BCG活化巨噬细胞膜表面新蛋白NMAAP1、Trim59的克
发布时间：2018-07-26 10:27
【摘要】： 巨噬细胞是一类广泛存在于组织中的单核吞噬细胞,它是机体内天然免疫的第一道屏障。同时,巨噬细胞在免疫监视中也发挥重要作用。巨噬细胞可以通过分泌如TNF、NO等杀伤性效应分子来杀伤肿瘤细胞;另一方面,巨噬细胞可以通过细胞间接触来直接杀伤肿瘤细胞,而对于巨噬细胞的直接杀伤活性的机制研究一直是肿瘤免疫的一个重要领域。通过对已经鉴定出来的细胞膜表面杀伤效应分子的研究发现,封闭这些效应分子后巨噬细胞仍然具有很强的肿瘤杀伤活性,这一现象提示说明巨噬细胞膜表面存在一些尚未为人所知的分子,这些分子在巨噬细胞杀伤肿瘤的进程中起关键作用。 本实验室前期研究发现,BCG刺激活化的巨噬细胞可以杀伤MCA207肿瘤细胞,这一过程与其细胞膜表面特有的蛋白分子密切相关,通过SDS-PAGE联合质谱检测的方法对BCG刺激活化和TGC诱导活化的巨噬细胞膜蛋白进行比对,我们得到了454个在BCG刺激活化的巨噬细胞膜表面上调表达的蛋白。为了揭示这些分子在巨噬细胞杀伤肿瘤过程中的作用,我们从中选取了两个新蛋白NMAAP1和Trim59进行基因克隆及其功能研究。 第一部分: 首先对本实验室的前期成果进行了总结,并利用生物信息学的手段从中筛选出了两个待研究的新蛋白,其名称分别是NMAAP1和Trim59,并对这两个蛋白的潜在功能做了预测。 结果:NMAAP1可以与DAPK结合,进而调控细胞凋亡的进程;另一方面,NMAAP1的同源家族成员kiaa1754可以与IP3R结合,进而调控钙离子通道,这一进程可能参与细胞分化;同时NMAAP1蛋白含有一个Mab-21结构域,在生物进化、系统发育等多方面都有这一结构的身影。另一个新蛋白Trim59的预测结果表明,这一蛋白含有几个可能参与分子间相互作用的结构,分别是ring finger、B-box以及卷曲螺旋,蛋白功能预测结果显示,Trim59可能与细胞间接触密切相关。 第二部分: 克隆并表达了这两个新蛋白,通过亲和层析的方法得到了两种纯化后的融合蛋白。然后利用这两个纯化后的蛋白作为抗原制备了多克隆抗体。利用Western-blotting检测了这两种抗体的特异性后,我们进行了免疫组化实验,对这两个蛋白在小鼠体内不同组织的分布进行了鉴定。 结果:通过原核表达的方式,得到了两个融合蛋白,并利用其制备了多克隆抗体,SDS-PAGE电泳以及western-blotting结果表明所纯化的两个蛋白正是我们所需要的目的蛋白,同时也证明了我们的抗体具有很强的特异性。通过免疫组化,证明了我们的抗体可以和天然构象下的蛋白正常结合。同时也分析了两个蛋白在机体内不同组织的分布情况,结果显示:NMAAP1在胸腺组织中的表达量较高,这可能与IP3R可以调节TCR敏感性进而参与T细胞的活化相关。Trim59在脾脏组织中的表达量较高,提示Trim59可能在免疫系统中扮演重要角色;另外,在卵巢组织中也检测到大量的Trim59表达,提示Trim59可能在生殖系统中也有作用。 第三部分: 利用自制的多克隆抗体进行了抗体封闭细胞毒实验,对我们所选取的两个新蛋白在巨噬细胞杀伤肿瘤过程中所发挥的作用进行了初步研究。另外,我们克隆了Trim59的全长基因,并将其稳定转染于Raw264.7细胞,通过对转染前后的Raw264.7细胞生物学性状的研究来鉴定Trim59的具体功能。 结果:通过抗体封闭细胞毒实验发现,我们所选取的蛋白Trim59在BCG刺激活化的巨噬细胞杀伤肿瘤的过程中起关键作用,而NMAAP1则在杀伤过程中几乎没有作用。为了进一步研究Trim59在杀伤过程中所扮演的角色,我们将Trim59转染后表达于Raw264.7细胞系,并检测了这一细胞的肿瘤杀伤活性。实验结果表明,Trim59并不直接参与杀伤。通过对转染后细胞的吞噬活力进行检测,发现Trim59可以促进巨噬细胞对异物的吞噬,这可能与Trim59可以促进巨噬细胞与其它分子之间相互作用有关。因而我们推断Trim59在巨噬细胞杀伤的过程中是一个辅助分子,它可以通过促进巨噬细胞与靶细胞接触来加强巨噬细胞的细胞毒作用。 综上所述,NMAAP1在胸腺组织中表达量较高,但其在巨噬细胞直接杀伤肿瘤的过程中没有明显作用,结合它的分子结构我们预测,NMAAP1可能参与巨噬细胞的特异性分化。Trim59在脾脏及卵巢中的表达量很高,提示其可能与机体免疫及生殖相关;Trim59是巨噬细胞杀伤肿瘤过程中的关键效应分子,但它是以一个辅助效应分子的身份在起作用,Trim59本身可以促进巨噬细胞对异物的吞噬作用。
[Abstract]:Macrophages are a kind of monocyte phagocyte, which is widely used in tissue. It is the first barrier of natural immunity in the body. Macrophages also play an important role in immune surveillance. Macrophages can kill tumor cells by secreting killer effects such as TNF, NO and so on; on the other hand, macrophages can pass through thin cells. The mechanism of the direct killing activity of macrophages has been an important area of tumor immunity. This phenomenon suggests that there are some unknown molecules on the surface of the macrophage membrane, which play a key role in the process of macrophage killing tumor.
