Rab蛋白調控巨噬細胞中TLR9信號轉導通路的研究
發(fā)布時間：2018-09-18 08:42
【摘要】：TLRs是一類重要的模式識別受體,天然免疫細胞通過識別病原微生物中保守的病原體相關分子模式,產生細胞因子和趨化因子,啟動天然免疫應答,從而構成了機體免疫反應的第一道防線。然而,TLRs的活化異�；蛘哌^分活化,可能使自身免疫疾病惡化,產生如系統(tǒng)性紅斑狼瘡、內毒素休克和自身免疫等疾病。因此,TLRs信號通路的活化必須得到嚴密控制。目的：構建Rab5a和Rab9真核表達載體,建立穩(wěn)定表達Rab5a及其突變體Rab5aN133I的巨噬細胞系,研究Rab5a及其突變體、Rab7及其突變體過表達后對CpG刺激的巨噬細胞中細胞因子的影響。方法：以小鼠巨噬細胞RAW264.7的cDNA為模板,根據GenBank中公布的小鼠Rab5a和Rab9全長序列設計引物,擴增Rab5a和Rab9的開放閱讀框,并與真核表達載體pcDNA3.1/Flag(-)B連接,轉化后挑取陽性克隆,酶切鑒定后測序。通過脂質體法(?)(?)Rab5a及其突變體Rab5aN133I的真核表達載體轉染到RAW264.7細胞中,用G418選擇性培養(yǎng)基進行篩選,建立穩(wěn)定表達Rab5a、Rab5aN133I的細胞系。通過RT-PCR, Real time-PCR和Western Blot方法鑒定穩(wěn)定表達的細胞系。用CpG刺激穩(wěn)定表達Rab5a、Rab5aN133I、Rab7及Rab7T22N的細胞系8小時后檢測細胞因子的表達量變化。結果：將Rab5a和Rab9的陽性克隆測序后與GeneBank中報道序列的同源性為100%和99%,均不存在突變。RAW264.7轉染Rab5a和Rab7真核表達載體后Rab5a和Rab7的表達量明顯高于對照質粒轉染細胞。Rab5a過表達后,在CpG的刺激作用下巨噬細胞分泌的TNF-α、IL-1β和IFN-β明顯升高,而Rab5aN133I過表達后上述細胞因子的分泌均有所恢復。CpG刺激過表達Rab7的細胞,其分泌的IL-6、IL-10及IFN-β顯著降低,而Rab7T22N過表達后卻促進了上述細胞因子的增殖。結論：成功構建Rab5a和Rab9真核表達載體。建立穩(wěn)定表達Rab5a、Rab5aN133I的細胞系。Rab5a可能是TLR9信號通路的正向調控蛋白,而Rab7可能是負向調控TLR9信號通路的蛋白。本研究為進一步了解Rab蛋白在TLRs信號轉導中的作用奠定了基礎。
[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.
【學位授予單位】：內蒙古農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：Q25;R392.12
本文編號：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.
【學位授予單位】：內蒙古農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：Q25;R392.12
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