NK细胞活化受体CD226的表达及功能研究
发布时间：2018-11-22 08:09
【摘要】： 固有免疫系统作为机体抵御外来微生物抗原入侵的第一道防线,能直接迅速的活化并对入侵机体的病原体进行清除,在获得性免疫系统肩动之前发挥了重要的屏障作用。天然杀伤细胞(natural killer cells,NK)是固有免疫系统的重要组成部分,在机体防御感染和癌症的免疫应答中起着重要作用。NK细胞的活化受到活化性与抑制性受体的共同调控,由于识别机制的多样化,近年来,NK细胞表面活化性受体得到了人们的广泛关注。 CD226分子是NK细胞表面活化性受体,近年来研究发现,CD226在NK细胞的活化并杀伤肿瘤细胞等生物学过程中发挥了重要作用,并与多种疾病的发生密切相关。虽然CD226的功能得到了人们的广泛关注,但其分子识别与活化机制仍然不是非常清楚。 本实验中,我们构建了CD226的胞外不同的结构域蛋白,包括含有N端19-129氨基酸序列的IgV样结构域ECD1(extracellular domain 1),以及含有两个IgV样结构域ECD2(extracellular domain 2)(19-243aa)。通过对两个长度不同的结构域与肿瘤细胞表面配体蛋白的相互作用进行研究,发现CD226蛋白与其配体可能的作用位点与其在肿瘤识别杀伤中的作用机理;进一步通过抗体介导的活化与蛋白阻断实验,探讨了CD226分子在NK细胞活化中的作用机制。 通过上述研究,我们初步获得了结果如下: 1.构建载体并原核表达CD226分子胞外结构域ECD1与ECD2,重组蛋白经复性纯化后,蛋白纯度达到95%以上,经Western Blot鉴定,蛋白成功表达。成功构建表达CD226胞外段蛋白的真核表达载体,经过转染并筛选获得稳定高表达可溶性CD226的CHO-K1细胞系。 2.发现CD226分子在NK细胞对肿瘤细胞识别杀伤中发挥重要作用,并且CD226分子与配体的识别与结合主要是通过胞外N端第一个结构域ECD1。蛋白结合实验发现,CD226胞外结构域可以识别并结合肿瘤细胞表面配体,单独的ECD1蛋白即可有效与靶细胞结合。CD226胞外结构域重组蛋白的加入可以有效阻断NK细胞对肿瘤细胞的杀伤,进一步研究发现,重组蛋白对效应细胞与靶细胞生长与增殖没有明显的影响,重组蛋白对杀伤的阻断主要是通过阻断NK细胞与靶细胞的结合实现的,提示CD226在NK细胞对靶细胞的识别中可能发挥了粘附分子的作用。 3.除粘附作用外,CD226传递的活化信号对NK细胞的活化有着重要的影响。抗体刺激CD226分子活化后可以引起NK细胞表面活化分子CD69的表达升高,细胞因子(IFNγ)与杀伤颗粒(CD107a,Granzyme B)的表达都明显升高。重组蛋白可以有效阻断K562引起的NK细胞的活化,包括CD69与颗粒酶B的表达降低,ERK1/2的磷酸化程度也有一定程度的降低。 上述结果证明,CD226分子作为NK细胞表面活化性受体,其与配体识别的主要位点位于胞外第一个免疫球蛋白样结构域ECD1。CD226分子传递的活化信号参与NK细胞与靶细胞的粘附,细胞因子分泌以及杀伤等多种免疫学过程,ERK1/2分子可能参与了CD226活化信号的传递。 淋巴细胞表面膜蛋白受体与炎症损伤触发密切相关,对淋巴细胞表面膜受体的研究与膜蛋白谱的描绘对研究淋巴细胞功能,维持机体的免疫平衡有着重要意义。为了能够快速有效筛选淋巴细胞表面低丰度的膜蛋白进行功能研究,在进一步工作中,我们建立了一种构建膜蛋白cDNA文库方法,并将其应用于小鼠肝脏淋巴细胞膜蛋白的特异性高通量筛选。
[Abstract]:The innate immune system, as the first line of defense for the body to resist the invasion of foreign microbial antigens, can activate and clear the pathogens of the invading organism directly, and play an important role in the protection of the immune system. Natural killer cells (NK) are an important part of the innate immune system and play an important role in the immune response of body defense infection and cancer. The activation of NK cells is co-regulated by the activation and inhibitory receptors. In recent years, the NK cell surface activating receptor has been widely concerned by the diversification of the recognition mechanism. CD226 is a receptor for NK cell surface activation. In recent years, it has been found that CD226 plays an important role in the activation of NK cells and killing of tumor cells, and has been associated with various diseases. The function of CD226 is closely related, but the molecular recognition and activation mechanism of CD226 is still not It is very clear that in this experiment we constructed the extracellular domain proteins of CD226, including the IgV-like domain ECD1 containing the amino acid sequence of the N-terminal 19-129, and the two IgV-like domains ECD2 (extacular domain 2) (19-243aa). Based on the study of the interaction of two different domains with the surface of the tumor cell surface, it was found that the role of the CD226 protein with its ligand might be related to the mechanism of its role in the identification and killing of the tumor. The experiments of activation and protein blocking were carried out, and the expression of CD226 in NK was discussed. A mechanism of action in the activation of a cell. The results are as follows: 1. Construction of the vector and prokaryotic expression of the extracellular domain ECD1 and ECD2 of the CD226, and the purity of the protein is over 95% after the renaturation and purification of the recombinant protein. Western Blot identification and successful expression of the protein. A true nuclear expression vector for expressing the extracellular domain of CD226 was successfully constructed, and a stable high-scale was obtained by transfection and screening. CHO-K1 cell line up to soluble CD226. 2. It was found that CD226 molecules play an important role in the identification and killing of tumor cells in NK cells, and the CD226 molecules and ligands It is found that the extracellular domain of the CD226 can recognize and bind to the tumor cells. the addition of the CD226 extracellular domain recombinant protein can effectively block the killing of the NK cells on the tumor cells, Histone has no obvious effect on the growth and proliferation of effector cells and target cells, and the blocking of the recombinant protein on the killing is mainly realized by blocking the combination of the NK cells and the target cells, and the prompt CD226 is NK cells may play an important role in the identification of target cells. In addition, the activation signal transferred by CD226 has an important effect on the activation of NK cells. After the antibody stimulates the activation of the CD226 molecules, the expression of the NK cell surface activating molecule CD69 can be increased, and the cytokine (IFN) and the killing can be caused. The expression of the injured particles (CD107a, Granzyme B) is significantly increased. The recombinant protein can effectively block the activation of NK cells caused by K562, including the CD69 and the particles. The expression of the enzyme B is reduced, and the phosphorylation of ERK1/ 2 is reduced to some extent. The above results show that the CD226 molecule acts as the surface activating receptor of the NK cell, and the main site for the ligand recognition is located in the first immunoglobulin-like junction outside the cell. The activation signal transferred by the molecule ECD2.CD226 is involved in the adhesion of NK cells to target cells, the secretion of cytokines and anti-killing. In many immunological processes, ERK1/ 2 may be involved in the transfer of CD226 activation signal. The study of membrane receptor and the expression of membrane protein spectrum are of great significance to study the function of lymphocytes and to maintain the immune balance of the body. In order to study the function of the membrane protein with low abundance in the surface of the lymphocytes quickly and effectively, we have set up a structure in the further work.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
