【作者】 张晶；

【导师】 段志军；

【作者基本信息】 大连医科大学 ， 内科学， 2012， 博士

【摘要】 目的：胃癌是世界上癌症致死的第二大病因。全球每年约有8.7万新增胃癌病例。该病的发病率存在显著的地域性，在东亚很高，而在欧洲、北美及非洲却很低。中国人的胃癌发病率仅低于韩国和日本。胃癌的预后与诊断的阶段关系十分密切。早期胃癌（early gastric cancer，EGC）患者的五年生存率超过90%，而晚期胃癌患者的预后却很差。因此，胃癌的早期诊断对于取得良好的预后至关重要。然而，由于大多数EGC患者没有特异性症状，将其与良性胃病变如胃炎鉴别是非常困难的。在许多国家中，EGC的诊断率仅为10%~20%，而在我国EGC病人检出所占比例更低，文献报道仅为4％~10％，且检出后多同进展期胃癌一样进行外科胃大部切除根治术及淋巴结清扫术。如何提高我国EGC的检出率、选择创伤最小且疗效等同外科手术的合理治疗，以改善胃癌患者预后及生活质量是当务之急。内镜结合病理仍是至今确诊胃癌的金标准，但很多癌前病变及EGC的内镜下改变相当细微，常规内镜检查容易漏诊，随着内窥镜成像技术的发展，某些内窥镜成像模式，如窄带成像（narrow-band imaging，NBI）内窥镜技术，已被用于对EGC的诊断。尽管有文献报道这一内窥镜成像模式能够通过增强形象化而提高EGC和癌前病变的诊断率，但关于将窄带成像与常规的内窥镜技术进行比较的研究尚少。分子生物学技术的发展给胃癌的早期诊断提供了有力的依据，有些肿瘤相关蛋白在癌前病变及EGC时出现明显变化，检测这些指标结合内镜检查有助于癌前病变及EGC的检出，可用于筛查高危人群并为临床综合诊断提供证据。在我们以往的研究中发现，用免疫组化方法检测到的Galectin-3蛋白表达从正常胃粘膜组到进展期胃癌组呈递增趋势，P27蛋白则呈递减趋势，两者表达正好相反，且在正常胃粘膜组织及癌前病变组织中表达与早期癌组织中表达都具有显著差异。本研究进一步加大病例数量，并应用RT--PCR方法从基因水平检测两者的表达情况，为寻求诊断EGC的有效指标提供更多的依据。胃癌治疗的关键是彻底切除病灶以及有可能转移的淋巴结。以往EGC治疗同进展期胃癌一样采用外科根治术及淋巴结清扫术，或胃局部切除术及节段切除术,还有留幽门及迷走神经功能的部分切除术，但都需切除部分胃，有较高的并发症发生率及死亡率，若术后病理发现有更深层的侵犯或切缘阳性时，还需再次手术。术后生活质量似进展期胃癌。EGC的治疗目标应当是在保证疗效的基础上将损伤降到最低以提高术后生活质量。近年来由于内镜技术的进步及治疗器械的开发，内镜下黏膜切除术(endoscopic mucosal resection，EMR)和内镜粘膜下剥离术(endoscopic submucosaI dissectjon，ESD)内镜技术应用于临床治疗EGC及癌前病变，该技术由于保留了消化道的完整性，显著提高了生活质量，在不手术的情况下获得完整的组织病理标本，也有助于临床选择正确的后续治疗，避免不必要的胃切除手术。尽管这种治疗方法有着外科手术无法比拟的优点，但关于EGC的内镜下粘膜切除治疗和外科剖腹手术治疗进行对比的临床研究较少。本研究的目的就是探讨在癌前病变及EGC阶段，放大窄带成像内镜下改变及Galectin-3及P27在胃癌进展中的变化，揭示其在EGC及癌前病变诊断中的价值，将内镜下粘膜切除术与外科手术治疗早期胃癌进行多角度对比分析，为寻找早期胃癌及癌前病变简便敏感、安全可行的诊治方法提供理论依据。本研究共分三部分第一部分运用放大窄带成像内窥镜诊断早期胃癌及癌前病变的研究第二部分Galectin-3及P27表达在胃癌进展中的意义第三部分内镜下粘膜切除术与外科手术治疗胃早期癌的多角度比较研究方法1、常规内镜下疑诊早期胃癌或癌前病变患者526例，经内镜切除或手术切除的组织标本病理确诊为早期胃癌或癌前病变122名，年龄最小16岁，最长94岁（包括83名男性和39名女性），平均年龄为63.5±14.1岁。122名患者均接受了包括常规内窥镜、放大NBI和放大色素内镜在内的检查，检查过程中对影像的质量进行了评价，并且分析了各种内镜模式下病变的形态、胃小凹分型及毛细血管形式。在组织切片中如果出现胃部恶性上皮肿瘤和高级别上皮内瘤变，在病理学上即视为阳性，用于对内窥镜诊断的准确性进行评价。2、采用免疫组织化学（S-P）方法及RT-PCR方法检测在正常胃粘膜组织40例、癌前病变组织50例、早期胃癌56例、进展期胃癌46例的Galectin-3、P27蛋白及mRNA表达情况。入组标准：（1）正常胃粘膜组：既往无胃肿瘤病史，内镜下见粘膜光滑，无凹陷及异常隆起，颜色正常，有光泽。（2）癌前病变：经内镜下活组织检查病理回报为癌前病变，再经内镜下粘膜切除或剥离术后病理证实除外癌变者。（3）早期胃癌组：1）内镜下病理诊断为癌，限期手术病例。2）经内镜下粘膜切除术或外科手术后组织切片病理证实为病变局限于黏膜或黏膜下层者，无论有无淋巴结转移。（4）进展期胃癌组：1）内镜下病理诊断为癌限期手术病例。2）外科手术后病理证实为病变突破黏膜下层者。排除标准：（1）既往恶性肿瘤病史者。（2）急诊手术者。（3）术前发现远处转移、周围脏器浸润者。（4）妊娠期、危重症及不能接受手术治疗者。（5）出凝血时异常者。3、回顾81例早期胃癌接受内镜下粘膜切除术及外科剖腹手术患者的临床和手术资料其中内镜下粘膜切除44例,外科剖腹手术37例，并比较两组的手术时间、手术费用、住院天数、术中术后并发症、术后随访等结果。结果1、就图像的清晰度而言，放大窄带成像和放大色素内镜在形态学、胃小凹分型及毛细血管形式方面，都要远远地优于传统放大内窥镜（P <0.01），而放大窄带成像在毛细血管形式方面，又要远远地优于放大色素内镜（P <0.01）。在患有高级别上皮内瘤形成的患者当中，检测到IV型、V1型和VI型胃小凹形态的患者分别为14例、43例和17例。在患有早期胃癌的患者中，V1型和VI型胃小凹形态的患者分别为9例和39例。有109例患者发现有不规则的微小血管和血管直径上的变化。对早期胃癌和癌前病变的诊断的准确率、敏感度、特异性、假阳性率和假阴性率分别为，常规内窥镜：68.9%、95.1%、63.1%、24.5%、32.4%，放大窄带成像：93.6%、92.7%、94.5%、5.7%、6.9%，色素内镜：91.3%、88.6%、93.2%、13.2%、21.48%。2、胃癌组织中G alectin-3蛋白阳性表达率为72.6%，在高中、低未分化胃癌中的阳性表达率分别为73.3%和71.4%，两组相比无显著差异性（p>0.05）；在TNM分期Ⅰ＋Ⅱ阳性表达率60.7%，在TNM分期Ⅲ＋Ⅳ阳性表达率80.4%，两组相比有显著差异性（p<0.05）；在有淋巴结转移和无淋巴结转移中阳性表达率分别为94.1%和61.8%,两组相比有显著差异性（p<0.05）；Galectin-3蛋白表达阳性率在正常粘膜组（30.0%）与癌前病变组（44.0%）无显著差异性（p>0.05），但明显低于早期胃癌组（64.3%）（p<0.05）和进展期胃癌组（82.6%）（p<0.01）；Galectin-3蛋白表达阳性率在癌前病变组（44.0%）与早期胃癌组（64.3%）无显著差异性（p>0.05），但明显低于进展期胃癌组（82.6%）（p<0.01）；Galectin-3蛋白表达阳性率在早期胃癌组（64.3%）与进展期胃癌组（82.6%）无显著差异性（p>0.05）。胃癌组织中P27蛋白阳性表达率为35.2%，在高中、低未分化胃癌中的阳性表达率分别为43.3%和23.8%，两组相比无显著差异性（p>0.05）；在TNM分期Ⅰ＋Ⅱ阳性表达率39.3%，在TNM分期Ⅲ＋Ⅳ阳性表达率30.4%，两组相比无显著差异性（p>0.05），在有淋巴结转移和无淋巴结转移中阳性表达率分别为58.8和23.5%，两组相比有显著差异性（p<0.05）。