【作者】 张志勉；

【导师】 高海青；

【作者基本信息】 山东大学 ， 老年医学， 2011， 博士

【摘要】 肿瘤是一类严重威胁人类健康的多发病和常见病。近三十年来,肿瘤的发病率和死亡率一直呈现上升趋势。食管癌是常见的消化道肿瘤,每年大约有三十万人死于此病。我国是食管癌发病大国,每年大约有十五万人因食管癌去世,其病死率约占所有肿瘤的22.34%,仅次于胃癌,居第二位。目前食管癌发病机制尚未完全明确,一般认为其发病与食物粗糙、饮食过快、饮酒、吸烟、精神作用,遗传以及食管炎症等关系密切。手术治疗对于原位癌十分有效,但患者常发生肿瘤转移或术后复发,而且,放、化疗对术后患者生存率亦无明显改善,术后五年生存率仅为25-40%。然而,研究发现,食管癌早期诊断患者五年生存率超过90%,因此,对于目前的医疗而言,早期诊断的意义尤为重要。目前对食管癌的诊断主要有食管拉网细胞学检查,食管钡餐造影,内镜检查及影像学检查等手段,但需要进行专业人员操作培训及配备相应的设备仪器,不适于基层和高发区食管癌的筛查,更不适用于大规模临床筛查。主要组织相容性复合物(major histocompability complex, MHC)是位于哺乳动物某一染色体上的一组紧密连锁的基因群,其蛋白产物的主要功能是参与抗原递呈和T细胞激活,在免疫应答和免疫调节中执行着重要而广泛的功能。其中MHC-Ⅰ基因编码的Ⅰ类分子主要参与排斥反应、抗体产生、细胞裂解和向CD8阳性T细胞提呈抗原。在肿瘤免疫研究领域,以往的研究多集中在肿瘤细胞表面的MHC-1分子上,肿瘤组织MHC-1分子表达降低或缺失,循环中的CTL细胞表面不能有效识别肿瘤抗原,癌组织中浸润淋巴细胞功能下降。本课题组对多种肿瘤,包括胃癌、肝癌、食管癌、乳腺癌等的研究表明,外周血单个核细胞表面的MHC-Ⅰ均表达降低,而且与肿瘤的分期存在一定的相关性,有望成为肿瘤早期诊断的标志物。基于此,本课题的研究内容包括以下三个部分：第一部分外周血单个核细胞MGC-Ⅰ表达在食管癌中的表达研究目的：比较食管癌患者、食管良性病变患者和正常个体外周血单个核细胞MHC-I mRNA和蛋白水平的表达变化,分析其与肿瘤分期、进展和转移的关系,探讨外周血单个核细胞MHC-I与食管癌临床病理特征的关系。材料和方法：收集2007年6月至2010年7月在山东大学齐鲁医院住院的食管鳞癌患者58例、良性食管疾病患者46例和健康志愿者65例,按照国际标准进行TNM分期,采用实时荧光定量PCR检测外周血单个核细胞MHC-I mRNA的表达变化,采用流式细胞术检测外周血单个核细胞表面MHC-I蛋白的变化,与食管癌分期、进展和转移相结合。结果：(1)食管癌患者、食管良性疾病患者和正常对照个体外周血单个核细胞MHC-I mRNA的表达水平分别为0.48±0.18,0.85±0.33和1.09±0.38。食管癌患者与正常对照个体MHC-I mRNA表达水平差异有统计学差异(P<0.05),食管癌晚期患者(ⅡⅢ、Ⅳ期患者)与正常对照个体间的差异更为明显(P<0.01)。(2)食管癌患者、食管良性疾病患者和正常对照个体外周血单个核细胞表面MHC-I分子分别为75.14±38.26,115.14±33.32和142.58±43.77,与mRNA表达水平的差异趋势一致。(3)外周血单个核细胞MHC-I的表达不受年龄和性别的影响,但在食管癌患者中,Ⅲ、Ⅳ期患者表达水平明显降低(p<0.01),Ⅰ、Ⅱ期患者与健康人相比差异亦有统计学意义(P<0.05)；而且,淋巴结转移的食管癌患者外周血单个核细胞表面MHC-I表达低于未转移患者(p<0.05)。(4)外周血单个核细胞MHC-I的表达与食管癌浸润深度、分化程度与否无关。结论：外周血单个核细胞MHC-I表达不受患者年龄、性别的影响,与食管癌浸润和分化程度无关,与食管癌临床分期和淋巴结转移有关,而且在食管癌进展早期即降低,表明外周血单个核细胞MHC-I可能作为食管癌发病早期的分子标志物,可能有助于食管癌的早期诊断。第二部分外周血单个核细胞MHC-I在食管癌高发区人群中的表达研究目的：通过选取食管癌高发区现场进行相关影响因素分析,并结合高发区人群肿瘤血清标志物与外周血中MHC-I表达的联合检测,研究各种相关因素在食管癌发病前期的影响,以及评价MHC-I作为反映机体免疫状态指标的可能性,并将宿主免疫状态低下与食管癌的易感性增加相联系,能够为食管癌的防治提供有效保护措施,也为食管癌的早期临床诊断提供新思路。方法：本研究采用人群为基础的病例对照研究,以食管癌高发区大汶河中下游流域自然村35-70岁无症状人群作为研究对象进行流行病学问卷调查和血样抽取,并以性别、年龄,居住情况为匹配条件,选取济南当地自然村中性别、年龄比例相当的群体作为对照,采取如下检测方法：(1)所有参加筛查的目标人群在签署知情同意书后方可进行一对一的流行病学问卷调查,内容主要包括基础信息、危险因素的收集。(2)通过拟合条件Logistic回归模型,采用单因素和多因素分析各暴露因素与食管癌的关系；(3)采集高发区现场与非高发区现场的土壤和水质样本送环保部门进行微量元素检测；(4)应用肿瘤血清标志物联合检测系统(TC-12)将高发区人群根据检测结果分为高危组(TC-12阳性)和低危组(TC-12阴性)；(5)采用流式细胞仪测定高发区人群(包括高危组和低危组)与正常对照人群外周血中单个核细胞表面MHC-I的表达情况。结果：(1)单因素分析：根据单因素分析初步筛选结果,以P<0.05进行变量筛选。文化水平低、饮水年数>20年、经常食用煎饼、经常食用热粥、经常空腹饮酒、新鲜蔬菜水果摄入较少等因素,经检验差异无统计学意义(P>0.05)；收入水平较低、室内油烟较多、粮食霉变后剔除部分霉变后继续食用、重盐口味、喜食腌菜、饮食干硬、吸自制烟、饮酒量每天>500ml、自我调节情绪差、不易适应环境以及疾病家族史等因素,经检验差异有统计学意义(P<0.05),可能成为食管癌的危险性因素：而经常食用葱姜蒜、饮茶,经检验差异有统计学意义(P<0.05),可能成为食管癌的保护性因素。(2)多因素分析：将单因素分析结果中P<0.05变量的因素,按照sle=0.3,sls=0.2逐步进入回归模型进行条件Logistic回归分析。结果显示：饮食干硬、吸自制烟、每天饮酒量>500ml、疾病家族史这4个因素有统计学意义(P<0.05),可能成为食管癌的危险性因素；同时饮茶也具有统计学意义(P<0.05),可能成为食管癌的保护性因素。(3)与非高发区相比,高发区现场的河水、地下水及土壤中总铬含量超标,差异有统计学意义(P<0.05)。(4)高发区组和正常对照组人群中,随着年龄的增加,T淋巴细胞和NK细胞在外周血淋巴细胞总数中所占比例无明显差异。(5)高发区组和正常对照组人群中,各个年龄段组中MHC-I在T淋巴细胞和NK细胞表面的表达差异无统计学意义(P>0.05),在男性和女性群体中MHC-I的表达水平差异也无统计学意义(P>0.05)。