【作者】 李吉学；

【导师】 王立东；

【作者基本信息】 郑州大学 ， 消化内科， 2003， 博士

【摘要】 食管癌是世界上最常见的六大恶性肿瘤之一，显著的地域性分布差异是食管癌流行病学突出特征，高低发区可相差500倍。并且食管癌预后很差，中晚期患者5年生存率仅为10％左右。中国是世界上食管癌发病率和死亡率最高的国家，每年全世界新增加的30万食管癌患者中，有一半发生在中国。在中国，河南省，特别是豫北地区的林州市(原林县)及其毗邻的辉县、安阳等地，是我国也是世界上食管癌发病率和死亡率最高的地区。过去几十年，老一代科学家对该地区人群进行了多学科、系统性、综合研究，在食管癌的发病因素，发病机制和防治方面做出了卓越的贡献。明确提出，食管癌变是一个多阶段进行性发展过程，确立了癌前病变的概念，提出亚硝胺及霉菌毒素等可能是该地区重要的致癌因素。但是，目前该地区人群食管癌发病率和死亡率仍未得到根本控制，甚至无明显改善。其主要原因是，对食管癌确切的发病因素和多阶段演进的分子学机制尚不十分清楚，缺乏有效的Ⅰ级预防措施和手段，缺乏早期诊断的生物学指标和方法。这也正是该研究领域所面临的重大研究课题。 目前普遍的观点认同食管癌变是多种基因变化综合作用(先后累积或叠加)的结果，食管癌变的不同阶段，分子变化可能不同，并是导致食管轻度癌前病变持续向癌的方向发展、决定食管癌前病变双向发展的不稳定特性的主要机制。纠正这些变化，或增强这些基因的正常功能，将有助于阻止或延缓癌变的发生。高发区人群普查和随访研究发现，食管癌前病变呈现明显的双向发展的不稳定特性，即这些病变可向癌的方向发展，也可退回到较轻度河南林州地区同一个体食管癌前病变和癌组织分子变化的特征的病变，或维持同一状态多年不变;很显然，单纯从形态学角度难以阐明这种现象。90年代开始，对食管癌变的分子变化有了较深入的了解。本实验室及其他学者的研究提示:食管癌变早期已发生许多重要的分子变化，主要包括某些肿瘤抑制基因的失活，如P53突变，Rb杂合缺失，P14和P15纯合缺失，以及某些癌基因的活化或过度表达，例如eyclin DI，C一myc，C一erbBZ等。这些分子变化可能是导致轻度癌前病变持续向癌的方向发展的重要机制。多数学者的研究是针对癌组织进行研究，对癌旁组织的研究较少，对同一个体癌组织及癌旁组织的研究报道更少。为了深入了解分子变化在食管癌变多阶段演进中的生物学意义，本研究利用Rl乍PCR、免疫组化及碘染色技术，系统分析同一个体手术切除组织的不同部位、不同组织类型的分子事件变化特征及规律，加深了对这些变化规律性及生物学意义的认识。1.材料与方法 1.1食管癌前病变组织、癌组织及无症状居民活检组织的收集及处理 45例癌和癌旁食管癌组织均来自河南食管癌高发区林州手术切除标本(男30例，女巧例，平均年龄57.37士8.34岁，年龄范围36一75岁)，所有患者术前均未接受放疗和化疗。手术切除标本病理学检查均为食管鳞状细胞癌。新鲜手术标本沿纵线切开分成两部分，一部分在癌肿组织和癌旁目测无病变部位(手术切端)分别取一对样品，做好标记，连同大体标本放入液氮中冷藏。另一部分在新鲜状态下进行卢戈氏(Lugol’s)液染色处理。 1.2色素镜(lugo一，s液染色)检查 在常规胃镜观察后，用洗涤喷雾管在食管表面均匀喷洒体积分数为2%的Lugofs液，1 05后立即温水冲洗，观察，对染色阳性及阴性区取活检粘膜。观察和记录染色结果，将碘染色区分为“染色区”，即碘染后食管粘膜呈棕褐色反应和“不染区”，即碘染后食管粘膜无呈色反应。将“不染区”细分为“点状不染区”。“花斑状不染区”，“条状不染区”和“片状不染区”等四种类型。每例碘染后，均认真冲洗并吸出冲洗液，尽量减少碘溶液的刺激作用。在上 郑州大学博士论文2003届述碘“不染区”和“染色区”分别取活检组织(所有不染区均取活检l一2块)，并常规在食管中段(距门齿30一32cm)和下段(食管赏门交界线上2一3cm)各取l一2块活检组织。 1.3食管癌手术切除大体标本粘膜碘染色 新鲜手术标本用生理盐水冲洗去粘液后，将粘膜面喷洒体积分数为1.2%Lugo，s碘液染色，105后立即在生理盐水冲洗，记录染色结果，并在所有片状不染区和染色区分别取材。共取样品43对，其中男30例，女13例，平均年龄5632士7.56岁，年龄范围36一75岁。 L4免疫组织化学 组织经85%乙醇固定、石蜡包埋、连续切片，用于组织病理学检查和免疫组化检查。免疫组化采用卵白素一生物素一辣根过氧化氢酶复合物(ABC)或过氧化物酶标志的链霉卵白素(SP)染色法。 1 .5 RT-PCR 选取16对癌和癌旁样品(其中男10例，女6例，平均年龄为56名7士6.48岁，年龄范围36一75岁)以及10例来自食管癌高发区无症状人群普查活检样品(其中男6例，女4例，平均年龄为41.2士3.34岁，年龄范围33一53岁)用于mRNA表达研究。新鲜组织经液氮冷藏后转入一70oC冰箱，RNA提取使用Promega公司sv Total RNA Isolation System试剂盒提取，反转录使用更多还原