正常粘膜组P27蛋白表达阳性率（85.0%）与癌前病变组（72.0%）相比无显著差异性（p>0.05），但显著高于早期胃癌组（42.6%）和进展期胃癌组（26.0%）（p<0.01）；癌前病变组P27蛋白表达阳性率（72.0%）显著高于早期胃癌组（42.6%）（p<0.05）及进展期胃癌组（26.0%）（p<0.01）；早期胃癌组P27蛋白表达阳性率（42.6%）与进展期胃癌组（26.0%）相比无显著差异性（p>0.05）。Galectin-3的mRNA在正常粘膜组的表达低于癌前病变组与早期胃癌组，癌前病变组与早期胃癌组之间无明显差异，但都明显低于进展期胃癌组；P27的mRNA表达在正常粘膜组，癌前病变组，早期胃癌组和进展期胃癌组中呈递减趋势。Galectin-3和P27的mRNA表达检测结果与其相应蛋白在组织中的表达结果一致。3、早期胃癌粘膜切除术治疗的手术时间、手术费用、住院天数、术后肛门排气时间及自主下床活动时间均少于外科剖腹手术组，具有统计学差异（P<0.05）。术中术后并发症发生率及术后镇痛剂使用率粘膜切除术均低于外科剖腹手术组，具有统计学意义（P<0.05）。术后随访二组暂均未发生残留、局部复发、异时病灶。在麻醉方式、手术入路、脏器损伤情况以及工作能力、静脉药物治疗时间、体力状况等术后生活质量评价上，粘膜切除术治疗明显优于外科手术治疗。结论1、放大窄带成像对于早期胃癌及癌前病变的诊断，要优于常规内窥镜，并且可被用于胃部恶性肿瘤的早期筛查。2、Galectin-3蛋白及mRNA表达的阳性率与胃癌发生发展相关，有可能作为早期胃癌筛查及判断胃癌淋巴结转移评估预后的重要指标；P27蛋白及mRNA表达的阳性率降低与胃癌发生发展相关， P27表达的减少有可能作为早期胃癌筛查及判断胃癌恶性程度、淋巴结转移及评估预后的有效指标。Galectin-3和P27的蛋白及mRNA在胃癌进展中的表达相反，Galectin-3和P27阳性表达在胃癌组织中无明显相关性，但同时检测两者可为胃癌的诊断提供更多的依据。3、内镜粘膜下切除术是治疗早期胃癌安全、可行、微创、有效的方法。更多还原

【Abstract】 Objective: Gastric carcinoma is one of the most common malignant tumors, and isthe second commonest cause of cancer deaths worldwide. About87000new gastriccancer cases are diagnosed every year on a global scale. There is a considerablegeographical variation in the incidence of this cancer. It is reported that incidence ratesare high in eastern Asia, and low in Europe, North America and Africa. Chinese havehigh gastric cancer rates, which are only less than those found in Korea and Japan. Theprognosis of gastric carcinoma is closely related to the stage of disease at the time ofdiagnosis. The survival rate is greater than90%in5years for early gastric cancer, butthe prognosis is very poor for advanced gastric cancer. Therefore, early diagnosis ofgastric cancer is very important for an excellent prognosis. However, since mostpatients with early gastric carcinoma do not have specific symptoms, it is difficult todistinguish early gastric carcinoma from benign peptic ulcer or gastritis, and only about10-20%of gastric cancers are diagnosed as early cancers in many countries, and onlyabout4-10%of patients with gastric cancer are diagnosed as early cancer in China asper the statistics, which is even lower. Moreover, most of those patients with gastriccancer who are diagnosed as early cancer in China have been treated with surgicalresection of subtotal gastrectomy and lymph node dissection same as for the advancedgastric cancer. It is a top urgent task to increase the rate of diagnosis of early gastriccancer in China and to select the rational therapy with minimum trauma but equivalenteffects of surgical operation so as to improve the prognosis and life quality of thepatients of gastric cancer.Endoscopy in combination with pathological diagnosis is still regarded as the goldstandard. But the expressions of many precancerous lesions and early gastric cancer are so subtle that they can be hardly distinguished from benign lesions. With thedevelopment of endoscopic imaging technology, some endoscopic imaging modalities,such as magnifying narrow-band imaging (NBI) endoscopy, have been used recently forthe diagnosis of early gastric cancer. Although many studies have reported that thisendoscopic imaging modality can increase the rate of diagnosis of early cancers andprecancerous lesions by enhancing visualization, there are few studies about comparisonbetween NBI and conventional endoscopy.The development of molecular biological technologies provides a powerful basisfor the early