【作者】 张义；

【导师】 汪运山；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2010， 博士

【摘要】 研究背景:胃癌是严重危害人类健康的恶性疾病,全球每年有超过93万新发的胃癌病例,胃癌死亡率约为29.34/10万(男性40.80/10万,女性18.60/10万),是世界范围高居第二位的癌症死亡原因。据卫生部疾控司2006年统计,我国每年新发胃癌患者约40万人,因胃癌死亡患者约30万人,患病率和死亡率超过世界平均水平的两倍。近年来,国内外学者对胃癌的发生及转移进行了大量的基础和临床研究,取得了部分成果。但在胃癌的发病机制、早期诊断、干预治疗和预后判断方面,仍处于积极的探索阶段。因此深入研究胃癌的发病机制,探讨胃癌发病过程中肿瘤和免疫细胞的功能及状态,对研究胃癌及干预治疗无疑具有重要的指导意义。胃癌的病因目前仍不十分清晰。流行病学研究发现,胃癌的发病与饮食因素、感染因素、遗传因素及其他环境因素有关。近年最新的研究认为,胃癌的发生发展是宿主免疫系统和胃癌细胞相互作用的结果,不仅涉及到宿主效应细胞多种功能(T淋巴细胞数量和功能异常、活化障碍及凋亡增加)及细胞因子(IFN-γ含量降低、IL-10含量升高等)的改变,还与胃癌细胞自身的异常(缺乏协同刺激分子、MHC表达降低或缺失、FasL高表达)及相关癌基因及抑癌基因异常密切相关。其中肿瘤细胞表面缺少共刺激分子的表达是免疫逃逸的主要原因。目前发现共刺激分子有两种,即正性共刺激分子和负性共刺激分子。正性共刺激分子通过与淋巴细胞的受体结合共刺激免疫反应,影响T细胞的活化、增殖及细胞因子分泌。而负性共刺激分子通过两种方法来诱导T细胞无能：一是诱导抑制因子表达在效应性免疫细胞上,二是诱导抑制性因子受体表达在肿瘤细胞上。免疫球蛋白样转录子(Ig-like transcripts,ILTs)或白细胞抑制受体是最近发现的一类HLA-I受体家族,目前发现有九个家族成员,在T淋巴细胞、单个核细胞、NK细胞及树突状细胞表面广泛表达。不同种类的ILTs通过传导抑制性或刺激性信号发挥广泛的免疫调节作用,在抑制免疫杀伤细胞的杀伤活性、调节树突状细胞与T细胞间的信号传递、参与树突状细胞的抗原俘获等方面具有重要的生物学意义。其中ILT2、ILT3、ILT4属抑制性受体,其结构共同特点是有一个长的膜内段,含有3个免疫受体酪氨酸抑制基序,有4个或2个膜外的IgSF区。该类因子的功能主要是抑制分子,但其在肿瘤细胞上的表达和功能尚不清楚。HLA-G分子是一种非经典HLA-I类抗原,该基因具有8个外显子和7个内含子。HLA-G分子在结构上胞内段很短,并且原始的HLA-G转录产物经选择性剪切产生7种mRNA,编码出7种HLA-G蛋白异构体。其中HLA-G1和HLA-G5可以高特异性结合ILT2、ILT4,且与ILT2、LT4结合亲和力比经典HLA-I与ILT2, ILT4亲和大3-4倍。HLA-G分子通过抑制免疫活性细胞的功能在免疫耐受的建立和维持中发挥关键作用。HLA-G可通过与NK细胞、T细胞、APC等细胞表面的抑制性受体ILT-2、ILT-4等结合发挥抑制性免疫调节作用。可溶性HLA-G(sHLA-G)还可与CD8+细胞受体结合,诱导NK细胞和T细胞的凋亡、抑制同种特异性T细胞的细胞周期进程。除此之外,研究显示,HLA-G还可诱导调节性T细胞和抑制性NK细胞的产生,并可在很大程度上通过移植细胞颗粒极化来保护靶细胞不被NK细胞和T细胞杀伤。HLA-G分子除表达于胎盘绒毛膜细胞、肿瘤细胞外,还可少量表达在众多组织细胞上,包括T淋巴细胞。而胃癌患者中淋巴细胞HLA-G表达变化如何?作为抑制性受体ILT2、ILT4等的主要配体,在不同分化程度胃癌发生过程中所起作用如何,目前尚不清晰。研究目的和方法：一、本研究采用实时荧光定量PCR-Sybr Green I和FACS流式细胞术等方法对人的高分化胃癌细胞株(MKN1)、中分化胃癌细胞株(SGC-7901、AGS)、低分化胃癌细胞株(MGC803)和未分化分化胃癌细胞株(HGC-27、BCG823)中的mRNA和蛋白表达进行了检测和分析,系统地探讨了ILT2、ILT3和ILT4在不同分化程度的人胃癌细胞中的表达变化,为后期试验和相关研究提供了试验依据。二、为了探讨不同表达程度的抑制性受体ILTs对免疫细胞的影响,我们通过体外细胞试验,将ILT2lowILT3lowILT4low胃癌细胞MKNI和ILT2highILT3highILT4high胃癌细胞HGC-27分别与PBMC和NK细胞共培养,通过检测细胞杀伤活性(MTT),CD4+、CD8+T淋巴细胞和NK细胞上HLA-G分子的表达改变探讨抑制性受体ILTs对效应细胞的调控作用。三、为进一步探讨抑制性受体ILT2、ILT3和ILT4在临床应用中的价值,采用免疫组织化学方法检测了临床不同分化程度胃癌患者病理组织中ILT2、ILT3和ILT4的表达,并探讨了其与肿瘤生物学行为和临床特征的关系。四、为了探讨作为抑制性受体ILTs主要配体的HLA-G在临床胃癌患者淋巴细胞中的表达变化,应用TaqMan探针FQ-PCR和FACS流式细胞术对不同分化胃癌患者外周血淋巴细胞HLA-G mRNA和CD4+、CD8+T淋巴细胞上HLA-G分子表达及其与临床进程的关系进行了初步分析。以上研究通过体外细胞试验和临床应用研究,系统的探讨了抑制性受体ILT2、ILT3和ILT4在不同分化程度的胃癌细胞中的表达变化及其对效应细胞的影响,期望为阐明胃癌发生和转移的分子机制提供实验依据,同时为临床胃癌的诊断、防治和新药开发寻找新的靶目标提供依据。结果及结论：一、胃癌细胞株抑制性受体ILTs mRNA和蛋白表达1.实时荧光定量PCR-Sybr Green I检测结果表明,6株不同分化程度的人胃癌细胞均不同程度表达了ILT2、ILT3和ILT4 mRNA,尤以ILT2表达最强。其中在高分化的胃癌细胞株MKN1中ILT2、ILT3和ILT4 mRNA表达最低,未分化胃癌细胞株HGC-27和BCG823的mRNA表达最高,显示随分化程度存在显著性差异。2.FACS流式细胞术检测显示,6株胃癌细胞中除一株未分化胃癌细胞BCG823表达ILT3、ILT4极低外,其余均检测到ILT2、ILT3和ILT4不同程度的蛋白表达,且其表达程度与肿瘤细胞分化相关,尤以ILT2表现最明显。上述实验结果表明,通过某种共同信号途径使恶性程度高的胃癌细胞高表达抑制性受体ILT2、ILT3和ILT4,并可能影响到效应细胞的功能及对肿瘤的杀伤。二、抑制性受体ILTs对效应细胞调控的实验研究1.ILT2highILT3highILT4high胃癌细胞HGC-27与PBMC、NK细胞共培养后效应细胞杀伤活性(MTT)较ILT2lowILT3lowILT4low胃癌细胞MKN1与PBMC、NK细胞共培养后效应细胞杀伤活性显著降低,初步表明胃癌细胞不同表达程度的ILT2、ILT3、ILT4影响PBMC、NK细胞的增殖和杀伤作用。