氧化應(yīng)激對(duì)NKG2D配體表達(dá)的影響
發(fā)布時(shí)間：2019-01-04 22:57
【摘要】： NKG2D是淋巴細(xì)胞活化型受體之一,主要表達(dá)在NK細(xì)胞、CD8~+αβT細(xì)胞、γδT細(xì)胞等免疫細(xì)胞表面,在免疫應(yīng)答中起著非常重要的作用。NKG2D有多種配體,現(xiàn)已發(fā)現(xiàn)的配體包括人的UL-l6結(jié)合蛋白(UL-l6 binding proteins,ULBPs)、非經(jīng)典性MHCⅠ類鏈相關(guān)分子A/B(MHC classⅠchain-related molecules A/B,MICA/B)和鼠的維甲酸誘導(dǎo)的基因(Retinoic acid inducible genes-1,RAE1,α、β、γ、δ、ε)、次要組織相容性抗原H60(the minor hitocompatibility antigen,H60,A,b,c)和鼠的ULBP樣轉(zhuǎn)錄產(chǎn)物1(murine ULBP-like transcript 1,MULT1)。NKG2D配體分子在正常組織中低表達(dá)或不表達(dá),但在腫瘤組織或處于氧化應(yīng)激、熱休克、病毒感染等應(yīng)激狀態(tài)時(shí)表達(dá)上調(diào)。據(jù)報(bào)道,氧化應(yīng)激可使氣道上皮細(xì)胞NKG2D配體的某些分子表達(dá)上調(diào)。本工作旨在研究氧化應(yīng)激對(duì)NKG2D配體表達(dá)及其功能的影響。主要工作如下: 一、氧化應(yīng)激對(duì)細(xì)胞NKG2D配體表達(dá)影響的研究 我們選取8種腫瘤細(xì)胞以及從正常人外周血中分選T細(xì)胞和單核來(lái)源的樹突狀細(xì)胞(the monocyte-derived dendritic cell,moDC),在培養(yǎng)基中加H_2O_2使其處于氧化應(yīng)激狀態(tài)。應(yīng)用RT-PCR、Real-time PCR和流式細(xì)胞儀等方法分別從mRNA水平和蛋白水平檢測(cè)腫瘤細(xì)胞ULBPs及MICA/B的表達(dá)變化,流式細(xì)胞儀分析氧化應(yīng)激對(duì)T細(xì)胞和moDC ULBPs及MICA/B表達(dá)的影響。并針對(duì)氧化應(yīng)激前后不同腫瘤細(xì)胞ULBPs及MICA/B表達(dá)的不同變化,選取五種細(xì)胞分析其變化對(duì)NK細(xì)胞功能的影響。結(jié)果顯示,無(wú)論是腫瘤細(xì)胞,還是正常細(xì)胞都僅表達(dá)眾多NKG2D配體中的一種或幾種,而且各種細(xì)胞的表達(dá)譜各不相同。細(xì)胞NKG2D配體在mRNA水平與細(xì)胞表面的蛋白表達(dá)并不完全一致;其中BGC823細(xì)胞表面蛋白與mRNA的表達(dá)較為一致。在氧化應(yīng)激之后各種配體的表達(dá)都有所升高,293T、Raji和K562細(xì)胞的大多數(shù)NKG2D配體在氧化應(yīng)激之后都有不同程度的提高。而Jurkat、HO8910、HeLa和Daudi細(xì)胞表面表達(dá)的配體種類很少,并且表達(dá)情況在應(yīng)激前后幾乎沒有變化。新鮮分離的T細(xì)胞應(yīng)激前僅表達(dá)低水平的ULBP2,而不表達(dá)MICA;氧化應(yīng)激后誘導(dǎo)出現(xiàn)MICA表達(dá)而ULBPs的表達(dá)沒有變化。moDC廣泛表達(dá)多種NKG2D配體,但應(yīng)激前后表達(dá)水平變化不大。氧化應(yīng)激增強(qiáng)了NK細(xì)胞對(duì)BGC823、Raji細(xì)胞的殺傷功能,且此效應(yīng)可被anti-NKG2D抗體阻斷。氧化應(yīng)激前后NK細(xì)胞對(duì)293T、Jurkat和HeLa細(xì)胞的殺傷作用變化不顯著。 二、小鼠氧化應(yīng)激模型中NKG2D配體的表達(dá)研究 建立小鼠吸入臭氧的動(dòng)物氧化應(yīng)激模型,利用RT-PCR、Real-time PCR和免疫組化方法分析氧化應(yīng)激對(duì)小鼠各組織Rae1和MULT1表達(dá)的影響。并用流式細(xì)胞儀分析氧化應(yīng)激前后小腸上皮細(xì)胞(iIEL)、脾臟和胸腺淋巴細(xì)胞的Rae1和MULT1的表達(dá)情況。Real-time PCR結(jié)果顯示,應(yīng)激后Rae1的表達(dá)在皮膚和淋巴結(jié)有所降低,在胸腺、小腸、脾臟表達(dá)升高。MULT1的表達(dá)在皮膚和小腸略有升高,但變化不明顯;MULT1在脾臟和淋巴結(jié)的表達(dá)下降,在胸腺基本沒有變化。而免疫組織化學(xué)的結(jié)果表明,小鼠氧化應(yīng)激后,MULT1在胸腺的表達(dá)降低,在淋巴結(jié)的表達(dá)略有升高,在脾臟和小腸的表達(dá)基本沒有變化;Rae1在胸腺表達(dá)略有升高,在淋巴結(jié)、脾臟和小腸的變化不明顯。 綜上所述,氧化應(yīng)激可以誘導(dǎo)細(xì)胞NKG2D不同配體的表達(dá),從而提高NK細(xì)胞對(duì)其殺傷活性。然而動(dòng)物氧化應(yīng)激模型中NKG2D配體表達(dá)的升高并不明顯,有的組織表達(dá)甚至降低。這提示體內(nèi)可能存在著更為復(fù)雜的NKG2D配體表達(dá)調(diào)節(jié)機(jī)制。
[Abstract]:NKG2D is one of the activated receptors of the lymphocyte, which is mainly expressed in the surface of the immune cells such as NK cells, CD8 ~ +, T-cells, and T-cells, and plays a very important role in the immune response. NKG2D has a variety of ligands, and it has been found that the ligands include human's UL-l6 binding proteins (ULBPs), non-classical MHC class I chain-related molecules A/ B (MHC class I chain-related molecules A/ B, MICA/ B), and a murine retinoic acid-induced gene (Retinoic acid-related genes-1, RAE1, IX, IX, IX, IX, IX), The minor histocompatibility antigen (H60, A, b, c) and the murine ULBP-like transscript 1 (MULT1). NKG2D ligand molecules are expressed or not expressed in normal tissues, but are up-regulated in the presence of tumor tissue or in the presence of oxidative stress, heat shock, viral infection, and the like. It is reported that oxidative stress may increase some of the molecular expression of the NKG2D ligand in the airway epithelial cells. The purpose of this work is to study the effect of oxidative stress on the expression of NKG2D ligand and its function. The main work is as follows: The effect of oxidative stress on the expression of NKG2D ligand in cells We selected 8 tumor cells as well as dendritic cells (the monocyte-derived drentic cell), which were isolated from the