diagnosis of gastric cancer. Some tumor markers have apparent changes inprecancerous lesions and early gastric cancer, so precancerous lesions and early gastriccancer can be found earlier with the detection of those markers combined withendoscopy, and this can provide evidence for screening high risk groups and clinicalcomprehensive diagnosis. In our previous studies, Galectin-3protein expressiondetected by immunohistochemistry was on the increase from the normal gastric mucosato advanced gastric cancer, while P27protein expression was on the decrease. So theexpressions of those two proteins were just opposite. Whether this result can be provenby other test methods will provide more bases for the search of the molecular biologicalindexes for the diagnosis of early gastric cancer.The key to gastric cancer treatment is the thorough resection of the focus and thelymph gland that tends to metastasis. In the past, the treatment of early gastric cancerwas same as that of the advanced gastric cancer in that the surgical radical resection andlymph node dissection were adopted, local excision of gastric lesion and segmentalresection, or partial resection preserving pylorus and vagus nerve function was used,while part of stomach must be excised in all cases with higher rate of complications andmortality. If deeper invasion or positive surgical margin is found by the post-operationpathological diagnosis, another operation must be carried out. So the life quality of thepatients after the operation is similar to that of the patients with advanced gastric cancer.The treatment of early gastric cancer should try to reduce the trauma as much aspossible while achieving the effects to improve the life quality of the patients. In recentyears, thanks to the progress of the endoscopic technology and the development of thetherapeutic devices, the use of EMR or ESD endoscopic technology for treatment hasbegun, which maintains the integrity of the digestive tract with significant improvementof life quality. Also, the complete tissue pathological sample can be obtained withoutsurgical operation, facilitating the selection of correct subsequent clinical therapy and avoiding unnecessary gastrectomy. Although this therapy has the advantages that areunmatched by the surgical operation, there are few clinical studies on the comparisonbetween the EGC endoscopic mucosal resection and the surgical laparotomy treatment.The objective of this study is to explore the magnifying narrow-band imagingendoscopic changes and the changes of the molecular biological indexes in theprecancerous lesion and early gastric cancer stage, to reveal their values in the diagnosisof precancerous lesion and early gastric cancer, to compare and analyze the treatment ofearly gastric cancer with endoscopic mucosal resection and the surgical laparotomy invarious aspects, and to provide the theoretical basis for the search of the reliablediagnosis and treatment methods of precancerous lesion and early gastric cancer.This thesis can be divided into three sections:First, the diagnosis of early gastric cancers and precancerous lesion by using theendoscope inspection of enlarging narrow band imaging (hereinafter to be referred asNBI);Second, the meanings of Galectin-3and P27expressions in the development ofgastric cancer;Third, a multi-angle comparative study between the mucosectomie