7 cells. After transfection of 24h LPS stimulation, Q PCR detection of cell cycle protein cyclin D, cyclin E, P21, P27 expression showed that LPS stimulated cyclin D, cyclin P21, P27 E were down regulated and up-regulated, indicating that LPS stimulation leads to the disorder of cell cycle, inhibiting cell proliferation, while transfection of MI R-322, LPS induced cyclin D, cyclin and E down P21, P27 upregulation was inhibited obviously, show that MI R-322 can affect cell cycle progression, followed by a further verify that MI R-322 can promote macrophage cycle process of flow cytometry, thus promoting cell proliferation, relieve cell injury. The MTT was used to detect the number of live cells, The results also proved that, after LPS stimulation, the number of live cells was significantly reduced, serious cell damage, and relieve cell injury in MI R-322 after transfection was significantly increased, the number of live cells. These results show that MI R-322 can promote macrophage proliferation, alleviate inflammatory injury. The predicted target gene prediction software: using mi the target gene of RNA (MI Randa targetscan) mi R-322 target gene prediction, found that MI R-322 can combine with complementary NF- kappa B1, suggesting that NF- kappa B1 may be verified the target gene.Mi R-322 target gene mi R-322: the NF- kappa B1 3'UTR was cloned into the dual luciferase plasmid, construct dual luciferase reporter vector and the mutant, with the MI R-322 mimics and MI R-322 (mimics) inhibitors (inhibitor) were transfected into 293T cells. Luciferase activity was detected, results showed that MI transfected with R-322 luciferase activity was significantly reduced, and the difference was significant (P0. 0001), MI R-322 group inhibitors luciferase activity was significantly increased, significant difference (P0.05), R-322 and NF- showed that MI kappa B1 interaction. Then the use of Q PCR and western of blot mi R-322 technique to detect the expression of NF- K B1 m RNA and protein level, results showed that the R-322 mimics NF- after transfection of MI kappa B1 m RNA and protein levels were significantly reduced after inhibitors R-322, transfection of MI in contrast to the results. The results indicated that NF- K B1 is the target gene of MI R-322, and Mi through the degradation of R-322 through the RNA to achieve m NF- kappa B1 on the expression of NF- kappa B1. Conclusion: Mi inhibited the expression of R-322 negatively regulates the inflammatory reaction mediated by LPS to inhibit NF- K B1 by target, MI and R-322 can promote cell proliferation, alleviate inflammatory injury.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.3

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相關(guān)期刊論文 前1條

1 史艷暉;盧圣棟;;轉(zhuǎn)錄因子NF-κB的研究現(xiàn)狀及其應(yīng)用前景[J];中國(guó)生物工程雜志;2007年04期

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本文編號(hào)：1770822