Rab蛋白調(diào)控巨噬細(xì)胞中TLR9信號(hào)轉(zhuǎn)導(dǎo)通路的研究
發(fā)布時(shí)間：2018-09-18 08:42
【摘要】：TLRs是一類(lèi)重要的模式識(shí)別受體,天然免疫細(xì)胞通過(guò)識(shí)別病原微生物中保守的病原體相關(guān)分子模式,產(chǎn)生細(xì)胞因子和趨化因子,啟動(dòng)天然免疫應(yīng)答,從而構(gòu)成了機(jī)體免疫反應(yīng)的第一道防線(xiàn)。然而,TLRs的活化異�；蛘哌^(guò)分活化,可能使自身免疫疾病惡化,產(chǎn)生如系統(tǒng)性紅斑狼瘡、內(nèi)毒素休克和自身免疫等疾病。因此,TLRs信號(hào)通路的活化必須得到嚴(yán)密控制。目的：構(gòu)建Rab5a和Rab9真核表達(dá)載體,建立穩(wěn)定表達(dá)Rab5a及其突變體Rab5aN133I的巨噬細(xì)胞系,研究Rab5a及其突變體、Rab7及其突變體過(guò)表達(dá)后對(duì)CpG刺激的巨噬細(xì)胞中細(xì)胞因子的影響。方法：以小鼠巨噬細(xì)胞RAW264.7的cDNA為模板,根據(jù)GenBank中公布的小鼠Rab5a和Rab9全長(zhǎng)序列設(shè)計(jì)引物,擴(kuò)增Rab5a和Rab9的開(kāi)放閱讀框,并與真核表達(dá)載體pcDNA3.1/Flag(-)B連接,轉(zhuǎn)化后挑取陽(yáng)性克隆,酶切鑒定后測(cè)序。通過(guò)脂質(zhì)體法(?)(?)Rab5a及其突變體Rab5aN133I的真核表達(dá)載體轉(zhuǎn)染到RAW264.7細(xì)胞中,用G418選擇性培養(yǎng)基進(jìn)行篩選,建立穩(wěn)定表達(dá)Rab5a、Rab5aN133I的細(xì)胞系。通過(guò)RT-PCR, Real time-PCR和Western Blot方法鑒定穩(wěn)定表達(dá)的細(xì)胞系。用CpG刺激穩(wěn)定表達(dá)Rab5a、Rab5aN133I、Rab7及Rab7T22N的細(xì)胞系8小時(shí)后檢測(cè)細(xì)胞因子的表達(dá)量變化。結(jié)果：將Rab5a和Rab9的陽(yáng)性克隆測(cè)序后與GeneBank中報(bào)道序列的同源性為100%和99%,均不存在突變。RAW264.7轉(zhuǎn)染Rab5a和Rab7真核表達(dá)載體后Rab5a和Rab7的表達(dá)量明顯高于對(duì)照質(zhì)粒轉(zhuǎn)染細(xì)胞。Rab5a過(guò)表達(dá)后,在CpG的刺激作用下巨噬細(xì)胞分泌的TNF-α、IL-1β和IFN-β明顯升高,而Rab5aN133I過(guò)表達(dá)后上述細(xì)胞因子的分泌均有所恢復(fù)。CpG刺激過(guò)表達(dá)Rab7的細(xì)胞,其分泌的IL-6、IL-10及IFN-β顯著降低,而Rab7T22N過(guò)表達(dá)后卻促進(jìn)了上述細(xì)胞因子的增殖。結(jié)論：成功構(gòu)建Rab5a和Rab9真核表達(dá)載體。建立穩(wěn)定表達(dá)Rab5a、Rab5aN133I的細(xì)胞系。Rab5a可能是TLR9信號(hào)通路的正向調(diào)控蛋白,而Rab7可能是負(fù)向調(diào)控TLR9信號(hào)通路的蛋白。本研究為進(jìn)一步了解Rab蛋白在TLRs信號(hào)轉(zhuǎn)導(dǎo)中的作用奠定了基礎(chǔ)。
[Abstract]:TLRs is an important type of pattern recognition receptor. Innate immune cells generate cytokines and chemokines by identifying conserved pathogen-associated molecular patterns in pathogenic microorganisms and initiate innate immune responses. This constitutes the body's immune response to the first line of defense. However abnormal or excessive activation of TLRs may worsen autoimmune diseases such as systemic lupus erythematosus endotoxic shock and autoimmune diseases. Therefore, the activation of TLRs signaling pathway must be strictly controlled. Aim: to construct eukaryotic expression vectors of Rab5a and Rab9, and to establish macrophage cell lines stably expressing Rab5a and its mutant Rab5aN133I, and to study the effect of overexpression of Rab5a and its mutant, Rab9, on cytokines in CpG stimulated macrophages. Methods: using the cDNA of mouse macrophage RAW264.7 as template, primers were designed according to the full-length sequence of mouse Rab5a and Rab9 published in GenBank. The open reading frame of Rab5a and Rab9 was amplified and ligated with the eukaryotic expression vector pcDNA3.1/Flag (-) B. The results were confirmed by enzyme digestion and sequenced. The eukaryotic expression vector of Rab5a and its mutant Rab5aN133I were transfected into RAW264.7 cells by liposome method. The cell lines stably expressing Rab5a,Rab5aN133I were established by G418 selective medium. The stably expressed cell lines were identified by RT-PCR, Real time-PCR and Western Blot. The expression of cytokines in cell lines which expressed Rab5a,Rab5aN133I,Rab7 and Rab7T22N stably by CpG was detected after 8 hours. Results: the homology of the positive clones of Rab5a and Rab9 was 100% and 99% with the reported sequences in GeneBank. The expression of Rab5a and Rab7 in the eukaryotic expression vector of Rab5a and Rab7 was significantly higher than that of the control plasmid transfected with. Rab5a. The secretion of TNF- 偽 -1 尾 and IFN- 尾 by macrophages stimulated by CpG was significantly increased, while the secretion of these cytokines recovered