endoscopiqueand early gastric cancer treatment in surgical operation.Methods:1、526patients were suspected to be early gastric cancers orprecancerous lesion in conventional endoscope inspection, including122who wereconfirmed as early gastric cancers or precancerous lesion evidenced by a tissuespecimen pathologically in endoscopic biopsy and endoscopic or surgical resection,among whom the youngest one was16years old and the oldest was94years old (83males and39females) with their average age at63.5±14.1years old. All122patientsaccepted inspections and diagnosis including the conventional endoscope, enlarged NBIand the magnifying chromoendoscopy. The imaging qualities were commented duringthe inspection process and the morphology features, gastric concave classifications andblood capillaries in different kinds of forms of endoscopy were also analyzed. If there isany stomach malignant epithelial neoplasm and high grade intraepithelial neoplasiaoccurred in the tissue slices, then it is to be considered as positive which can be used tocomment the correctness of endoscopic diagnosis.2、By using immunohistochemistry (S-P) and RT-PCR methods, the expressions ofGalectin-3, P27proteins and mRNA were inspected among40cases of normal gastricmucosa,50cases of atypical hyperplasia,56cases of early gastric cancers and46cases of advanced gastric carcinoma. The selected standards for each group are as follows:(1)Cases of normal gastric mucosa group: no stomach neoplasms history, smoothendoscopic mucosa can be seen without any cave or protruding, in normal color, glossy.(2) Cases of atypical hyperplasia group: atypical hyperplasia reported by the endoscopicbiopsy pathological mechanism, and then cases confirmed as canceration except thoseevidenced by endoscopic mucosal resection or sub mucosal dissection;(3) Cases ofearly gastric cancers group:1) cancer confirmed by endoscopic pathology with anoperation limit cases, and2) tissue slices pathology proved as canceration after theendoscopic mucosal resection or surgery operations but limited to mucous membrane orsubmucosal layer and high grade intraepithelial neoplasia no matter how the lymphnode metastasis is.(4) Cases of advanced gastric carcinoma group:1) cases of cancerwith an operation limit in endoscopic pathology diagnosis, and2) canceration is notlimited to submucosal proved by surgery operation pathology. Excluding standards:(1)one has a malignant tumor history,(2) one has an emergency operation,(3) whendistant metastases or infiltration of adjacent organs are found before an operation,(4)one can not be operated if during one’s trimester of pregnancy, or one is critically illand in any other cases, and (5) one is coagulation abnormal.3、81patients with EGC (44for endoscopic mucosal resection,37for surgicaltreatment) from the year2001to2011in the Central hospital of Dalian were collectedfor this analysis.And compared with two groups of operation time, operation expense,length of stay in hospital, operative complications, follow-up and so on.Results:1、For image resolution, magnifying NBI and magnifying chromoendoscopy weresignificantly superior tomagnifying conventional endoscopy in morphology, pit patternand blood capillary form (P <0.01), andmagnifying NBI was significantly superior tomagnifying chromoendoscopy in blood capillary form (P <0.01). IV, V1,and VI type ofgastric pit pattern were