BCG活化巨噬細(xì)胞膜表面新蛋白NMAAP1、Trim59的克
發(fā)布時(shí)間：2018-07-26 10:27
【摘要】： 巨噬細(xì)胞是一類廣泛存在于組織中的單核吞噬細(xì)胞,它是機(jī)體內(nèi)天然免疫的第一道屏障。同時(shí),巨噬細(xì)胞在免疫監(jiān)視中也發(fā)揮重要作用。巨噬細(xì)胞可以通過分泌如TNF、NO等殺傷性效應(yīng)分子來殺傷腫瘤細(xì)胞;另一方面,巨噬細(xì)胞可以通過細(xì)胞間接觸來直接殺傷腫瘤細(xì)胞,而對(duì)于巨噬細(xì)胞的直接殺傷活性的機(jī)制研究一直是腫瘤免疫的一個(gè)重要領(lǐng)域。通過對(duì)已經(jīng)鑒定出來的細(xì)胞膜表面殺傷效應(yīng)分子的研究發(fā)現(xiàn),封閉這些效應(yīng)分子后巨噬細(xì)胞仍然具有很強(qiáng)的腫瘤殺傷活性,這一現(xiàn)象提示說明巨噬細(xì)胞膜表面存在一些尚未為人所知的分子,這些分子在巨噬細(xì)胞殺傷腫瘤的進(jìn)程中起關(guān)鍵作用。 本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),BCG刺激活化的巨噬細(xì)胞可以殺傷MCA207腫瘤細(xì)胞,這一過程與其細(xì)胞膜表面特有的蛋白分子密切相關(guān),通過SDS-PAGE聯(lián)合質(zhì)譜檢測(cè)的方法對(duì)BCG刺激活化和TGC誘導(dǎo)活化的巨噬細(xì)胞膜蛋白進(jìn)行比對(duì),我們得到了454個(gè)在BCG刺激活化的巨噬細(xì)胞膜表面上調(diào)表達(dá)的蛋白。為了揭示這些分子在巨噬細(xì)胞殺傷腫瘤過程中的作用,我們從中選取了兩個(gè)新蛋白NMAAP1和Trim59進(jìn)行基因克隆及其功能研究。 第一部分: 首先對(duì)本實(shí)驗(yàn)室的前期成果進(jìn)行了總結(jié),并利用生物信息學(xué)的手段從中篩選出了兩個(gè)待研究的新蛋白,其名稱分別是NMAAP1和Trim59,并對(duì)這兩個(gè)蛋白的潛在功能做了預(yù)測(cè)。 結(jié)果:NMAAP1可以與DAPK結(jié)合,進(jìn)而調(diào)控細(xì)胞凋亡的進(jìn)程;另一方面,NMAAP1的同源家族成員kiaa1754可以與IP3R結(jié)合,進(jìn)而調(diào)控鈣離子通道,這一進(jìn)程可能參與細(xì)胞分化;同時(shí)NMAAP1蛋白含有一個(gè)Mab-21結(jié)構(gòu)域,在生物進(jìn)化、系統(tǒng)發(fā)育等多方面都有這一結(jié)構(gòu)的身影。另一個(gè)新蛋白Trim59的預(yù)測(cè)結(jié)果表明,這一蛋白含有幾個(gè)可能參與分子間相互作用的結(jié)構(gòu),分別是ring finger、B-box以及卷曲螺旋,蛋白功能預(yù)測(cè)結(jié)果顯示,Trim59可能與細(xì)胞間接觸密切相關(guān)。 第二部分: 克隆并表達(dá)了這兩個(gè)新蛋白,通過親和層析的方法得到了兩種純化后的融合蛋白。然后利用這兩個(gè)純化后的蛋白作為抗原制備了多克隆抗體。利用Western-blotting檢測(cè)了這兩種抗體的特異性后,我們進(jìn)行了免疫組化實(shí)驗(yàn),對(duì)這兩個(gè)蛋白在小鼠體內(nèi)不同組織的分布進(jìn)行了鑒定。 