(6)与正常对照组人群相比,高发区人群外周血T淋巴细胞和NK细胞表面MHC-I表达水平低下,NK细胞表面MHC-I表达水平减低更为显著(P<0.001)；在高发区人群中的高危组(TC-12阳性)与低危组(TC-12阴性)相比较,前者外周血T淋巴细胞和NK细胞表达MHC-I的水平较后者亦有降低,差异均有统计学意义(P<0.05)。(7)自食管癌高发区和正常对照地区采集的374例样本经由后期数据整理分析表明,在26例肿瘤血清标志物(TC-12)阳性个体中,有22例个体同时暴露于我们筛选出的食管癌相关危险因素(即方法(三)中单因素及多因素条件Logistic分析),从而说明了食管癌众多高危因素可能影响肿瘤血清标志物的异常阳性表达,并进一步导致了机体外周血中PBMCs中MHC-I的表达降低。结论：本研究中食管癌高发区人群与正常对照组人群外周血中T细胞和NK细胞表面的MHC-I降低,而在高发区个体中肿瘤标志物TC-12阳性组以及阳性组中大部分暴露于食管癌高危因素的个体其外周血中MHC-I表达降低更为显著,从而说明了食管癌众多高危因素可能影响肿瘤血清标志物的异常阳性表达,并进一步导致了机体外周血PBMCs表面MHC-I的表达降低。提示可将外周血淋巴细胞的MHC-I的表达水平初步作为反映宿主免疫状态的指标之一,其表达高低影响了宿主的肿瘤易感性,并通过长期临床随访及最终发病情况对该指标的准确性和特异性进行客观评价,由此可为食管癌大规模人群早期筛查提供准确无创的检测手段。第三部分外周血单个核细胞MHC-I在食管癌中表达改变机制的初步探讨目的：1.检测食管癌患者血清中Th1、Th2型细胞因子的表达水平,探讨食管癌患者Th1/Th2漂移与PBMCs表面MHC-I类分子表达的关系；2.观察食管癌细胞株EC9706、EC9706培养上清对PBMCs表面MHC-I类分子的表达的影响,并检测此时PBMCs中APMmRNA的变化；3.干扰PBMCs表面MHC-I类分子的表达,观察其受抑制后T淋巴细胞生物学活性的改变。方法：1.收集食管癌患者、食管良性疾病患者和正常人血清,应用双抗体夹心ELISA法检测IL-2、IFN-γ、IL-4和IL-10；2.将EC9706培养上清、EC9706与正常人PBMCs共培养,流式细胞术检测PBMCs表面MHC-Ⅰ类分子的表达；3.收集与食管癌细胞株培养上清共培养后的PBMCs,提取其mRNA,检测TAP1、TAP2、LMP2和LMP7表达的改变；4.应用RNAi技术,通过抑制β2m的表达抑制MHC-I类分子的表达,并在10-200nM浓度范围内,结合细胞死亡率与转染率,检测最佳siRNA转染浓度；在成功转染后,检测T淋巴细胞活化、增殖和凋亡活性在MHC-Ⅰ类分子受抑制后的改变。结果：1.食管癌患者外周血IL-2、IFN-γ降低,IL-4、IL-10表达升高,Thl型细胞因子/Th2型细胞因子浓度比值与MHC-I类分子的表达呈负相关。2.食管癌细胞株EC9706和其培养上清均可降低PBMCs表面MHC-Ⅰ类分子的表达,两者降低程度间的差异无统计学意义。3.EC9706可以降低MHC-I的APM分子TAP1和LMP2 mRNA的表达(P<0.05),但对另外两种APM分子TAP2和LMP7 mRNA的表达无影响。4.有效干扰PBMCs表面MHC-I类分子表达的p2m浓度为100nM,此时转染率为59.01%,细胞存活率为62.17%。成功抑制MHC-I后,T淋巴细胞活化、增殖均受抑制,凋亡细胞数增多。结论：1.食管癌患者Thl型细胞因子表达降低,Th2型细胞因子表达升高,即发生了Th1/Th2漂移。2.食管癌对PBMCs表面MHC-I的抑制作用主要通过分泌的细胞因子实现的。3.TAP1和LMP2参与了EC9706对MHC-I类分子的抑制作用,而TAP2和LMP7不参与这一过程。4.T细胞表面的MHC-I类分子表达降低可以使T淋巴细胞的活化、增殖受抑,凋亡增加。更多还原

【Abstract】 Esophageal cancer (EC) is a common digestive tract malignant cancer and the sixth most common cause of cancer-related death, the majority of which is squamous cell carcinoma. There are approximately 300,000 people dying of this disease every year1. China is one of the high incidence countries in the world, and number of death is about 150,000 people per year2. Surgery is the standard treatment for localized and resectable EC, but patients often experience distant metastasis or local recurrence even after curative operation. Furthermore, combing preoperative chemotherapy and/or radiotherapy with surgery have often shown no significant survival benefit3-5. Consequently, long-term outcome remains unfavorable and overall 5-year survival after surgery is only 25-40%. However,5-year survival of early cases exceeds 90%6. Thus, to improve patients’ prognosis, novel strategies need to be established for early diagnose.MHC-I molecules are cell surface glycoproteins that play critical roles in the regulation of immune responses. These molecules are expressed on the surface of all nucleated cells, necessary for the presentation of peptide antigens to CTLs7 and for