detected in14cases,43cases, and17cases in patients with highgradeintraepithelial neoplasia, respectively. V1and VI type of gastric pit pattern weredetected in9cases and39cases inpatients with early gastric cancer, respectively. Thepresence of irregular minute vessels and variation in the caliberof vessels was found in109cases. The accuracy, sensitivity, specificity, false positive rate and false negativerate fordiagnosis of early gastric cancer and precancerous gastric lesions were68.9%,95.1%,63.1%,24.5%, and32.4%forconventional endoscopy,93.6%,92.7%,94.5%,5.7%, and6.9%for magnifying NBI, and91.3%,88.6%,93.2%,13.2%,and21.48%for magnifying chromoendoscopy, respectively.2、The positive expression percentages of Galectin-3protein in gastric canceroustissues is72.6%, and the positive expression percentages of high and middle, and lowanaplastic carcinoma are73.3%and71.4%respectively. There is no significantdifference compared between the two groups (p>0.05); The positive expression in TNMperiod I+II is60.7%, while in TNM period III+IV is80.4%, there is great differencebetween these two groups (p<0.05); the positive expression of lymph node metastasisand no lymph node metastasis are94.1%and61.8%respectively, there is greatdifference between this two groups (p<0.05). There is no obvious difference (p>0.05)between the normal gastric mucosa group (30.0%) and atypical hyperplasia group(44.0%) of the positive expression of Galectin-3protein, however it is distinctly lowerthan that of early gastric cancers group (64.3%)(p<0.05) and advanced gastriccarcinoma group (82.6%)(p<0.01). There is no obvious difference (p>0.05) betweenatypical hyperplasia group (44.0%) and early gastric cancers group (64.3%) of thepositive expression of Galectin-3protein, however it is evidently lower than that ofadvanced gastric carcinoma group (82.6%)(p<0.01). There is no obvious difference(p>0.05) between early gastric cancers group (64.3%) and advanced gastric carcinomagroup (82.6%) of the positive expression of Galectin-3protein.The positive expression of P27protein in gastric cancerous tissues is35.2%, andthe positive expression percentages of high and middle, and low anaplastic carcinomaare43.3%and23.8%respectively. There is no significant difference compared betweenthe two groups (p>0.05); The positive expression in TNM period I+II is39.3%, whilein TNM period III+IV is30.4%, there is no obvious difference between this twogroups (p>0.05); the positive expression of lymph node metastasis and no lymph nodemetastasis are58.8%and23.5%respectively, there is great difference between thesetwo groups (p<0.05). There is no obvious difference (p>0.05) between the normalgastric mucosa group (85.0%) and atypical hyperplasia group (72.0%) of the positiveexpression of P27protein, however it is distinctly higher than that of early gastriccancers group (42.6%) and advanced gastric carcinoma group (26.0%)(p<0.01). Thepositive expression of P27protein in atypical hyperplasia group (72.0%) is distinctivelyhigher than that of early gastric cancers group (42.6%)(p<0.05)and advanced gastriccarcinoma group (26.0%)(p<0.01); There is no obvious difference (p>0.05) betweenearly gastric cancers group (42.6%) and advanced gastric carcinoma group (26.0%) ofthe positive expression of P27protein. The positive expression of mRNA of Galectin-3in the cases of normal gastricmucosa group