2.ILT2highILT3highILT4high的HGC-27细胞和ILT2lowILT3lowILT4low的MKNl细胞分别与PBMC和NK细胞共培养24h后,与各自对照相比,T细胞和NK细胞表面HLA-G的表达明显变化,且ILT2highILT3highILT4high的HGC-27细胞对HLA-G的影响较ILT2lowILT3lowILT4low的MKN1细胞更明显。其中T淋巴细胞HLA-G主要表达于CD4+T细胞,CD8+T细胞表达很低或基本不表达；CD56+NK细胞表达结果显示同样的趋势,但总体表达水平较CD4+细胞低。实验结果提示胃癌细胞上ILT2、ILT3和ILT4分子表达对效应细胞的杀伤活性有影响,并可能对HLA-G的表达有一定的调控作用。三、胃癌患者ILT表达的临床应用研究免疫组织化学分析显示,50例不同组织类型、不同分化程度的胃癌患者病理组织中ILT2、ILT3和ILT4分子有不同程度的表达。在肿瘤细胞上的表达形式是散在的,可位于胃癌细胞膜或胞浆内或两者皆有,病灶周围的基质细胞也有部分表达。ILT2、ILT3和ILT4表达阳性率在50例胃癌组织标本中差异较大。其中ILT2表达阳性率最高,为62.0%(31/50),ILT3、ILT4表达阳性率分别为18.0%(9/50)和34%(17/50),正常胃组织中未见ITL2,ILT3, ILT 4的表达。提示抑制性受体ILTs,尤其是ILT2有可能成为胃癌临床诊断的有价值指标,并为胃癌临床干预治疗提供新的靶点。四、胃癌患者淋巴细胞非经典HLA-G的表达1.通过TaqMan探针FQ-PCR检测发现,胃癌患者及健康对照淋巴细胞HLA-G mRNA,总体表达水平较弱。其中,胃癌临床分级Ⅰ+Ⅱ期的患者与健康对照相比相对表达量无显著性差异(P>0.05)；临床分级Ⅲ+Ⅳ期患者转录水平较Ⅰ+Ⅱ期相对表达量低(P<0.05)。而从临床病理组织分化程度分析,分化良好型与低分化型胃癌患者HLA-G mRNA表达未见明显改变。2.通过FACS流式细胞仪检测发现,无论健康对照和胃癌患者,淋巴细胞HLA-G分子表达阳性率均较低。与同型对照相比,CD8+T细胞基本不表达,HLA-G分子表达主要集中于CD4+T淋巴细胞,且表达阳性率在不同胃癌患者中变化幅度较大,可能与肿瘤微环境及相关影响有关。更多还原

【Abstract】 Background:Gastric Cancer is one of the most frequent malignant tumors which are harmful to human health.There are more than 9.3 million newly discovered cases annually in the world. The death rate of Gastric Cancer is approximately 29.34 per million (man 40.80 per million and woman 18.60 per million respectively) that is second larger in the world. According to Ministry of Health statistics in 2006, the number of Gastric Cancer patients is as many as 4 million, and 3 million people die of this disease every year in our country. Recently many scholars study the cause and diversion of Gastric Cancer and acquire some success. However, we are still in double regarding the mechanism of cancer, early diagnosis and estimate of prognostic. Thus, it is significant to research the mechanism of cancer and to discuss the state and function of immunocyte during the process of tumor. There is no doubt that it will be beneficial to understand and cure Gastric Cancer.At present, the pathogen of this cancer is not clear. Epidemiology research suggests the cause of Gastric Cancer relates to many factors such as food, infection and inheritance. Recently, it is considered that the process of Gastric Cancer is an interaction between immune system and cancer cells in host. It involves not only change of effector cell functions (abnormal quantity and function of T lymphocyte) and some cytokines (IFN-y and IL-10), but also the abnormal tumor cell self (high expression of FasL and lack of co-stimulatory molecules and decrease of MHC) and relevant oncogene and anti-oncogene modification. Among all the factors above, the lack of co-stimulatory molecules which are expressed on the cancer cell surface is one of the most important reasons that results in immune evasion. Positive stimulatory molecules influence activation