【作者】 郭军辉；

【导师】 王立东；

【作者基本信息】 郑州大学 ， 内科学， 2009， 硕士

【摘要】 1研究背景河南林州市及其比邻的安阳、辉县等地区是我国,也是世界上食管癌(EC)发病率和死亡率最高的地区,该病目前仍是本地区肿瘤相关死亡的主要原因。既往已研究、发现许多食管癌变相关分子改变,但EC发生发展的分子改变机制仍不十分清楚,缺乏用于高危人群食管癌筛查和早期诊断的敏感且特异的分子指标。近年研究表明,肿瘤细胞多阶段癌变演进过程中,会表达胚胎细胞发育过程中出现的蛋白,而这些蛋白在成熟分化的细胞中很少表达,利用这些蛋白作为肿瘤分子标志物,已被应用到肿瘤的早期诊断以及疗效、预后评价。胎儿食管上皮发育由幼稚单层柱状上皮逐渐发育成熟为复层鳞状上皮,分化由不成熟逐渐成熟,上皮细胞由分裂增殖活跃的上皮干细胞逐渐成熟分化为复层鳞状上皮,而成人食管上皮癌变是由成熟分化的上皮组织在致癌因素作用下,出现过度增生和异常分化最终发展成为癌。显然,两者具有相反的分化过程,从而,比较胎儿食管上皮发育和成人食管上皮癌变二个相反过程中的上皮细胞形态和分子变化特征及其与细胞增殖和分化的关系,将为进一步阐明食管癌变的分子机制,寻找用于筛查和早期发现的分子指标提供重要的理论基础。既往国内外曾有对胎儿食管上皮发育过程形态学研究,但对胎儿食管上皮细胞的分子学研究较少。本实验室前期研究中发现胎儿食管上皮基底层细胞旺盛的增殖状态同同癌前病变有相似之处,并利用蛋白质组技术发现同胎儿食管上皮发育过程相关蛋白。本研究正是在上述研究的基础上,利用光镜、组织病理学以及免疫组化技术,对比分析来自河南辉县、获嘉等食管癌高发区3-10个月不同胎龄食管上皮成熟分化和成人食管上皮癌变多阶段演进失分化过程中的组织形态学和分子变化差异及其与细胞增生和分化的关系,旨在筛选增生和分化异常蛋白,加深对食管癌变机理的了解,为确定高危人群筛查和早期发现的分子标志物提供重要理论依据。2材料与方法2.1研究对象2.1.1胎儿食管164例人胎儿食管组织标本(男性59例,女性104例,1例性别不详)来自河南省辉县、获嘉计划生育服务中心,其中胎龄3个月29例(18%)、4个月50例(30%)、5个月29例(1 8%)、6个月24例(15%)、7个月15例(9%)、8个月12例(7%)、9个月4例(2%)、10个月1例(1%)。2.1.2成人食管93例成人食管上皮标本(男性54例,女性39例,中位年龄58岁)均来自河南省林州市姚村食管癌医院,所有患者在术前均未接受任何的化疗或放疗。2.2标本的收集与处理2.2.1胎儿食管胎儿食管标本取自合法流产及米索针剂(前列腺素类)引产正常胎儿,根据孕妇提供末次月经时间结合测量胎儿顶跟长度及手、足形态核实胎龄。胎儿引出后2个小时内,解剖出整个食管和胃上1/3部,沿食管纵轴将食管全层1/2切开,一半经85%酒精固定,剩余的一半入液氮中保存,后转入-80℃冰箱保存,提DNA用。2.2.2成人食管食管癌手术切除标本离体后,均在2小时内沿纵轴剖开。根据卢戈氏液对食管上皮增生活跃组织着色浅或不着色的原理,每例手术标本的一半先经卢戈氏液染色后对染区和不染区食管粘膜分别取材,尽量于肿瘤实体5cm之外或手术切缘,以后的处理及另一半标本的处理方法同2.2.1。2.3组织病理学诊断标准胎儿食管上皮分析:取组织结构清晰,染色均匀的HE切片,光镜下记录上皮的厚度,细胞层数以及食管固有腺、贲门腺的出现等。成人食管上皮分析:采用本实验室已建立的诊断标准,根据细胞形态、组织结构和分化程度,将成人食管上皮分为正常上皮(Normal,NOR)、基底细胞过度增生(Basic cell hyperplasia,BCH)、不典型增生(Dysplasia,DYS)、原位癌(Carcinoma in situ,CIS)和食管鳞癌(esophageal squamous cellcarcinoma,ESCC)。2.4胎儿食管上皮的形态学分析通过显微镜观察,对HE染色的食管上皮进行计量并列表统计。2.5免疫组织化学免疫组化采用卵白素-生物素-辣根过氧化氢酶复合物(Avidin BiotinComplex,ABC)法。2.6统计分析采用SPSS10.0统计软件,进行Kruskal-Wallis和Mann-Whitney检验,检验水准为α=0.05。3结果3.1组织病理学观察3.1.1不同胎龄胎儿食管上皮组织学变化光镜下观察164例不同胎龄(3～10个月)胎儿食管上皮组织发现:3月份胎儿食管上皮薄厚不均(1～3层细胞),单层高柱状上皮、复层矮柱状和复层纤毛柱状上皮,伴有柱状上皮出现。4～5月上皮细胞层数3～5层,细胞边界清晰,基底层细胞出现,有1-2层细胞,细胞呈矮柱状,胞质呈强嗜碱性;可见大量纤毛柱状上皮细胞;食管的中段可见复层鳞状上皮。6～8月上皮细胞层数6～9层,基底层、棘层以及浅表层分层明显。基底细胞层1～3层,细胞排列整齐,3-5层上中下厚薄分布不均,仍有少量纤毛柱状上皮。9-10个月上皮细胞层数8-10层,中间有5-6层多边形细胞,浅层有2-3层嗜酸性的扁平细胞,纤毛已基本脱落。4个月胎儿食管上皮内见到食管腺导管。6个月可见大量食管固有腺。3.1.2贲门腺的发生及变化在3和6个月胎龄时,在食管下段可见柱状上皮岛,7个月时这些柱状上皮逐渐向粘膜固有层下陷,并向周围蔓延,逐渐形成食管贲门腺。3.1.3食管下段食管粘膜皱襞的发生及变化在4个月胎龄胎儿食管下段可见固有层突然出现凹陷,取而代之的是增厚的分化不清的上皮,层数可达20层以上。在7个月胎龄胎儿食管下段可见粘膜和粘膜下层突入管腔,形成巨大的纵行皱襞。3.1.4食管上、中、下三段上皮细胞分化的非同步性在7个月胎龄胎儿食管中可以看出食管上皮上、中、下三段细胞层数不一,上端5-6层,而到中下段可达10-15层;在6个月胎龄胎儿食管中可以看出食管上皮上、中、下三段细胞分化不一,上段和下段均为柱状和纤毛柱状,而中段则呈鳞状。3.1.5食管柱状上皮岛的发生及变化在食管粘膜上皮中常可见到小片状的移位的胃黏膜上皮,在食管上段尤为多见,有学者认为这种移位的胃黏膜上皮与食管憩室、囊肿、溃疡及癌的发生有密切关系。3.1.6成人食管取经病理学证实93例带有癌旁组织的食管癌,根据病理学标准将其分为NOR、BCH、DYS、CIS和ESCC,又根据分化程度的不同将ESCC分为高、中、低分化三组。3.2免疫组织化学分析胎儿食管上皮组织中Prx6阳性表达率为87%(34/39),在3、7、8三个月胎龄中Prx6蛋白的免疫染色强度明显低于4、5、6、9四个月份;在92%(34/37)的NOR中有Prx6蛋白表达,随着病变加重其表达下降,CIS时Prx6蛋白完全失表达的比例达到了79%(22/28)。高分化SCC时Prx6蛋白表达又升高,而后随着SCC失分化表达逐渐降低。sPLA2-ⅡA蛋白在胎儿食管上皮中表达的总阳性率为98%(45/46),随胎龄增加sPLA2-ⅡA蛋白的表达逐渐降低。在食管癌及癌旁组织中从NOR→BCH→DYS→CIS→SCC演变过程中,随着病变程度加重,sPLA2-ⅡA蛋白表达逐渐升高,在高分化SCC组织中sPLA2-ⅡA蛋白的阳性表达率为100%(14/14),而后随着SCC失分化sPLA2-ⅡA蛋白的表达逐渐降低。4结论4.1本研究中发现的胎儿食管基底细胞层旺盛的增殖状态与成人食管上皮不典型增生,胎儿食管上皮基底膜的变化与成人食管癌变时基底膜的变化,以及胎儿食管上皮粘膜层大量淋巴滤泡与成人食管癌变时淋巴滤泡的出现等比较,提示成人食管上皮癌变过程中存在与胎儿食管上皮成熟发育过程有极相似的形态变化特征。4.2本研究中发现的胎儿食管下段柱状上皮细胞逐步分化的过程可能为食管贲门腺的发生、发展过程。4.3食管中段上皮鳞化的靠前性,提示中段食管上皮分化较为活跃。4.4 Prx6蛋白表达增强或降低很可能与食管上皮细胞的增殖与分化相关,Prx6在快速增殖的食管上皮中可能起到抗氧化、保护细胞免受损伤的作用,可能为评价细胞分化程度的一个理论依据。4.5 sPLA2-ⅡA蛋白与食管癌分化程度有关,可能在食管上皮癌变中起重要作用,但与胎儿食管上皮成熟分化无明显关系。更多还原