after Rab5aN133I overexpression. The IL-6,IL-10 and IFN- 尾 secreted by the cells stimulated by Rab5aN133I were significantly decreased. However, Rab7T22N overexpression promoted the proliferation of these cytokines. Conclusion: the eukaryotic expression vectors of Rab5a and Rab9 were successfully constructed. The establishment of a cell line with stable expression of Rab5a,Rab5aN133I. Rab5a may be a positive regulatory protein for the TLR9 signaling pathway, while Rab7 may be a protein that negatively regulates the TLR9 signaling pathway. This study laid a foundation for further understanding the role of Rab protein in TLRs signal transduction.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：Q25;R392.12
本文編號(hào)：2247368
[Abstract]:TLRs is an important type of pattern recognition receptor. Innate immune cells generate cytokines and chemokines by identifying conserved pathogen-associated molecular patterns in pathogenic microorganisms and initiate innate immune responses. This constitutes the body's immune response to the first line of defense. However abnormal or excessive activation of TLRs may worsen autoimmune diseases such as systemic lupus erythematosus endotoxic shock and autoimmune diseases. Therefore, the activation of TLRs signaling pathway must be strictly controlled. Aim: to construct eukaryotic expression vectors of Rab5a and Rab9, and to establish macrophage cell lines stably expressing Rab5a and its mutant Rab5aN133I, and to study the effect of overexpression of Rab5a and its mutant, Rab9, on cytokines in CpG stimulated macrophages. Methods: using the cDNA of mouse macrophage RAW264.7 as template, primers were designed according to the full-length sequence of mouse Rab5a and Rab9 published in GenBank. The open reading frame of Rab5a and Rab9 was amplified and ligated with the eukaryotic expression vector pcDNA3.1/Flag (-) B. The results were confirmed by enzyme digestion and sequenced. The eukaryotic expression vector of Rab5a and its mutant Rab5aN133I were transfected into RAW264.7 cells by liposome method. The cell lines stably expressing Rab5a,Rab5aN133I were established by G418 selective medium. The stably expressed cell lines were identified by RT-PCR, Real time-PCR and Western Blot. The expression of cytokines in cell lines which expressed Rab5a,Rab5aN133I,Rab7 and Rab7T22N stably by CpG was detected after 8 hours. Results: the homology of the positive clones of Rab5a and Rab9 was 100% and 99% with the reported sequences in GeneBank. The expression of Rab5a and Rab7 in the eukaryotic expression vector of Rab5a and Rab7 was significantly higher than that of the control plasmid transfected with. Rab5a. The secretion of TNF- 偽 -1 尾 and IFN- 尾 by macrophages stimulated by CpG was significantly increased, while the secretion of these cytokines recovered after Rab5aN133I overexpression. The IL-6,IL-10 and IFN- 尾 secreted by the cells stimulated by Rab5aN133I were significantly decreased. However, Rab7T22N overexpression promoted the proliferation of these cytokines. Conclusion: the eukaryotic expression vectors of Rab5a and Rab9 were successfully constructed. The establishment of a cell line with stable expression of Rab5a,Rab5aN133I. Rab5a may be a positive regulatory protein for the TLR9 signaling pathway, while Rab7 may be a protein that negatively regulates the TLR9 signaling pathway. This study laid a foundation for further understanding the role of Rab protein in TLRs signal transduction.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：Q25;R392.12
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相關(guān)期刊論文 前4條
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