【作者】 张晶；

【导师】 赵志华；

【作者基本信息】 郑州大学 ， 临床病理学（专业学位）， 2017， 硕士

【摘要】 背景与目的食管癌在全球恶性肿瘤中排名第四位,病理类型以鳞状细胞癌(squamous cell carcinoma,SCC)最为多见。据大数据统计报道,食管癌的5年生存率在30%左右(15%~42%)。我国河南省林县是食管癌的高发区,相对于其他恶性上皮源性的肿瘤,该区域的食管癌死亡率明显高于全国平均水平。就肿瘤的发病机制来讲,肿瘤的异质化是由多元素导致的,例如外界化学物质刺激、基因突变、内环境紊乱或者是机体自身正常代谢停滞、异常代谢活跃等。某种因子在机体代谢活动中生成增多会影响正常的代谢过程,导致在细胞水平或者是基因水平上对整个机体的稳态构成威胁,进而出现肿瘤样变。因此,确定肿瘤基因突变位点是研究肿瘤发展进程的关键。M2型丙酮酸激酶(pyruvate kinase M2,PKM2)作为糖酵解途径中最重要的调节酶,能够对糖酵解过程产生的大分子物质的数量起到了关键性的掌控作用,同时为肿瘤细胞的生长代谢提供了所需的能量和原料,也对肿瘤细胞的生长周期起到了调控作用。泛素特异性蛋白酶18(Ubiquitin specific protease 18,USP18)作为一种干扰素诱导基因,在人体内环境中主要扮演去泛素化的作用,受损组织被机体识别后会与ISG15(类泛素干扰素刺激基因)形成泛素化复合物,进而被吞噬细胞或水解酶降解清除。而USP18阻断ISG15发挥泛素化作用,进而防止损伤的靶细胞被吞噬细胞降解或被水解酶水解,导致损伤细胞在体内堆积不能及时清除,使肿瘤细胞处在营养丰富的环境内,达到促进肿瘤异质化进程的效果,进而对肿瘤的生长代谢起到推动作用。近年来寻找生物学标记物成为了了解食管癌的肿瘤发病机制以及探索食管癌的早期筛查指标和靶向治疗位点的研究热点,既能在探索食管癌发生发展的同时对食管癌做出早期诊断,又能进行靶向对症治疗以提高生存率。明确肿瘤的突变点能够从根本上遏制食管癌的发生,也能够从分子角度扭转肿瘤的单克隆现象,促进有肿瘤化趋势的细胞发生凋亡,调节正常细胞进行常规的新陈代谢。进而,能够响应“早发现、早诊断、早治疗”的医疗目标,对于提高食管癌患者的生存率、减少患者的致死率起到决定性的作用,也对食管癌的临床治疗效果和结局起到显著改善和指导的效果。本研究通过免疫组织化学法观察PKM2和USP18在食管鳞状细胞癌组织、正常食管黏膜组织以及食管鳞状上皮内瘤变组织(低级别和高级别)中的表达情况,以及分析在不同分化程度、浸润深度、年龄、性别、淋巴结转移等情况下的食管鳞癌组织中的差异性表达状况,观察其表达差异性是否与食管鳞状上皮的异型增生的进程以及各临床指标有关,对于探讨PKM2和USP18在推进食管癌发生过程中的作用有很大的帮助作用。方法1.筛选60例于2015年01月~2015年12月间由郑州大学第一附属医院病理科诊断为食管鳞状细胞癌的病例,其中高分化、中分化、低分化鳞状细胞癌组织各20例。病例标本均为手术切除后的食管大标本,经常规病理取材,病理报告中有明确的肿物大小、位置、分化程度、浸润深度、淋巴结转移以及切缘是否取干净等情况。另外选取60例正常食管黏膜组织,以及60例经ESD或EMR切除后诊断为鳞状上皮内瘤变的组织,其中高级别、低级别鳞状上皮内瘤变组织各30例。2.实验运用免疫组织化学技术对食管鳞癌组织、食管鳞状上皮内瘤变组织以及正常食管黏膜组织进行USP18和PKM2的染色,严格按照SP免疫组化二步染色法的步骤进行。免疫标记物PKM2的阳性对照采用胃腺癌组织,免疫标记物USP18的阳性对照采用肝癌组织,两者阳性染色位于细胞核和细胞质中,阴性对照均为不加一抗染色的食管鳞癌组织。3.统计学处理:运用SPSS 17.0统计软件进行分析处理,对癌组织、食管鳞状上皮内瘤变组织、正常食管黏膜组织以及不同临床指标下的食管癌组织进行独立的两组二分类和多组二分类比较,检验水准α=0.05。结果1、PKM2在食管鳞癌组织、食管鳞状上皮内瘤变组织以及正常食管黏膜组织中的阳性表达率分别为63.3%、30%、11.7%,且3组间比较,差异具有统计学意义(χ2=36.19,P<0.05)。可见,PKM2在食管鳞癌组织中的表达显著高于其在食管鳞状上皮内瘤变组织以及食管正常黏膜组织中的表达。另外在低级别和高级别鳞状上皮内瘤变组织中PKM2的表达阳性率分别是16.7%和43.3%,差异具有统计学意义(χ2=5.08,P<0.05)。2、USP18在食管鳞癌组织、食管鳞状上皮内瘤变组织以及正常食管黏膜组织中的阳性表达率分别为65%、31.7%、3.3%,3组间比较,差异具有统计学意义(χ2=51.45,P<0.05)。可见,USP18在食管鳞癌组织中的表达程度也是明显高于其在食管鳞状上皮内瘤变组织以及正常食管黏膜组织中的表达。USP18在低级别和高级别鳞状上皮内瘤变组织中的阳性表达率分别为23.3%和40.0%,虽然阳性表达有逐渐增加的趋势,但是与PKM2不同,USP18在低级别和高级别鳞状上皮内瘤变组织中的阳性表达差异性很小,差异无统计学意义(χ2=1.93,P>0.05)。3、将诊断为食管鳞癌的病例根据不同的临床指标(肿瘤浸润深度、分化程度、年龄、性别、有无淋巴结转移)进行分类,PKM2和USP18分别在不同临床指标的食管鳞癌组织中表达差异均无统计学意义(P>0.05),说明不同临床参数对于PKM2和USP18在食管鳞癌中的表达高低影响不大。结论PKM2和USP18与食管鳞状上皮异质化进程呈正相关,提示PKM2和USP18对于食管癌的发生发展有关,PKM2和USP18有望作为食管鳞癌的病理诊断指标。更多还原

【Abstract】 Background and ObjectiveEsophageal cancer is the fourth leading global malignancy,according to the data statistics report,5-year survival rate of esophageal cancer at 30%(15% ~ 42%).Esophagus with coating squamous epithelium is given priority to,so the primary tumor in squamous cell carcinoma,squamous cell carcinoma,SCC)is the most rare.Lin County is a high incidence of esophageal cancer in henan province in China,relative to other incidence on malignant tumor,esophageal cancer mortality is significantly higher than the average level of the region.In terms of the pathogenesis of esophageal cancer,increased formation of a certain factor in the body’s metabolic activity will affect the normal metabolic processes,resulting in cellular level or the gene level pose a threat to the whole body of the steady state,thus appear tumor samples.As a result,the tumor gene mutations is the key to the study of tumor development,this study selects the PKM2 and USP18 as immune index for exploratory research on development of esophageal squamous carcinoma.M2 pyruvate kinase(PKM2)as the most important enzyme