the immune regulatory activity exerted by NK cells8. Under physiological conditions, the expression of MHC-I might be regulated in a tissue-specific, differentiation-dependent, cell cycle-controlled manner and modulated by the microenvironment, thereby altering the repertoire of T cell epitopes presented. MHC-I molecules in esophageal squamous cell carcinoma (ESCC) cells are often down-regulated at both the protein and mRNA levels, which is considered as an early event in carcinogenesis9. Many factors have the ability to reduce the expression of MHC-I on tumor surface. For instance, hypermethylation of the promoter regions is a major mechanism of transcription inactivation of MHC-I genes10. Expression levels of MHC-I molecules on EC cells are related to lymph nodes metastasis and patient’s survival11. Furthermore, polymorphisms of MHC-A and -B genes affect the genetic susceptibility to EC12. However, invasive surgery or biopsy is necessary in order to examine the MHC-I expression on EC cell surface. Thus, the expression of MHC-I molecules on tumor cells can not meet the need of early diagnosis of EC. MHC-I molecules are plentiful on the surface of peripheral blood mononuclear cells (PBMCs), but their clinical significance in tumor progression remains unclear.Chapter I. Investigation of MHC-I expression on PBMCs in esophageal cancer patientsObjective: To compare the expression of MHC-I on PBMCs at mRNA and protein levels in the serum of patients with esophageal cancer, patients with benign esophageal diseases and healthy individuals, and analyse the correlations of MHC-I with tumor stage, advance and metastatis, then investigate the relationship of MHC-I on PBMCs with clinicopathological characteristics of esophageal cancer.Materials and Methodes:ESCC patients who were histopathological confirmed in Qilu Hospital of Shandong University, Jinan, China (June 2007 to July 2010) were enrolled in this study. None of them had been treated with radiotherapy, chemotherapy prior to sampling. A total of 58 EC patients (23 females and 35 males, age range 56-69 years, median age 63.2 years) were involved in this study. According to the tumor, node and metastasis (TNM) classification,21 patients had tumors of histological grades I and II, and 37 grades III and IV. Expression levels of MHC-I protein and mRNA on PBMCs were determined by flow cytometry and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), respectively.Results:(1) The expression level of MHC-I mRNA on PBMCS of EC patients, benign disease patients and healthy volunteers was 0.48±0.18,0.85±0.33 and 1.09±0.38, respectively, which showed that MHC-I mRNA on PBMCS was significantly down-regulated in EC patients (P<0.05). And this down-regulation was more significant in EC patients with stage III and IV (P<0.01).