is not less than that of cases of atypical hyperplasia group and cases ofearly gastric cancers group. There is no obvious difference between atypical hyperplasiagroup and early gastric cancers group, however both of them are distinctly lower thanthat of advanced gastric carcinoma group; the positive expression of mRNA of P27innormal gastric mucosa group, atypical hyperplasia group, early gastric cancers groupand advanced gastric carcinoma group shows a decreasing trend. The expressioninspection results of mRNA in Galectin-3and P27are respectively in accordance withthe realities of their protein expressions in tissues.3、For endoscopic mucosal resection group, the operation time, operation expense,length of stay in hospital, postoperative anus exhaust time and independent activitytime under the bed were all less than surgical resection group, and there was asignificant difference between the two groups (P <0.05).About operative complicationsoccurring rate and postoperative painkillers utilization rate, endoscopic mucosalresection group were lower than those of the surgical resection group, and there was asignificant difference between them (P <0.05). Follow-up of both two groups did nothappen with residue, local recurrence and heterochrony lesions. The anesthesia andoperative approach, injury of organs as well as working ability, veneous drug therapy,physical condition and so on postoperative life quality evaluation, endoscopic mucosalresection group was better than surgical group.Conclusion:1、 This study demonstrates that magnifying NBI is superior toconventionalendoscopy in the diagnosisof early gastric cancer and precancerous gastriclesions, and can be used for screening early malignancies of thestomach.2、The positive expression percentages of mRNA and Galectin-3proteins arerelated to the occurrences and development of gastric carcinoma, where the normalgastric mucosa group and atypical hyperplasia group are obviously lower than earlygastric cancers group and advanced gastric carcinoma group, where it is notsignificantly related to the TNM periods or the differentiation carcinoma, where it isrelated to lymph node metastasis, and it is probably to be an important index ofevaluation prognosis for early gastric cancers screening and judgments of lymph nodemetastasis in early gastric cancer.The positive expression percentages of mRNA and P27proteins are related to theoccurrences and development of gastric carcinoma, where the normal gastric mucosa group and atypical hyperplasia group are obviously higher than early gastric cancersgroup and advanced gastric carcinoma group, where it is likely related to the TNMperiods and the differentiation carcinoma, where it is related to lymph node metastasis,and its expression reduction of P27is probably to be an effective indicator of evaluationprognosis for early gastric cancers screening and judgments of lymph node metastasis inearly gastric cancer and lymph node metastasis.The expression of Galectin-3and P27protein are opposite to that of mRNA in thedevelopment of gastric cancers. There is no significant difference between the positiveexpressions of Galectin-3and P27, but inspections of both Galectin-3and P27at thesame time can provide more bases for the gastric cancers diagnosis.3、Endoscopic mucosal resection treatment for EGC is safe, feasible, minimallyinvasive and effective.更多还原