peripheral blood of the normal human. moDC), H _ 2O _ 2 is added to the culture medium to make it oxygen The expression of ULBPs and MICA/ B in tumor cells were detected from mRNA level and protein level by RT-PCR, Real-time PCR and flow cytometry. The changes of the expression of ULBPs and MICA/ B in different tumor cells before and after oxidative stress were studied. The results show that both tumor cells and normal cells express only one or more of a plurality of NKG2D ligands, and the expression of various cells The cell NKG2D ligand is not exactly the same as the protein expression of the cell surface at the mRNA level, and the expression of the surface protein and mRNA of the BGC823 cell is different from that of the mRNA. The expression of various ligands increased after oxidative stress, and most of the NKG2D ligands of the 293T, Raji, and K562 cells were different after oxidative stress The number of ligands expressed on the surface of Jurkat, HO8910, HeLa and Daudi cells was very small, and the expression was a few before and after the stress. There was no change. Only a low level of ULBP2 was expressed prior to the freshly isolated T-cell stress without expressing MICA; the expression of MICA was induced after oxidative stress and the table of ULBPs There is no change. moDC is widely used to express a variety of NKG2D ligands, but the expression of water before and after stress The level change is not large. The oxidative stress enhances the killing function of NK cells on the BGC823 and Raji cells, and the effect can be anti-NKG2 The anti-killing effect of NK cells on 293T, Jurkat and HeLa cells before and after oxidative stress The change is not significant. 2. NKG in the oxidative stress model of mice The oxidative stress model of mice was established by means of RT-PCR, Real-time PCR and immunohistochemistry. and the influence of the expression of MULT1 was analyzed by flow cytometry, and the Ra1 of the small intestinal epithelial cells (iIEL), spleen and thymus lymphocytes was analyzed by flow cytometry. and the expression of muLT1. Real-time PCR results show that the expression of Ra1 after stress is reduced in the skin and lymph nodes, and in the chest, The expression of MULT1 increased slightly in the skin and the small intestine, but the change was not significant; and the expression of MULT1 in the spleen and the lymph node The results of the immunohistochemical study showed that the expression of MULT1 in the thymus decreased after the oxidative stress of the mice, the expression of the lymph node increased slightly, the expression of the spleen and the small intestine was not changed, and the expression of Ra1 in the thymus was slightly increased, in the lymph node, In conclusion, oxidative stress can induce the expression of NKG2D different ligands, in that oxidative stress model of the animal, the expression of the NKG2D ligand is increase and It is not clear that some of the tissue is expressed or even decreased. This suggests that there may be more complex in the body
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
[Abstract]:NKG2D is one of the activated receptors of the lymphocyte, which is mainly expressed in the surface of the immune cells such as NK cells, CD8 ~ +, T-cells, and T-cells, and plays a very important role in the immune response. NKG2D has a variety of ligands, and it has been found that the ligands include human's UL-l6 binding proteins (ULBPs), non-classical MHC class I chain-related molecules A/ B (MHC class I chain-related molecules A/ B, MICA/ B), and a murine retinoic acid-induced gene (Retinoic acid-related genes-1, RAE1, IX, IX, IX, IX, IX), The minor histocompatibility antigen (H60, A, b, c) and the murine ULBP-like transscript 1 (MULT1). NKG2D ligand molecules are expressed or