【Abstract】 Esophageal carcinoma (EC) is one of the six most common malignant diseases worldwide. The dramatic geographic distribution is the striking characteristic for EC, the ratios for EC incidence between the high- and low-risk areas could be as great as 500:1. Moreover, EC has a very poor prognosis; the five-year survival rate for the middle and advanced EC is only 10%. Linzhou (formerly Linxian) and Huixian of Henan Province, northern China, have been well recognized as the highest incidence areas for EC in the world. EC remains a leading cause of cancer-related deaths in these area. The reason for this stable incidence pattern are because of that: the exact etiological factors for EC have not been identified; the mechanism of esophageal carcinogenesis is not very clear; and there are lack of useful reagents for chemoprevention, sensitive biomarkers for early detection and high-risk subject screening.Studies by us and other laboratories indicate that an early indicator for the subjects predisposed to EC is the abnormal hyperproliferation of esophageal epithelial cells, morphologically manifested as basal cell hyperplasia (BCH), especially at the papilla region, dysplasia and cacinoma in situ, all these lesions could be considered as preacancerous lesions for EC. Our field work has showed that these lesions appear unstable, i.e., of the lesions with the similar morphology, some may progress to more severe type, or remain at that stage for long time, or return to less severe lesion, even to normal. These phenomena raise some important questions. For example, what are the key factors to address the mild lesions to more severe lesions? What is the mechanism to determine thereversibility of these lesions? Apparently, these questions could not be answered only by morphological analysis. To further characterize the molecular changes and their correlations with morphological alterations at the different stages of carcinogenesis may provide important clues to elucidate these questions: mass survey and follow-up studies to the population at high-incidence area for EC with linear interval biopsies could not be overemphasized in elucidating the natural history for EC, accumulating important biological material bank for molecular analysis and evaluating the effects of chemoprevention.The knowledge about the molecular alterations of esophageal carcinogenesis has been greatly improved in 1990s. The studies finished by ours and the other research groups showed that many important molecular alterations have occurred in the early stage of esophageal carcinogenesis, mainly including some tumor inhibition genes inactive, such as p53 mutation, Rb LOH, p14 and p15 loss, and some oncogene, such as Cyclin D1, C-myc, C-erbB2 active or overexpression. The molecular alterations may be the key mechanism to address the mild precancerous lesions into cancer. Most of the studies has focused on the cancer tissues but not the adjacent tissues, and there are few reports on molecular changes in both cancer tissue and adjacent tissue from the same EC patient. To further characterize the molecular changes in the multistage progress in esophageal carcinogenesis from the same patient, we designed this multi-profile and multi-molecular study focusing on the molecular events in cancer and adjacent tissue from the same EC patient.1 Materials and Methods1.1 Surgical and biopsy tissue collection and processingAll the surgically resected primary EC specimens were collected from 45 EC patients in Linzhou, Henan, the high incidence area for EC (30 men and 15 women, 36 to 75 years of age, with a mean ±SD of 57 37 ±8.34 years,) . All the patients had not received either chemotherapy or radiotherapy before surgery. All the specimens were confirmed by pathology as esophageal squamous cell carcinoma. Within one hour after excision, each specimen was cut into two segments, thecancer tissue and its adjacent tissue were collected as one case in one segment and stored in liquid nitrogen for further study The other segment was stain更多还原