and proliferation of T lymphocyte while negative ones inhibit the function of T lymphocyte through the follow two ways:they induce inhibitory factors to express on effect cells and tumor cells.Ig-like transcript (ILT) which is also called leukocyte inhibitory receptor(LIR) is a kind of newly discovered receptor family which recognize HLA-I. At present,9 members of the ILT family are known and they are expresses on a wide range of cells which include T lymphocyte, monocyte, NK and dendrite cells. Different kinds of ILTs play a great role in immunologic modification by transmitting inhibitory or simulative signals, for instance, in term of inhibiting activity of killer cells and regulating signal transmission between dendrite cells and T lymphocytes and so on. Inhibitory receptors (ILT2, ILT3, ILT4) consist 4 or 2 extracellular C-2 type Ig-SF domains and possess long cytoplasm tails that contain 3 immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the expression and function of these receptors have not been well understood on tumors.HLA-G molecule is one kind of nonclassical antigen and possesses a shore cytoplasm domain. HLA-G gene contains 8 extrons and 7 introns. Alternative splicing of the primary transcripts results in seven different mRNA, encoding four membrane-anchored (HLA-G1,G2,G3,G4) and three soluble (HLA-G5,G6,G7) isoforms, in which HLA-G 1 and HLA-G5 can combine ILT2 and ILT4 with high specificity. HLA-G plays an important part in establishing and maintaining immune tolerance by inhibiting the function of immune cells. HLA-G molecule exerts suppressive role via integrating with ILT2 and ILT4 which be expressed in NK cells, T lymphocytes and APC. Soluble HLA-G(sHLA-G) molecule also can link cells receptor with CD8+thus induces apoptosis of NK and T lymphocytes. Besides, the latest research suggests that HLA-G molecule can tempt the produce of Tregs and inhibitory NK cells, and protect the target cells from being damaged by NK and T lymphocytes to a large extent. HLA-G molecule can be primarily expressed on the surface of trophoblast cells and cancer cells. There are also a few HLA-G molecules which can be expressed on the surface of histiocytes, such as T lymphocyte. However, we are still wondering in the variety of HLA-G on T lymphocytes of patients with Gastric Cancer, which is main ligand for inhibitory receptor ILT2 and ILT4.Objectives and Methods:Ⅰ. We analyzed the mRNA and protein expression of well-differentiated Gastric Cancer cell lines (MKN1), moderate differentiation Gastric Cancer cell lines (SGC-7901、AGS), poorly differentiated cell lines(MGC803) and undifferentiation cell lines (HGC-27、BCG823) respectively by fluorescent quantization PCR-Sybr GreenⅠand FACS flow cytometer. And we discussed the variety of ILT2, ILT3, ILT4 in diverse differentiation of Gastric Cancer cell lines systemically, which provide experimental evidence for succedent research.