Previous studies in our laboratory found that BCG stimulated activated macrophages can kill MCA207 tumor cells. This process is closely related to the specific protein molecules on the surface of the cell membrane. By SDS-PAGE combined mass spectrometry, the BCG stimulation activation and TGC induced activation of macrophage membrane proteins are compared. We have obtained 454 in BC G prickly activated protein on the surface of macrophage membrane was up-regulated. In order to reveal the role of these molecules in the process of killing tumor in macrophages, we have selected two new proteins, NMAAP1 and Trim59, for gene cloning and functional study.
Part one:
First of all, the preliminary results of the laboratory were summarized, and two new proteins were screened by means of bioinformatics. Their names were NMAAP1 and Trim59, and the potential functions of the two proteins were predicted.
Results: NMAAP1 can be combined with DAPK to regulate the process of apoptosis; on the other hand, NMAAP1 family members, kiaa1754, can bind to IP3R, and then regulate the calcium channel. This process may participate in cell differentiation; at the same time, the NMAAP1 protein contains a Mab-21 domain, in many aspects such as biological evolution, phylogeny and so on. The prediction of another new protein Trim59 shows that the protein contains several structures that may participate in intermolecular interaction, namely, ring finger, B-box and curl spiral, and the protein function prediction results show that Trim59 may be closely related to the indirect contact with the cell.
The second part:
The two new proteins were cloned and expressed, and two purified fusion proteins were obtained by affinity chromatography. Then the two purified proteins were used as antigen to prepare polyclonal antibodies. The specificity of these two antibodies was detected by Western-blotting. We performed immunohistochemical experiments on these two proteins. The distribution of different tissues in the mice was identified.
Results: two fusion proteins were obtained by prokaryotic expression, and polyclonal antibodies were prepared by them. The results of SDS-PAGE electrophoresis and Western-blotting showed that the purified two proteins were the target proteins we needed, and also proved that our antibody has a strong specificity. The antibodies can be combined with the natural conformation proteins. Meanwhile, the distribution of the two proteins in different tissues is also analyzed. The results show that the expression of NMAAP1 in the thymus is high, which may be associated with the IP3R regulation of TCR sensitivity and further participation in the expression of.Trim59 in the spleen tissues of the T cells. High, suggesting that Trim59 may play an important role in the immune system; in addition, a large number of Trim59 expressions have also been detected in the ovarian tissue, suggesting that Trim59 may also play a role in the reproductive system.
The third part:
Using the homemade polyclonal antibody, the antibody closed cytotoxicity test was carried out. The effect of two new proteins on the killing of macrophages in the process of killing the tumor was preliminarily studied. In addition, we cloned the full length gene of Trim59 and transfected it into Raw264.7 cells steadily, through the Raw264.7 fine before and after transfection. The study of cell biological characteristics will identify the specific functions of Trim59.
Results: the antibody closed cytotoxicity test showed that the protein Trim59 we selected played a key role in the process of BCG stimulating activated macrophages to kill the tumor, while NMAAP1 had little effect during the killing process. In order to further study the role of Trim59 in the killing process, we expressed Trim59 after transfection to Raw. The 264.7 cell line and the tumor killing activity of this cell were detected. The results showed that Trim59 was not directly involved in killing. By detecting the phagocytic activity of the transfected cells, it was found that Trim59 could promote the phagocytosis of the foreign body by macrophages, which may be related to the interaction between macrophages and other molecules by Trim59. So we infer that Trim59 is an auxiliary molecule in the process of macrophage killing, which can enhance the cytotoxic effect of macrophages by promoting the contact of macrophages with the target cells.