(2) MHC-I expression on PBMCs from EC patients, benign disease patients and healthy volunteers was 75.14±38.26,115.14±33.32 and 142.58±43.77, respectively. The decreased expression of MHC-I protein on PBMCs surface was similar to that of MHC-I mRNA, with more significant than that of MHC-I mRNA.(3) The decreased MHC-I expression on PBMCs of EC patients was not influenced by age and gender, but in EC patients, this parameter was reduced significantly (p<0.05), especially in stage III and IV EC patients (p<0.01). Furthermore, MHC-I on PBMCs in EC patients with lymph node metastasis was lower than that with no lymph node metasis.(4) There was no relationship between MHC-I on PBMCs with invasive depth and differentiation degree of EC.Conclusions: MHC on PBMCs was not influenced by age and gender of patients, and had no relationship with invasion and EC differentiation, but had a negative correlation with clinical stage of EC, and was downregulated at early stage of EC. Thus, MHC-I on PBMCs of EC patients might be used as a potential biomarker of EC, and useful to early diagnosis of EC.Chapter II. Investigation of MHC-I expression on PBMCs in individuals from high-incidence region of esophageal cancerObject: To explore the relevant influencing factors in region of high-incidence esophageal cancer, and combine with detections of serum tumor markers and the MHC-I expression in peripheral blood. The present study made an evaluation of MHC -I as an indicator to reflect host immune status.Method:By the method of populmion-based case-control study, the epidemiological data and blood samples was collected from the residents aged range 35 to 70 in Dawen River Basin as region of high incidence of esophageal cancer. To make the gender, age, living conditions matched, we selected the local villages in Jinan as a normal control group.(1) All participants in the investigation were asked to sign an informed consent before the epidemiological survey, which mainly include basic information, impact factors etc.(2) Single- and multi-factors analysises were used to analysis the relationship between exposure factors and esophageal cancer.(3) The soil and water samples collected from the regions of high-incidence esophageal cancer and normal control group were for Trace elements detection.(4) The population in region of high-incidence esophageal cancer were divided into high-risk group (TC-12-positive) and low-risk group (TC-12 negative) according to result of serum tumor marker detection system (TC-12).(5) MHC-I expression on peripheral blood mononuclear cell surface in region of high incidence (including high-risk groups and low-risk group)and normal control using flow cytometryResult:(1)Single-factors analysis:According to single-factors analysis with P<0.05, the results indicated that lower income, more indoor cooking fumes, removed some food mildew before eating, tasted salty, dry and hard eating, self-made tobacco, alcohol>500 ml/day, drinking alcohol with xihu, poor self-regulation emotional, difficult to adapt to enyironmental, and family history of cancer had statistically significants, All these described above may be the risk factors. While the regular consumption of Congj iang Suan and the tea inspection may be the protective factors for esophageal cancer(P<0.05).