Ⅱ. In order to explore the effect of the different degree expression of inhibiting ILTs to immune cells, we try to investigate that different leveles of inhibitory ILTs influence immunocytes in vitro. We cultureed well-differentiated Gastric Cancer cell lines (MKN1), which express slight ILT2/ILT3/ILT4, and undifferentiation Gastric Cancer cell lines (HGC-27), which express substantial ILT2/ILT3/ILT4, with PBMC and NK together respectively, and tested MTT and HLA-G in CD4+、CD8+T lymphocytes and NK to discuss the role that inhibitory ILT regulate effector cells.Ⅲ. Immunohistochemistry was used to detect the expression of ILT2/ILT3/ILT4 in pathologic tissue with diverse differentiation from patients who surfer Gastric Cancer to make further efforts to explore the value of ILT2/ILT3/ILT4 in clinical application.Ⅳ. We also introduced TaqMan probe FQ-PCR and FACS flow cytometer to inspect the expression of HLA-G that is ligand for ILTs in PBMC and CD4+、CD8+T lymphocyte, and to research the changes of HLA-G and analyse the relation between Gastric Cancer phase and the expression of HLA-G molecule preliminarily. In a word, from this part of research we discussed systemically the variety of ILT2, 3,4 in diverse differentiation of Gastric Cancer cell lines and their effect on immunocytes. It is expected to provide experimental evidence to illustrate the mechanism for the growth and metastasis of tumor and theoretical basis simultaneously for the diagnosis and treatment of Gastric Cancer and the exploiture of new drugs.Results and Conclusions:I. Expression of inhibitory ILTs mRNA and protein of human gastric cancer:an experiment study in vitro1. Using fluorescent quantization PCR-Sybr Green I, we detected the expression of ILT2,3,4 mRNA from 6 Gastric Cancer cell lines with diverse differentiation. From the results, we can see that all the differentiation groups express various extents ILT mRNA, and ILT2 mRNA is expressed the highest especially. In the mean time, well-differentiated Gastric Cancer cell lines (MKN1) express the lowest amount of ILTs among them and, on the contrary, undifferentiation Gastric Cancer cell lines (HGC-27 and BCG823) express the highest amount of ILTs. There is significant difference in 6 cell lines with diverse differentiation.2. We found that ILT2,3,4 are expressed with different extent in nearly all differentiation groups except undifferentiation Gastric Cancer cell lines (BCG823), which express little ILT3 and ILT4, and the different levels of ILTs relates to the differentiation of tumor cells by FACS flow cytometer.Experimental results above demonstrate that malignant Gastric Cancer cells can express ILT2/ILT3/ILT4 through some common pathway, which influence the function of effector cells and their killer activity to tumor cells.II Inhibitory ILTs regulation to effector cells:an