NK細胞活化受體CD226的表達及功能研究
發(fā)布時間：2018-11-22 08:09
【摘要】： 固有免疫系統(tǒng)作為機體抵御外來微生物抗原入侵的第一道防線,能直接迅速的活化并對入侵機體的病原體進行清除,在獲得性免疫系統(tǒng)肩動之前發(fā)揮了重要的屏障作用。天然殺傷細胞(natural killer cells,NK)是固有免疫系統(tǒng)的重要組成部分,在機體防御感染和癌癥的免疫應(yīng)答中起著重要作用。NK細胞的活化受到活化性與抑制性受體的共同調(diào)控,由于識別機制的多樣化,近年來,NK細胞表面活化性受體得到了人們的廣泛關(guān)注。 CD226分子是NK細胞表面活化性受體,近年來研究發(fā)現(xiàn),CD226在NK細胞的活化并殺傷腫瘤細胞等生物學(xué)過程中發(fā)揮了重要作用,并與多種疾病的發(fā)生密切相關(guān)。雖然CD226的功能得到了人們的廣泛關(guān)注,但其分子識別與活化機制仍然不是非常清楚。 本實驗中,我們構(gòu)建了CD226的胞外不同的結(jié)構(gòu)域蛋白,包括含有N端19-129氨基酸序列的IgV樣結(jié)構(gòu)域ECD1(extracellular domain 1),以及含有兩個IgV樣結(jié)構(gòu)域ECD2(extracellular domain 2)(19-243aa)。通過對兩個長度不同的結(jié)構(gòu)域與腫瘤細胞表面配體蛋白的相互作用進行研究,發(fā)現(xiàn)CD226蛋白與其配體可能的作用位點與其在腫瘤識別殺傷中的作用機理;進一步通過抗體介導(dǎo)的活化與蛋白阻斷實驗,探討了CD226分子在NK細胞活化中的作用機制。 通過上述研究,我們初步獲得了結(jié)果如下: 1.構(gòu)建載體并原核表達CD226分子胞外結(jié)構(gòu)域ECD1與ECD2,重組蛋白經(jīng)復(fù)性純化后,蛋白純度達到95%以上,經(jīng)Western Blot鑒定,蛋白成功表達。成功構(gòu)建表達CD226胞外段蛋白的真核表達載體,經(jīng)過轉(zhuǎn)染并篩選獲得穩(wěn)定高表達可溶性CD226的CHO-K1細胞系。 2.發(fā)現(xiàn)CD226分子在NK細胞對腫瘤細胞識別殺傷中發(fā)揮重要作用,并且CD226分子與配體的識別與結(jié)合主要是通過胞外N端第一個結(jié)構(gòu)域ECD1。蛋白結(jié)合實驗發(fā)現(xiàn),CD226胞外結(jié)構(gòu)域可以識別并結(jié)合腫瘤細胞表面配體,單獨的ECD1蛋白即可有效與靶細胞結(jié)合。CD226胞外結(jié)構(gòu)域重組蛋白的加入可以有效阻斷NK細胞對腫瘤細胞的殺傷,進一步研究發(fā)現(xiàn),重組蛋白對效應(yīng)細胞與靶細胞生長與增殖沒有明顯的影響,重組蛋白對殺傷的阻斷主要是通過阻斷NK細胞與靶細胞的結(jié)合實現(xiàn)的,提示CD226在NK細胞對靶細胞的識別中可能發(fā)揮了粘附分子的作用。 3.除粘附作用外,CD226傳遞的活化信號對NK細胞的活化有著重要的影響。抗體刺激CD226分子活化后可以引起NK細胞表面活化分子CD69的表達升高,細胞因子(IFNγ)與殺傷顆粒(CD107a,Granzyme B)的表達都明顯升高。重組蛋白可以有效阻斷K562引起的NK細胞的活化,包括CD69與顆粒酶B的表達降低,ERK1/2的磷酸化程度也有一定程度的降低。 上述結(jié)果證明,CD226分子作為NK細胞表面活化性受體,其與配體識別的主要位點位于胞外第一個免疫球蛋白樣結(jié)構(gòu)域ECD1。CD226分子傳遞的活化信號參與NK細胞與靶細胞的粘附,細胞因子分泌以及殺傷等多種免疫學(xué)過程,ERK1/2分子可能參與了CD226活化信號的傳遞。 淋巴細胞表面膜蛋白受體與炎癥損傷觸發(fā)密切相關(guān),對淋巴細胞表面膜受體的研究與膜蛋白譜的描繪對研究淋巴細胞功能,維持機體的免疫平衡有著重要意義。為了能夠快速有效篩選淋巴細胞表面低豐度的膜蛋白進行功能研究,在進一步工作中,我們建立了一種構(gòu)建膜蛋白cDNA文庫方法,并將其應(yīng)用于小鼠肝臟淋巴細胞膜蛋白的特異性高通量篩選。
[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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392
【引證文獻】
相關(guān)期刊論文 前2條
1 羅闊;陳曉品;;腫瘤靶向治療的新型細胞載體分析[J];檢驗醫(yī)學(xué)與臨床;2014年04期
2 李季;官杰;;CD226與腫瘤關(guān)系的研究進展[J];齊齊哈爾醫(yī)學(xué)院學(xué)報;2014年15期
，本文編號：2348639
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