in glycolytic pathway,can play a key role of control in the process of glycolysis,at the same time supply energy and raw materials for the growth of tumor cell,also played a regulation role in the growth of tumor cell cycle.Ubiquitin specific protease 18(USP18)as a kind of interferon induced genes,in the human body environment mainly plays the role of ubiquitin in the human body environment,damaged tissues can be connected with ISG15(class ubiquitin interferon stimulated genes)as a formation of ubiquitin compounds after recognition by the body,which are cleared by phagocytes or hydrolytic enzyme degradation,and USP18 would block ISG15 to give a play to the role of the ubiquitin-proteasome change,to prevent the target cells from the damage,causing damaged cells in the body accumulation not clear in time,the tumor cells are in enriched environment,to promote the process of tumor heterogeneity effect,thus to promote the growth of tumor metabolism.Looking for biological markers in recent years becomes the understanding of the tumor pathogenesis of esophageal cancer and to explore the early screening indexes and targeted therapy for esophageal cancer research focus in the site,it can make early diagnosis of esophageal cancer and at the same time explore the origin and development of esophageal cancer at the same time to make early diagnosis of esophageal cancer,then to improve the survival rate.So we can fundamentally curb the occurrence of esophageal cancer,if we make it clear the abrupt change point of the tumor.And we can also reverse the phenomenon of monoclonal hyperplasia of tumor cells,from the perspective of molecular monoclonal phenomenon.It would has a trend of tumor cells apoptosis,adjust the metabolism of normal cells for regular.We are able to respond to the medical goal of "early detection,early diagnosis,early treatment",and it play a decisive role for improving the survival rates of patients with esophageal cancer,reduce the fatality rate in patients.It is also have guidance effect to the clinical treatment effect of esophageal cancer.By means of the immunohistochemical technique to observe the expression of type M2 pyruvate kinase(pyruvate kinase M2,PKM2)and Ubiquitin specific protease 18(Ubiquitin specific protease 18,USP18)in esophageal squamous cell carcinoma tissues,normal esophageal mucosa tissues and squamous intraepithelial lesion tissues(Low-grade and High-grade).Analysis USP18 and PKM2 in each phase of the squamous epithelium and different differentiation degree,infiltration depth,such as age,sex,lymph node metastasis cases to express the difference of the situation,whether the expression difference was observed with the process of esophagus hyperplasia and clinical indicators,to explore the PKM2 and USP18 in advancing its effect in the process of esophageal cancer has a great help.Methods1.First of all,60 cases of esophageal squamous cell carcinoma diagnosed by pathology in the First Affiliated Hospital of Zhengzhou University from January 2015 to December 2015 were selected,all of the cases were esophageal specimens after operation,routine pathological examination,There were clear tumor size,location,degree of differentiation,depth of invasion,lymph node metastasis,and whether or not the edge was clean in this report.In addition,selecte 60 cases of normal esophageal mucosa and 60 cases were diagnosed as squamous intraepithelial lesion tissues(30 cases of low grade squamous intraepithelial lesion tissues,30 cases of