結(jié)果:通過原核表達(dá)的方式,得到了兩個(gè)融合蛋白,并利用其制備了多克隆抗體,SDS-PAGE電泳以及western-blotting結(jié)果表明所純化的兩個(gè)蛋白正是我們所需要的目的蛋白,同時(shí)也證明了我們的抗體具有很強(qiáng)的特異性。通過免疫組化,證明了我們的抗體可以和天然構(gòu)象下的蛋白正常結(jié)合。同時(shí)也分析了兩個(gè)蛋白在機(jī)體內(nèi)不同組織的分布情況,結(jié)果顯示:NMAAP1在胸腺組織中的表達(dá)量較高,這可能與IP3R可以調(diào)節(jié)TCR敏感性進(jìn)而參與T細(xì)胞的活化相關(guān)。Trim59在脾臟組織中的表達(dá)量較高,提示Trim59可能在免疫系統(tǒng)中扮演重要角色;另外,在卵巢組織中也檢測(cè)到大量的Trim59表達(dá),提示Trim59可能在生殖系統(tǒng)中也有作用。 第三部分: 利用自制的多克隆抗體進(jìn)行了抗體封閉細(xì)胞毒實(shí)驗(yàn),對(duì)我們所選取的兩個(gè)新蛋白在巨噬細(xì)胞殺傷腫瘤過程中所發(fā)揮的作用進(jìn)行了初步研究。另外,我們克隆了Trim59的全長(zhǎng)基因,并將其穩(wěn)定轉(zhuǎn)染于Raw264.7細(xì)胞,通過對(duì)轉(zhuǎn)染前后的Raw264.7細(xì)胞生物學(xué)性狀的研究來鑒定Trim59的具體功能。 結(jié)果:通過抗體封閉細(xì)胞毒實(shí)驗(yàn)發(fā)現(xiàn),我們所選取的蛋白Trim59在BCG刺激活化的巨噬細(xì)胞殺傷腫瘤的過程中起關(guān)鍵作用,而NMAAP1則在殺傷過程中幾乎沒有作用。為了進(jìn)一步研究Trim59在殺傷過程中所扮演的角色,我們將Trim59轉(zhuǎn)染后表達(dá)于Raw264.7細(xì)胞系,并檢測(cè)了這一細(xì)胞的腫瘤殺傷活性。實(shí)驗(yàn)結(jié)果表明,Trim59并不直接參與殺傷。通過對(duì)轉(zhuǎn)染后細(xì)胞的吞噬活力進(jìn)行檢測(cè),發(fā)現(xiàn)Trim59可以促進(jìn)巨噬細(xì)胞對(duì)異物的吞噬,這可能與Trim59可以促進(jìn)巨噬細(xì)胞與其它分子之間相互作用有關(guān)。因而我們推斷Trim59在巨噬細(xì)胞殺傷的過程中是一個(gè)輔助分子,它可以通過促進(jìn)巨噬細(xì)胞與靶細(xì)胞接觸來加強(qiáng)巨噬細(xì)胞的細(xì)胞毒作用。 綜上所述,NMAAP1在胸腺組織中表達(dá)量較高,但其在巨噬細(xì)胞直接殺傷腫瘤的過程中沒有明顯作用,結(jié)合它的分子結(jié)構(gòu)我們預(yù)測(cè),NMAAP1可能參與巨噬細(xì)胞的特異性分化。Trim59在脾臟及卵巢中的表達(dá)量很高,提示其可能與機(jī)體免疫及生殖相關(guān);Trim59是巨噬細(xì)胞殺傷腫瘤過程中的關(guān)鍵效應(yīng)分子,但它是以一個(gè)輔助效應(yīng)分子的身份在起作用,Trim59本身可以促進(jìn)巨噬細(xì)胞對(duì)異物的吞噬作用。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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
相關(guān)博士學(xué)位論文 前1條
1 張領(lǐng)兵;巨噬細(xì)胞接觸依賴性殺傷機(jī)制的研究及活化相關(guān)膜蛋白的鑒定[D];吉林大學(xué);2007年
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