experiment study in vitrol.By coculture ILT2highILT3highILT4high HGC-27 cell and ILT2lowILT3lowILT4low MKN1 cell with PBMC、NK respectively, we found that the killer activity of effector cells was reduced distinctly, and the former was more prominence than latter. Thus, we considered that varying degree of ILT2/ILT3/ILT4 that were expressed by human stomach cancer cell influences the proliferation and killer activity of PBMC and NK cells.2. Coculture were obtained as described above for 24 hours, we conclued that HLA-G at the surface of T. and NK cells varies markedly and ILT2highILT3highILT4hig HGC-27 had a more significant effect on HLA-G than those by ILT2lowILT3lowILT4low MKN1. Inaddition, HLA-G that is expressed at the surface of T lymphocytes mainly located CD4+T lymphocytes, and there was little even no HLA-G at the surface of CD8+T lymphocytes. Besides, HLA-G expressed on CD56+NK cells is lower than that in T lymphocytes.Experimental results indicated that ILT2/ILT3/ILT4 have an impact on the killer activity of effector cells, and may regulate the expression of HLA-G..III. Expression of inhibitory ILTs on gastric cancers:a clinical studyThe expression of ILT2/ILT3/ILT4 from 50 Gastric Cancer pathological tissues were tested. From the results, we can see that it is dissimilar with diverse differentiation and type of tumor, and ILT2/ILT3/ILT4 cound be expressed either in cytoplasm or membrane of Gastric Cancer cells and even both. The positive rate of ILT2,ILT3 and ILT4 expression among 50 Gastric Cancer pathological tissues also varies a lot, and the positive rate of ILT2 is the highest-62.0%(31/50), while the positive rate of ILT3 and ILT4 is 18.0%(9/50) and 34%(17/50) respectively. There was no ILT2/ILT3/ILT4 on normal tissues, which indicated that inhibitory ILTs, especially ILT2, may be a valuable index for diagnosing Gastric Cancer and provide a new target to treat gastric cancer.IV. Expression of non-classical HLA-G on PBLs of gastric cancers:a clinical study1. By Fluorescent quantitation RT-PCR, we considered that HLA-G mRNA in both lymphocytes from Gastric Cancer and healthy control was weak, and there was no significant difference betweenⅠ+ⅡGastric Cancer and healthy control (P>0.05) However, HLA-G mRNA inⅢ+ⅣGastric Cancer was lower than that inⅠ+Ⅱ(P< 0.05). Also, there is no distinct change between well-differentiated and poorly differentiated Gastric Cancer in term of clinical differentiation.2. By FACS Flow Cytometry, we found that the expression of HLA-G on lymphocytes both from Gastric Cancer and healthy control was low, and there was nearly no HLA-G expression on CD8+T lymphocytes of patients of Gastric Cancer. On the contrary, HLA-G molecules were mainly expressed on CD4+T lymphocytes, and the positive rate was also different in diverse patients, which was associated with tumor microenvironment and other influence factor.更多还原