【Abstract】 1 BACKGROUNDLinzhou City and its nearby counties,Anyang,Huixian and so on in Henan Province,have the highest incidence and mortality rates for esophageal cancer(EC) in China as well as in the world.EC remains the leading cause of cancer-related death in these areas.Although studies have shown the changes of some EC-associated molecules,the key molecular mechanisms in esophageal carcinogenesis are largely unknown.Furthermore,high specific and sensitive biomarkers for high-risk subject screening and early detection of EC haven’t been identified.Recent studies suggest that,during multi stages carcarcinogenesis,epithelial cells could abnormally express some proteins that often exist only in embryo development other than in the well differentiated tissues,which could be clinically used as molecular markers for early tumor diagnosis,therapy and evaluating the prognosis.The genesis of fetal esophageal epithelia has been proposed to develop from nascent simple columnar epithelia cells to stratified squamous epithelium in which the well differentiation is gradually formed.In this process,the proliferous nascent epithelia cells tend to be differentiated and partitioned to basal stem cells,middle proliferous cells,epithelia cells.This genesic process is quite different from that of epithelia carcinogenesis in which the well differentiated epithelia cells extraordinarily undergo hyperproliferation, loss of differentiation and finally malignant transformation.The comparative study of fetal and adult esophagus epithelia on the morphology,the molecular changes and their relationships with cell proliferation and differentiation will contribute to the illumination of the exact mechanisms of epithelial carcinogenesis,the identification of molecular biomarkers for early diagnosis and high-risk subject screening.However, the comparative studies on the molecular changes in multiple stages of fetal and adult esophageal epithelia in the processes of cancerization are poor.Previous research reports the serum protein analysis also indicates the remarkable protein level variations in EC and precancerous lesions.Refer to these studies,the present studies to take proteomic analysis,immunohistochemistry,microscopy and histopathology to characterize the molecular and morphological changes related with cell proliferation in mature differentiation of fetal esophageal epithelia with different ages from 3-10 months and in loss of differentiation of adult esophageal multistage carcinogenesis. to screening and build the molecular biomarkers with abnormal differentiation and cell proliferation,to further understanding of the mechanisms of human esophageal carcinogenesis,to supply important theory basic for high-risk subject screening and early detection.2 MATERIALS and METHODS2.1 Subjects2.1.1 Fetal esophagusThe 164 cases of fetal esophageal epithelial samples(male 59,female104,1 Nonsexsuality) were obtained from Huixian and Huojia Family Planning Centers, including 29 cases(18%) at 3 months,50(30%) at 4 months,29(18%) at 5 months, 24(15%) at 6 months,15(9%) at 7 months,12(7%) at 8 months,4(2%) at 9 months, and 1(1%) at 10 months.2.1.2 Adult esophagusThe 93 cases(54 males,39 females,a mean age of 58 years) of adult esophageal epithelia samples were obtained from Linzhou Central Hospital and Yaocun Esophageal Tumor Hospital of Henan Province.All the patients were not received any chemotherapy or radiotherapy before surgery.2.2 Sample collection and