树突状细胞内源性递呈途径相关Rab分子的鉴定研究
发布时间：2018-11-12 18:25
【摘要】： 树突状细胞(dendritic cell, DC)是目前所知的体内功能最强的专职抗原递呈细胞。通过MHC I类内源性抗原递呈途径,它不仅能够递呈病毒抗原,有效活化初始性CD8+T细胞,启动抗病毒免疫,同时此过程也是机体诱导自身抗原外周及中枢耐受的重要机制。 关于内源性抗原递呈途径的研究数据大多来源于B细胞,目前认为内源性表达蛋白在胞浆生成后,可被蛋白酶体消化降解成8-10个氨基酸的多肽,经抗原加工相关转运体(TAP)转运进入内质网(endoplasmic reticulum, ER),在众多分子伴侣如钙联蛋白(calnexin)、钙网蛋白(calreticulin)、tapasin等的帮助下,与新生成的MHC I类分子形成稳定复合物,通过高尔基体转运至细胞膜,递呈给CD8+T细胞。此过程可视为一个连续囊泡转运过程,而关于此过程涉及的囊泡转运机制,我们现在还所知甚少。 细胞内膜性结构的转运过程包括:囊泡的形成,囊泡的移动,囊泡与靶膜的拴缚(tethering)。近来细胞生物学研究表明,此生物学过程受到Rab蛋白家族及其效应分子的调节,并且不同Rab蛋白高度局限性地定位在各种囊泡的膜表面,故可被用于描述特定的内吞体系和特定的转运途径。例如:Rab4可作为早期内吞体的标志,Rab5调节笼形蛋白(clathrin)衣被小泡在质膜和早期内吞体间的转运,Rab7则与晚期内吞体的形成、转运密切相关。因此,Rab分子可以作为“路标”来描述抗原呈递途径中的复杂转运过程。同时,国外及我室相关实验提示某些Rab蛋白与抗原递呈相关,故进一步鉴定与DC内源抗原递呈囊泡转运相关的Rab蛋白,研究这些Rab蛋白与DC内源抗原递呈的相关性具有重要意义,可为理解DC内源抗原递呈的分子调节机制奠定基础。 近年大规模siRNA干扰技术的进展,为系统性研究与DC内源性抗原递呈相关的Rab蛋白提供了可能。本研究利用基于慢病毒的siRNA干扰技术,构建了针对57个Rab家族蛋白的siRNA慢病毒库。在建立的DC内源性抗原递呈检测系统基础上,发展了大规模筛选策略,从而观察干扰Rab家族蛋白表达对DC内源性抗原递呈的影响。结果显示一些Rab分子(Rab1B, Rab4A, Rab5B, Rab5C, Rab8A, Rab9,Rab11A,Rab19,Rab24)与DC内源递呈相关。 结合这些Rab分子的特殊胞内定位特征,我们认为:(1)干扰Rab1B、Rab8A,Rab24这些控制胞内物质由高尔基体往胞膜上转运的Rab蛋白,能够抑制DC细胞的内源抗原递呈,这与经典的内源性抗原递呈理论一致;(2)Rab4A, Rab5B, Rab5C,Rab11A主要是控制早期内吞体与胞膜间转运的Rab蛋白, Rab9则主要与晚期内吞体的转运相关,干扰它们的表达抑制DC细胞的内源抗原递呈,提示DC的内源递呈途径可能存在经典途径以外的其他途径。(3)Rab19蛋白功能目前研究并不清楚,可能在DC内源抗原递呈中也发挥一定作用。以上结果为进一步解释DC内源抗原递呈可能机制及Rab蛋白功能奠定了重要基础。 基于以上研究,我们推测DC细胞可能存在区别于经典内源性抗原递呈的其他途径,并与早期内吞体相关。据此我们选择与早期内吞体回运相关的Rab4蛋白进行进一步验证siRNA干扰库筛选的结果。 为证实上述推测,我们首先采用化学合成siRNA转染DC-OVA细胞,发现瞬时下调Rab4基因表达对DC内源抗原递呈的抑制作用更为明显,并且转染细胞表面Kb分子也有一定程度下调。随后,我们构建了Rab4野生型,活化型和抑制型突变体的慢病毒表达载体,转染DC-OVA细胞后,抗原递呈检测结果显示其抑制型的突变体可明显抑制DC内源性抗原递呈,而活性突变体及野生型蛋白则对DC内源性抗原递呈具有促进作用。同时,我们采用共聚焦显微镜观察突变体蛋白在细胞中的表达情况,结果发现Rab4蛋白主要分布在细胞胞膜周围的囊泡结构中,而对照蛋白则呈弥散表达,野生型和活化型突变体Rab4Q67L主要聚集在胞膜下囊泡中,抑制型突变体Rab4S22N则主要聚集表达在核周囊泡中。这些结果与库筛选的结果一致,并且说明Rab4主要通过其活性的变化和定位改变参与内源性抗原递呈的调节。 Rab4蛋白作为早期内吞体的标志,与Rab5B, Rab5C,Rab11A一起调节早期内吞体与质膜之间的转运,它们对于DC内源性抗原递呈的影响提示,DC内源性抗原递呈胞内囊泡的转运可能与早期内吞体有关,因此可能与外源抗原的交叉递呈具有共同途径,而Rab蛋白很可能因此在DC内源性抗原递呈与外源抗原的交叉递呈的调节中,起着关键性作用,为今后调节抗原递呈从而有效激活肿瘤或感染中细胞免疫应答提供了靶标。 总之,本研究利用针对Rab家族蛋白的siRNA库筛选出与DC内源抗原递呈相关的Rab蛋白,选择Rab4蛋白对库筛选的结果进行进一步的验证,并提示Rab4蛋白的活性参与DC细胞内源性抗原递呈。然而,有关DC内源性抗原递呈的途径以及相关Rab蛋白的调节机制,都有待进一步的研究证实。
[Abstract]:Dendritic cells (DC) are the most powerful antigen-presenting cells with the strongest in vivo function. By the way of MHC class I endogenous antigen presenting, it is not only capable of presenting the virus antigen, effectively activating the initial CD8 + T cell, and starting the antiviral immunity, and the process is also an important mechanism for the body to induce the peripheral and central tolerance of the autoantigen. Most of the data on the pathway of endogenous antigen presentation are derived from B cells, and it is believed that the endogenous expression protein can be degraded into 8-10 amino acids by the proteasome digestion after the cytoplasm is generated, and the endoplasmic reticulum (endoplasmic reticulum) can be transported into the endoplasmic reticulum through the trans-processing related transporter (TAP). and with the help of a plurality of molecular chaperones such as calcinin, calreticulin, tapasin and the like, a stable