本文编号：2348639
[Abstract]:The innate immune system, as the first line of defense for the body to resist the invasion of foreign microbial antigens, can activate and clear the pathogens of the invading organism directly, and play an important role in the protection of the immune system. Natural killer cells (NK) are an important part of the innate immune system and play an important role in the immune response of body defense infection and cancer. The activation of NK cells is co-regulated by the activation and inhibitory receptors. In recent years, the NK cell surface activating receptor has been widely concerned by the diversification of the recognition mechanism. CD226 is a receptor for NK cell surface activation. In recent years, it has been found that CD226 plays an important role in the activation of NK cells and killing of tumor cells, and has been associated with various diseases. The function of CD226 is closely related, but the molecular recognition and activation mechanism of CD226 is still not It is very clear that in this experiment we constructed the extracellular domain proteins of CD226, including the IgV-like domain ECD1 containing the amino acid sequence of the N-terminal 19-129, and the two IgV-like domains ECD2 (extacular domain 2) (19-243aa). Based on the study of the interaction of two different domains with the surface of the tumor cell surface, it was found that the role of the CD226 protein with its ligand might be related to the mechanism of its role in the identification and killing of the tumor. The experiments of activation and protein blocking were carried out, and the expression of CD226 in NK was discussed. A mechanism of action in the activation of a cell. The results are as follows: 1. Construction of the vector and prokaryotic expression of the extracellular domain ECD1 and ECD2 of the CD226, and the purity of the protein is over 95% after the renaturation and purification of the recombinant protein. Western Blot identification and successful expression of the protein. A true nuclear expression vector for expressing the extracellular domain of CD226 was successfully constructed, and a stable high-scale was obtained by transfection and screening. CHO-K1 cell line up to soluble CD226. 2. It was found that CD226 molecules play an important role in the identification and killing of tumor cells in NK cells, and the CD226 molecules and ligands It is found that the extracellular domain of the CD226 can recognize and bind to the tumor cells. the addition of the CD226 extracellular domain recombinant protein can effectively block the killing of the NK cells on the tumor cells, Histone has no obvious effect on the growth and proliferation of effector cells and target cells, and the blocking of the recombinant protein on the killing is mainly realized by blocking the combination of the NK cells and the target cells, and the prompt CD226 is NK cells may play an important role in the identification of target cells. In addition, the activation signal transferred by CD226 has an important effect on the activation of NK cells. After the antibody stimulates the activation of the CD226 molecules, the expression of the NK cell surface activating molecule CD69 can be increased, and the cytokine (IFN) and the killing can be caused. The expression of the injured particles (CD107a, Granzyme B) is significantly increased. The recombinant protein can effectively block the activation of NK cells caused by K562, including the CD69 and the particles. The expression of the enzyme B is reduced, and the phosphorylation of ERK1/ 2 is reduced to some extent. The above results show that the CD226 molecule acts as the surface activating receptor of the NK cell, and the main site for the ligand recognition is located in the first immunoglobulin-like junction outside the cell. The activation signal transferred by the molecule ECD2.CD226 is involved in the adhesion of NK cells to target cells, the secretion of cytokines and anti-killing. In many immunological processes, ERK1/ 2 may be involved in the transfer of CD226 activation signal. The study of membrane receptor and the expression of membrane protein spectrum are of great significance to study the function of lymphocytes and to maintain the immune balance of the body. In order to study the function of the membrane protein with low abundance in the surface of the lymphocytes quickly and effectively, we have set up a structure in the further work.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
相关期刊论文 前2条
1 罗阔;陈晓品;;肿瘤靶向治疗的新型细胞载体分析[J];检验医学与临床;2014年04期
2 李季;官杰;;CD226与肿瘤关系的研究进展[J];齐齐哈尔医学院学报;2014年15期
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