high grade squamous intraepithelial lesion).2.Using the immunohistochemical technique to esophageal squamous carcinoma tissues,squamous intraepithelial lesion tissues and normal esophageal mucosa tissues of USP18 and PKM2 dyeing experiment,in strict accordance with the SP immunohistochemical staining method steps of two.PKM2 immune markers positive control in gastric adenocarcinoma tissue and immune markers USP18 positive control in liver cancer tissue and the positive staining in the nucleus and cytoplasm,negative control were without a stain resistance of esophageal squamous carcinoma tissues.3.Statistical processing: using SPSS 17.0 statistical software analysis processing,to carcinoma,esophageal squamous intraepithelial lesion tissue,normal esophageal mucosa tissue and the esophageal tissue under different clinical indicators to separate two sets of binary classification and multiple sets of binary classification comparison,inspection level will be subject to α=0.05.Results1.The positive expression rate of PKM2 in esophageal squamous carcinoma,esophageal squamous intraepithelial lesion tissue and normal esophageal mucosa tissues were respectively 63.3%,30% and 11.7%,and comparison between the three groups,the difference is statistically significant(χ2=36.19,P<0.05).Thus it can be seen,the expression of PKM2 in esophageal squamous carcinoma tissues were significantly higher in the squamous intraepithelial lesion tissues,and also which is expressed in normal esophageal mucosa tissues.In addition,the The positive expression rate of PKM2 in the low level and high level of squamous epithelial hyperplasia tissues were 16.7% and 43.3%,respectively,the difference is statistically significant(χ2=5.08,P<0.05).2.The positive expression rate of USP18 in esophageal squamous carcinoma,esophageal squamous intraepithelial lesion tissues and normal esophageal mucosa tissues were respectively 65%,31.7% and 3.3%,and comparison between the three groups,the difference is statistically significant(χ2=51.45,P<0.05).It follows that,the expression of USP18 in esophageal squamous carcinoma tissues were significantly higher in the squamous intraepithelial lesion tissues,and also which is expressed in normal esophageal mucosa tissues.furthermore,the The positive expression rate of USP18 in the low level and high level of squamous intraepithelial lesion tissues were 23.3% and 40.0%.Although positive expression has a tendency to increase gradually,but unlike PKM2,the positive expression of USP18 at low level and high level in squamous intraepithelial lesion tissue has a small difference,there was no statistically significant difference(χ2=1.93,P>0.05).3.Will be diagnosed cases of esophageal squamous carcinoma according to the different clinical indicators(tumor infiltration depth,differentiation degree,age,gender,presence of lymph node metastasis)to classify.PKM2 and USP18 respectively in different clinical indicators of esophageal squamous carcinoma tissues expression differences had no statistical significance(P>0.05),showed that different clinical parameters for PKM2 and USP18 expressed in esophageal squamous cell carcinomas of the high and low impact is not big.ConclusionPKM2 and USP18 positively correlated with esophageal squamous epithelium heterogeneous process.This is explained that PKM2 and USP18 has a great role on the development of esophageal cancer,PKM2 and USP18 can both be immune markers as indicators of the pathological diagnosis of esophageal cancer.更多还原