Rab蛋白调控巨噬细胞中TLR9信号转导通路的研究
发布时间：2018-09-18 08:42
【摘要】：TLRs是一类重要的模式识别受体,天然免疫细胞通过识别病原微生物中保守的病原体相关分子模式,产生细胞因子和趋化因子,启动天然免疫应答,从而构成了机体免疫反应的第一道防线。然而,TLRs的活化异常或者过分活化,可能使自身免疫疾病恶化,产生如系统性红斑狼疮、内毒素休克和自身免疫等疾病。因此,TLRs信号通路的活化必须得到严密控制。目的：构建Rab5a和Rab9真核表达载体,建立稳定表达Rab5a及其突变体Rab5aN133I的巨噬细胞系,研究Rab5a及其突变体、Rab7及其突变体过表达后对CpG刺激的巨噬细胞中细胞因子的影响。方法：以小鼠巨噬细胞RAW264.7的cDNA为模板,根据GenBank中公布的小鼠Rab5a和Rab9全长序列设计引物,扩增Rab5a和Rab9的开放阅读框,并与真核表达载体pcDNA3.1/Flag(-)B连接,转化后挑取阳性克隆,酶切鉴定后测序。通过脂质体法(?)(?)Rab5a及其突变体Rab5aN133I的真核表达载体转染到RAW264.7细胞中,用G418选择性培养基进行筛选,建立稳定表达Rab5a、Rab5aN133I的细胞系。通过RT-PCR, Real time-PCR和Western Blot方法鉴定稳定表达的细胞系。用CpG刺激稳定表达Rab5a、Rab5aN133I、Rab7及Rab7T22N的细胞系8小时后检测细胞因子的表达量变化。结果：将Rab5a和Rab9的阳性克隆测序后与GeneBank中报道序列的同源性为100%和99%,均不存在突变。RAW264.7转染Rab5a和Rab7真核表达载体后Rab5a和Rab7的表达量明显高于对照质粒转染细胞。Rab5a过表达后,在CpG的刺激作用下巨噬细胞分泌的TNF-α、IL-1β和IFN-β明显升高,而Rab5aN133I过表达后上述细胞因子的分泌均有所恢复。CpG刺激过表达Rab7的细胞,其分泌的IL-6、IL-10及IFN-β显著降低,而Rab7T22N过表达后却促进了上述细胞因子的增殖。结论：成功构建Rab5a和Rab9真核表达载体。建立稳定表达Rab5a、Rab5aN133I的细胞系。Rab5a可能是TLR9信号通路的正向调控蛋白,而Rab7可能是负向调控TLR9信号通路的蛋白。本研究为进一步了解Rab蛋白在TLRs信号转导中的作用奠定了基础。
[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- 伪 -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.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：Q25;R392.12
本文编号：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- 伪 -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.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：Q25;R392.12
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