processing2.2.1 Fetal esophagusThe fetal esophagus specimens were obtained from legal abortion or induced abortion with prostaglandin.The gestational ages were determined by the last catamenia time before pregnancy,and verified by measuring the full length of the embryo and observation of the morphology of fetal hands and feet.The whole esophagus and one third of upper stomach were collected within 2 hours after abortion or induced abortion and splitted vertically.One half of the sample was fixed with 85%ethanol.The other half was frozen in nitrogen and then stored at -80℃for extraction of protein,DNA.2.2.2 Adult esophagusThe surgically resected esophageal cancer specimen was splitted vertically within 2 hours.Based on the theory that the actively hyperplastic esophageal epithelium was lightly stained or negative for Lugol solution staining,half of the specimen was stained with Lugol solution and sampled from stained and unstained areas 5 cm away,or from the cutting edge.The following treatments for both half of the specimen were the same as fetal esophagus.2.3 Criteria for histopathologieal diagnosisFor fetal esophagus,the thickness of epithelium,layers of cells and apoptotic cell fraction were examined under microscopy after HE staining.For adult esophagus,with the diagnosis criteria established previously by us, based on cellular morphology,tissue structure and cell differentiation,epithelia were classified into normal(NOR),basal cell hyperplasia(BCH),dysplasia(DYS), carcinoma in situ(CIS),well differentiated squamous cell carcinoma(SCC), moderately differentiated SCC and poorly differentiated SCC.2.4 Morphometrical analysis on fetal esophageal epitheliaMicroscopical sizing apparatus was used to measure the fetal esophageal epithelium after stained with HE.All the data were statistically analyzed.2.5 ImmunohistochemistryImmunohistochemistry was performed with Avidin Biotin Complex(ABC) method.2.6 Statistical analysisData was analyzed with SPSS10.0 software for Kruskal-Wallis and Mann-Whitney tests.P＜0.05 was considered statistically significant.3 RESULTS3.1 Histopathological observation3.1.1 Growth of fetal esophageal epitheliumIn the 3 month,the esophageal epithelium is stratified columnar,simple high columnar and multi-layered ciliated cells,being in general 1-3 cells deep,the epithelium varies in thickness,and nuclei are scattered irregularly,without cells differentiation distinctly.In the 4 and 5 month,being in general 3-5 cells deep,and epithelium cells proliferation actively,the basal layer were up to 1-2 cells deep with out-of-order arrangement and exhibited columnar and cuboid morphology,these cells was strongly basophilic,and the karyon was big and dark granular colored.There are patches of ciliated cells in an otherwise non-ciliated epithelium.In the 6-8 month,the epithelium cells arrange regularly,there are plenty of stratified squamous epithelium, and some patches of ciliated cells,and the epithelium up to 6-9 cells deep.In the 9-10 month,the middle layer were up to 5-6 cells deep,the supper layer were 2-3 cells deep with squamous epithelium.In 4 month fetal esophageal,esophageal gland ducts could be found,and we can see lot of deep submucous glands in 6-month fetus.3.1.2 Genesis of esophageal cardiac glandsColumnar epithelium persists in the lower portion esophageal epithelium in 3 and 6 month,and these columnar cells gradually sank into the lamina propra in the cardio-esophageal