complex is formed with newly generated MHC class I molecules, CD8 + T cells. This process can be seen as a continuous vesicle transport process, and the vesicle transport mechanism involved in this process, we It is also known that the transport of the endometrial structure involves the formation of the vesicles, the movement of the vesicles, the vesicle and the target, Recent cell biology studies have shown that this biological process is regulated by the Rab protein family and its effector molecules, and the different Rab proteins are highly localized on the membrane surface of the various vesicles and can therefore be used to describe Specific endocytosis and specific transport pathways. For example, Rab4 can be used as a marker for early endocytosis, and Rab5 regulates the transport of the cage-shaped proteins between the plasma membrane and the early endocytosis, Rab7 is associated with The formation and transport of the late endocytosis are closely related. Thus, the Rab molecules can be traced as a 鈥渞oad sign鈥，
本文编号：2327861
[Abstract]:Dendritic cells (DC) are the most powerful antigen-presenting cells with the strongest in vivo function. By the way of MHC class I endogenous antigen presenting, it is not only capable of presenting the virus antigen, effectively activating the initial CD8 + T cell, and starting the antiviral immunity, and the process is also an important mechanism for the body to induce the peripheral and central tolerance of the autoantigen. Most of the data on the pathway of endogenous antigen presentation are derived from B cells, and it is believed that the endogenous expression protein can be degraded into 8-10 amino acids by the proteasome digestion after the cytoplasm is generated, and the endoplasmic reticulum (endoplasmic reticulum) can be transported into the endoplasmic reticulum through the trans-processing related transporter (TAP). and with the help of a plurality of molecular chaperones such as calcinin, calreticulin, tapasin and the like, a stable complex is formed with newly generated MHC class I molecules, CD8 + T cells. This process can be seen as a continuous vesicle transport process, and the vesicle transport mechanism involved in this process, we It is also known that the transport of the endometrial structure involves the formation of the vesicles, the movement of the vesicles, the vesicle and the target, Recent cell biology studies have shown that this biological process is regulated by the Rab protein family and its effector molecules, and the different Rab proteins are highly localized on the membrane surface of the various vesicles and can therefore be used to describe Specific endocytosis and specific transport pathways. For example, Rab4 can be used as a marker for early endocytosis, and Rab5 regulates the transport of the cage-shaped proteins between the plasma membrane and the early endocytosis, Rab7 is associated with The formation and transport of the late endocytosis are closely related. Thus, the Rab molecules can be traced as a 鈥渞oad sign鈥，
本文编号：2327861
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