not expressed in normal tissues, but are up-regulated in the presence of tumor tissue or in the presence of oxidative stress, heat shock, viral infection, and the like. It is reported that oxidative stress may increase some of the molecular expression of the NKG2D ligand in the airway epithelial cells. The purpose of this work is to study the effect of oxidative stress on the expression of NKG2D ligand and its function. The main work is as follows: The effect of oxidative stress on the expression of NKG2D ligand in cells We selected 8 tumor cells as well as dendritic cells (the monocyte-derived drentic cell), which were isolated from the peripheral blood of the normal human. moDC), H _ 2O _ 2 is added to the culture medium to make it oxygen The expression of ULBPs and MICA/ B in tumor cells were detected from mRNA level and protein level by RT-PCR, Real-time PCR and flow cytometry. The changes of the expression of ULBPs and MICA/ B in different tumor cells before and after oxidative stress were studied. The results show that both tumor cells and normal cells express only one or more of a plurality of NKG2D ligands, and the expression of various cells The cell NKG2D ligand is not exactly the same as the protein expression of the cell surface at the mRNA level, and the expression of the surface protein and mRNA of the BGC823 cell is different from that of the mRNA. The expression of various ligands increased after oxidative stress, and most of the NKG2D ligands of the 293T, Raji, and K562 cells were different after oxidative stress The number of ligands expressed on the surface of Jurkat, HO8910, HeLa and Daudi cells was very small, and the expression was a few before and after the stress. There was no change. Only a low level of ULBP2 was expressed prior to the freshly isolated T-cell stress without expressing MICA; the expression of MICA was induced after oxidative stress and the table of ULBPs There is no change. moDC is widely used to express a variety of NKG2D ligands, but the expression of water before and after stress The level change is not large. The oxidative stress enhances the killing function of NK cells on the BGC823 and Raji cells, and the effect can be anti-NKG2 The anti-killing effect of NK cells on 293T, Jurkat and HeLa cells before and after oxidative stress The change is not significant. 2. NKG in the oxidative stress model of mice The oxidative stress model of mice was established by means of RT-PCR, Real-time PCR and immunohistochemistry. and the influence of the expression of MULT1 was analyzed by flow cytometry, and the Ra1 of the small intestinal epithelial cells (iIEL), spleen and thymus lymphocytes was analyzed by flow cytometry. and the expression of muLT1. Real-time PCR results show that the expression of Ra1 after stress is reduced in the skin and lymph nodes, and in the chest, The expression of MULT1 increased slightly in the skin and the small intestine, but the change was not significant; and the expression of MULT1 in the spleen and the lymph node The results of the immunohistochemical study showed that the expression of MULT1 in the thymus decreased after the oxidative stress of the mice, the expression of the lymph node increased slightly, the expression of the spleen and the small intestine was not changed, and the expression of Ra1 in the thymus was slightly increased, in the lymph node, In conclusion, oxidative stress can induce the expression of NKG2D different ligands, in that oxidative stress model of the animal, the expression of the NKG2D ligand is increase and It is not clear that some of the tissue is expressed or even decreased. This suggests that there may be more complex in the body
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
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