junction in 7 month.3.1.3 Growth of the mucosal fold at the lower of the esophagusIn the lower end of the esophagus the epithelium varies greatly in thickness in adjacent areas,thicker areas of stratified un-differentiation epithelium being intermingled with thinner areas of stratified columnar ciliated epithelium in 4 month; and in the lower end of the esophagus the submucosa run into the lumen,being the mucosa in the 7 month.3.1.4 The different differentiation and cell deep epithelium in the upper、middle and lower of the same case esophagusIn the upper of the esophagus epithelium was 5-6 cells deep and the middle was 10-15 cells deep in one 7 month case.In the upper the esophagus epithelium were columnar and ciliated cell,however,the middle esophageal epithelium were stratified squamous cell.3.1.5 Patches of columnar cells occur in the upper of esophageal epitheliumPatches of one or two layers of columnar cell islands occur in the upper of esophageal epithelium in 4、5 and 7 month cases.In adult esophagus,some expert reported the columnar cell islands were related with the esophageal diverticulum、cyst and ulcer、cancer.3.1.6 Adult esophagusBased on lugol staining,two pieces of tissue(one from cancer,another from the adjacent to cancer) were obtained from each SCC specimen(n=93).Based on precancerous lesions detected,these samples were divided into NOR、BCH、DYS、CIS and ESCC.And ESCC was sub-grouped to well,moderately and poorly differentiated SCC. 3.2 Immunohistochemical analysisPrx6 positive rate in fetal esophageal epithelium specimens was 87.2%(34/39), the Prx6 protein expression in 3、7、8 months are lower than in 4、5、6 and 9 months. Prx6 protein expressed in 91.9%(34/37) normal esophageal epithelium and decreased with SCC progression.In carcinoma in situ(CIS),21.4.0%foci lost Prx6 protein expression.The expression of Prx6 protein was increased in well differentiated SCC and decreased with loss differentiation of SCC.sP1A2 positive rate in fetal esophageal epithelium specimens was 97.8%(45/46),the expression of sP1A2 protein was decreased with the fetus growth,sP1A2 expression was increased with the progression from NOR→BCH→DYS→CIS→W-SCC.The sP1A2 positive rate was 100%(14/14) in well differentiated SCC and decreased with loss differentiated of SCC.4 CONCLUSINS4.1 This study have showed that there are active proliferated cells,the change of basement membrane,and piles of lymph follicle in fetal esophageal epithelia,all these results suggest a similar morphology changes in adult esophageal multistage carcinogenesis and fetal esophageal epithelial development.4.2 The differentiation process of columnar epithelium at the end of fetal esophagus maybe the base of the cardiac gland.4.3 This study showed the appearance of squamous epithelium at the middle of fetal esophageal epithelium,point out that the actively differentiation of middle of fetal esophageal epithelium.4.4 The expressed Prx6 be related with the proliferation and differentiation of the esophageal epithelium,Prx6 could eliminate the peroxide that produced while metabolizing and protect the cells from injury as an antioxidase,the protein maybe a theory basis to determine the differentiation.4.5 The expressed sP1A2 protein is related with well-differentiated squamous cell carcinoma,sP1A2 maybe act an important role in the development of esophageal carcinogenesis,sP1A2 is no apparently related with fetal esophageal maturities development.更多还原