氧化应激对NKG2D配体表达的影响

发布时间：2019-01-04 22:57

【摘要】： NKG2D是淋巴细胞活化型受体之一,主要表达在NK细胞、CD8~+αβT细胞、γδT细胞等免疫细胞表面,在免疫应答中起着非常重要的作用。NKG2D有多种配体,现已发现的配体包括人的UL-l6结合蛋白(UL-l6 binding proteins,ULBPs)、非经典性MHCⅠ类链相关分子A/B(MHC classⅠchain-related molecules A/B,MICA/B)和鼠的维甲酸诱导的基因(Retinoic acid inducible genes-1,RAE1,α、β、γ、δ、ε)、次要组织相容性抗原H60(the minor hitocompatibility antigen,H60,A,b,c)和鼠的ULBP样转录产物1(murine ULBP-like transcript 1,MULT1)。NKG2D配体分子在正常组织中低表达或不表达,但在肿瘤组织或处于氧化应激、热休克、病毒感染等应激状态时表达上调。据报道,氧化应激可使气道上皮细胞NKG2D配体的某些分子表达上调。本工作旨在研究氧化应激对NKG2D配体表达及其功能的影响。主要工作如下: 一、氧化应激对细胞NKG2D配体表达影响的研究我们选取8种肿瘤细胞以及从正常人外周血中分选T细胞和单核来源的树突状细胞(the monocyte-derived dendritic cell,moDC),在培养基中加H_2O_2使其处于氧化应激状态。应用RT-PCR、Real-time PCR和流式细胞仪等方法分别从mRNA水平和蛋白水平检测肿瘤细胞ULBPs及MICA/B的表达变化,流式细胞仪分析氧化应激对T细胞和moDC ULBPs及MICA/B表达的影响。并针对氧化应激前后不同肿瘤细胞ULBPs及MICA/B表达的不同变化,选取五种细胞分析其变化对NK细胞功能的影响。结果显示,无论是肿瘤细胞,还是正常细胞都仅表达众多NKG2D配体中的一种或几种,而且各种细胞的表达谱各不相同。细胞NKG2D配体在mRNA水平与细胞表面的蛋白表达并不完全一致;其中BGC823细胞表面蛋白与mRNA的表达较为一致。在氧化应激之后各种配体的表达都有所升高,293T、Raji和K562细胞的大多数NKG2D配体在氧化应激之后都有不同程度的提高。而Jurkat、HO8910、HeLa和Daudi细胞表面表达的配体种类很少,并且表达情况在应激前后几乎没有变化。新鲜分离的T细胞应激前仅表达低水平的ULBP2,而不表达MICA;氧化应激后诱导出现MICA表达而ULBPs的表达没有变化。moDC广泛表达多种NKG2D配体,但应激前后表达水平变化不大。氧化应激增强了NK细胞对BGC823、Raji细胞的杀伤功能,且此效应可被anti-NKG2D抗体阻断。氧化应激前后NK细胞对293T、Jurkat和HeLa细胞的杀伤作用变化不显著。二、小鼠氧化应激模型中NKG2D配体的表达研究建立小鼠吸入臭氧的动物氧化应激模型,利用RT-PCR、Real-time PCR和免疫组化方法分析氧化应激对小鼠各组织Rae1和MULT1表达的影响。并用流式细胞仪分析氧化应激前后小肠上皮细胞(iIEL)、脾脏和胸腺淋巴细胞的Rae1和MULT1的表达情况。Real-time PCR结果显示,应激后Rae1的表达在皮肤和淋巴结有所降低,在胸腺、小肠、脾脏表达升高。MULT1的表达在皮肤和小肠略有升高,但变化不明显;MULT1在脾脏和淋巴结的表达下降,在胸腺基本没有变化。而免疫组织化学的结果表明,小鼠氧化应激后,MULT1在胸腺的表达降低,在淋巴结的表达略有升高,在脾脏和小肠的表达基本没有变化;Rae1在胸腺表达略有升高,在淋巴结、脾脏和小肠的变化不明显。综上所述,氧化应激可以诱导细胞NKG2D不同配体的表达,从而提高NK细胞对其杀伤活性。然而动物氧化应激模型中NKG2D配体表达的升高并不明显,有的组织表达甚至降低。这提示体内可能存在着更为复杂的NKG2D配体表达调节机制。
[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
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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