(2) Multi-factors analysis:The results in multi-factors analysis(P<0.05)were enter the regression model for Logistic regression analysis according to sle=0.3 and sls=0.2. The results showed that drinking tea may be a protective factors for esophageal cancer,while dry and hard eating, Self-made tobacco,alcohol consumption of>500ml/day, family history of cancer may be risk factors for esophageal cancer.(3)Compared with non-high incidence, high incidence of on-site river, ground water and soil content of total chromium exceeded, the difference was statistically significant (P<0.05)(4) With the increase of age, percentages of T lymphocytes and NK cell did not show a statistically significant difference. In the same age group, there were no significant changes of percentages of T lymphocytes and NK cells between high incidence villagers and normal controls.(5) With the increase of age, there was no significant difference in the expression of MHC class I on T lymphocytes and NK cells (P>0.05). Gender had no effect on the expression of MHC class I on T lymphocytes and NK cells(P>0.05). In the same group by age or gender, there were no significant differences between high incidence villagers and normal controls.(6) Compared with normal controls, MHC class I on T lymphocytes in Lijiadian villagers were significantly lower (P<0.05). The levels of MHC class I on NK cells was even lower (P<0.001). In high incidence villagers, subjects were divided into two groups according to the C-12 levels. Expression of MHC class I on T lymphocytes and NK cells in high-risk groups were significantly lower than in the low-risk group (P<0.05).(7) By the post-data analysis showed that:in 26 cases of tumor markers in serum (TC-12) positive individuals, there were 22 cases of individual exposure to esophageal cancer related risk factors simultaneously, which explains a number of risk factors for esophageal cancer may affect tumor serum markers for abnormal expression, and further lead to reduce the expression of MHC-I in PBMCs.Conclusion:To make an evaluation of MHC-I as an indicator to reflect host immune status. MHC-I expression on peripheral blood mononuclear cell surface may provide accurate non-invasive early detection methods for esophageal cancer screening large populations, and will substantially improve the early diagnosis of esophageal cancer. Chapter III Pilot studies of the mechanisms of MHC-I changes on PBMCs in esophageal cancer patientsObject:(1) To measure the expression of cytokines secreted by Thl/Th2 lymphocytes in the serum of EC patients, in an attempt to investigate the relationship between cytokines and MHC-I molecules on the surface of PBMCs.