In summary, NMAAP1 is highly expressed in the thymus, but it does not play a significant role in the direct killing of the tumor by macrophages. Combining its molecular structure, we predict that NMAAP1 may participate in the specific differentiation of.Trim59 in the spleen and ovary, suggesting that it may be associated with the immune and reproductive phase of the body. Trim59 is a key effector in the process of macrophage killing and tumor, but it is acting as an auxiliary effect molecule, and Trim59 itself can promote the phagocytosis of the foreign body by macrophages.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：2145702
[Abstract]:Macrophages are a kind of monocyte phagocyte, which is widely used in tissue. It is the first barrier of natural immunity in the body. Macrophages also play an important role in immune surveillance. Macrophages can kill tumor cells by secreting killer effects such as TNF, NO and so on; on the other hand, macrophages can pass through thin cells. The mechanism of the direct killing activity of macrophages has been an important area of tumor immunity. This phenomenon suggests that there are some unknown molecules on the surface of the macrophage membrane, which play a key role in the process of macrophage killing tumor.
Previous studies in our laboratory found that BCG stimulated activated macrophages can kill MCA207 tumor cells. This process is closely related to the specific protein molecules on the surface of the cell membrane. By SDS-PAGE combined mass spectrometry, the BCG stimulation activation and TGC induced activation of macrophage membrane proteins are compared. We have obtained 454 in BC G prickly activated protein on the surface of macrophage membrane was up-regulated. In order to reveal the role of these molecules in the process of killing tumor in macrophages, we have selected two new proteins, NMAAP1 and Trim59, for gene cloning and functional study.
Part one:
First of all, the preliminary results of the laboratory were summarized, and two new proteins were screened by means of bioinformatics. Their names were NMAAP1 and Trim59, and the potential functions of the two proteins were predicted.
Results: NMAAP1 can be combined with DAPK to regulate the process of apoptosis; on the other hand, NMAAP1 family members, kiaa1754, can bind to IP3R, and then regulate the calcium channel. This process may participate in cell differentiation; at the same time, the NMAAP1 protein contains a Mab-21 domain, in many aspects such as biological evolution, phylogeny and so on. The prediction of another new protein Trim59 shows that the protein contains several structures that may participate in intermolecular interaction, namely, ring finger, B-box and curl spiral, and the protein function prediction results show that Trim59 may be closely related to the indirect contact with the cell.
The second part:
The two new proteins were cloned and expressed, and two purified fusion proteins were obtained by affinity chromatography. Then the two purified proteins were used as antigen to prepare polyclonal antibodies. The specificity of these two antibodies was detected by Western-blotting. We performed immunohistochemical experiments on these two proteins. The distribution of different tissues in the mice was identified.
Results: two fusion proteins were obtained by prokaryotic expression, and polyclonal antibodies were prepared by them. The results of SDS-PAGE electrophoresis and Western-blotting showed that the purified two proteins were the target proteins we needed, and also proved that our antibody has a strong specificity. The antibodies can be combined with the natural conformation proteins. Meanwhile, the distribution of the two proteins in different tissues is also analyzed. The results show that the expression of NMAAP1 in the thymus is high, which may be associated with the IP3R regulation of TCR sensitivity and further participation in the expression of.Trim59 in the spleen tissues of the T cells. High, suggesting that Trim59 may play an important role in the immune system; in addition, a large number of Trim59 expressions have also been detected in the ovarian tissue, suggesting that Trim59 may also play a role in the reproductive system.
The third part:
Using the homemade polyclonal antibody, the antibody closed cytotoxicity test was carried out. The effect of two new proteins on the killing of macrophages in the process of killing the tumor was preliminarily studied. In addition, we cloned the full length gene of Trim59 and transfected it into Raw264.7 cells steadily, through the Raw264.7 fine before and after transfection. The study of cell biological characteristics will identify the specific functions of Trim59.
Results: the antibody closed cytotoxicity test showed that the protein Trim59 we selected played a key role in the process of BCG stimulating activated macrophages to kill the tumor, while NMAAP1 had little effect during the killing process. In order to further study the role of Trim59 in the killing process, we expressed Trim59 after transfection to Raw. The 264.7 cell line and the tumor killing activity of this cell were detected. The results showed that Trim59 was not directly involved in killing. By detecting the phagocytic activity of the transfected cells, it was found that Trim59 could promote the phagocytosis of the foreign body by macrophages, which may be related to the interaction between macrophages and other molecules by Trim59. So we infer that Trim59 is an auxiliary molecule in the process of macrophage killing, which can enhance the cytotoxic effect of macrophages by promoting the contact of macrophages with the target cells.
In summary, NMAAP1 is highly expressed in the thymus, but it does not play a significant role in the direct killing of the tumor by macrophages. Combining its molecular structure, we predict that NMAAP1 may participate in the specific differentiation of.Trim59 in the spleen and ovary, suggesting that it may be associated with the immune and reproductive phase of the body. Trim59 is a key effector in the process of macrophage killing and tumor, but it is acting as an auxiliary effect molecule, and Trim59 itself can promote the phagocytosis of the foreign body by macrophages.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 ;Proteomic Analysis of Macrophages:A Potential Way to Identify Novel Proteins Associated with Activation of Macrophages for Tumor Cell Killing[J];Cellular & Molecular Immunology;2007年05期
相关博士学位论文 前1条
1 张领兵;巨噬细胞接触依赖性杀伤机制的研究及活化相关膜蛋白的鉴定[D];吉林大学;2007年
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