(2) To observe the effect of culture supernant of EC cell line on MHC-I molecules expression on PBMCs surface.(3) To oberve the changes of T lymphocytes biological behaviors after MHC-I molecules were inhibited by RNAi technique.Methods:(1) Serum from EC patients, patients with benign esophageal cancer diseases and healthy individuals was collected and double antibodies sandwich ELISA were used to measure the expression levels of IL-2, IFN-y, IL-4 and IL-10.(2) PBMCs from healthy individuals were co-cultured with supernants from EC9706, or with EC9706, respectively, and MHC-I on PBMCs surface was measured by FACS.(3) PBMCs were collected, and mRNA was extracted to detect the changes of APM mRNA.(4) Using RNAi technique, MHC-I molecules were inhibited by inhibiting the expression of (32m. Then combining cell death rate and transfection rate, the optimal transfection concentration of siRNA was determined between 10 and 200 nM. After successfully inhibited, biological behaviors of T lymphocytes, such as proliferation and apoptosis, were measured.Results:(1) In the serum of EC patients, IL-2 and IFN-γwere down-regulated, and IL-4 and IL-10 were up-regulated. The ratio of Thl type cytokines and Th2 type cytokines had a negative correlation with the expression of MHC-I molecules on the surface of PBMCs.(2) EC9706 and its cultured supernant had the same ability to reduce the expreesion of MHC-I on the surface of PBMCs.(3) EC9706 could reduce the expression of TAP 1 and LMP2 Mrna (p<0.05), but had no effect on TAP2 and LMP7. (4) The optimal concentration ofβ2m to interfere with the expression of MHC-I molecules on the surface of PBMCs was 100nM. At the moment, transfection rate was 59.01%, and cell survival rate was 62.17%. Activation, proliferation of T lymphocytes were inhibited, and apoptosis was promoted after MHC-I on the surface of PBMCs was successfully inhibited.Conclusions:(1) Then levels of Thl type cytokines weakened and Th2 type cytokines enhanced. That is, Th1/Th2 shift happened in EC.(2) Cytokines secreted by EC played a major role in inhibiting MHC-I on PBMCs.(3) TAP1 and LMP2 were involved in the inhibiting role of MHC-I on PBMCs by EC, but nor TAP2 and LMP7.(4) Downregulation of MHC-I molecules on T lymphocytes could weaken the activation and proliferation, and increase the apoptosis.Creative Points in This Study1. To clarify the changes of MHC-I on PBMCs in EC for the first time, and put forward the hypothesis that this parameter may be a potential marker for the early diagnosis of EC.2. Thl/Th2 shift in EC could inhibit the expression of MHC-I on PBMCs, and TAP1 and LMP2 were involved in this process.3. Downregulation of MHC-I on T lymphocyte surface could inhibit immune activities of T lymphocytes.更多还原