| 1 BACKGROUND AND OBJECTIVEEsophageal cancer(EC) is one of the six most frequent malignant tumors.It is its evident epidemiologic character that it has notable regionally distribution difference, poor prognosis too that only has 10%five-year survival rate.It is because that the key molecular mechanisms in esophageal carcinogenesis remain largely unknown and 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 stage carcinogenesis,epithelial cells could abnormally express some proteins that often exist only in embryo development other than in well differentiated tissues, which could be clinically used as molecular markers for early tumor diagnosis,such as AFP in liver cancer,etc.Previously our laboratory made the comparative analysis on fetal esophageal epithelia,ESCC,tumor adjacent tissue with 2-DE based proteomics,and we found that S100A9,PRXⅥand other correlative proteins gave expression both in ESCC and fetal esophageal epithelia.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.This genesic process is contrary to that of epithelia carcinogenesis in which the well differentiated epithelia cells extraordinarily undergo hyperproliferation,loss of differentiation and finally malignant transformation.So by comparing the molecular changes and morphological changes of the esophagus we can make some understanding of carcinogenesis of EC.Our studies probe into the character that S100A9 and PRXⅥexpress in fetal esophageal epithelia of different ages(3-9 months),EC and precancerous lesions,study the relation of esophageal epithelium proliferation and differentiation by using LM and immunohistochemistry, and then to facilitate the understanding of the mechanisms of human esophageal carcinogenesis.We can offer theoretical reference for the early diagnosis and prevention of EC.2 MATERIAS AND METHODSThe 102 cases of fetal esophagus samples(male 32,female 70) were obtained from Henan Key Laboratory for Esophageal Cancer research(obtained in 2006-2007), and the fetal esophagus specimen were obtained from legal abortion or induced abortion with prostaglandin,including 15 cases at 3 months,32 at 4 months,12 at 5 months,18 at 6 months,12 at 7 months,9 at 8 months,4 at 9 months.The 30 cases (23 males and 7 females with a mean age of 61.1±8.31) of adult esophageal epithelia samples were obtained from Yaocun Esophageal Tumor Hospital(Linzhou, Henan).All the patients were not received any chemotherapy or radiotherapy before surgery.All of the surgically resected specimen was fixed with formalin,paraffin embedded,and serially sectioned for histopathological observation.Adult esophageal epithelia samples and 32 fetal esophagus samples(15 males,17 females,fetal esophagus 4 at 3 months,5 at 4 months,5 at 5 months,5 at 6 months,5 at 7 months,4 at 8 months,4 at 9 months ) were used with immunohistochemistry(ABC method). Each sample was examined by five eye-fields under high power magnification(×400) by random selection and adopted the average value.All the samples were grouped in the percentage of the positive cells.The number of positive cells＞5%was considered as positive,the number of positive cells＞25%was considered as high expression,the number of positive cells＜25%was considered as low expression.The data were analyzed by SPSS10.0 statistical software using Fisher's exact test of probabilities. The test used for the statistics(P＜0.05) was considered significant.3 RESULTS3.1 Histopathologieal observation3.1.1 Histopathological diagnosis of ESCCAdult esophagus:30 cases of EC with adjacent tissue after pathological confirmation were obtained,7 cases of well differentiated SCC,15 cases of moderately differentiated SCC and 8 cases of poorly differentiated SCC,two cases of them showed Basic cell hyperplasia(BCH) BCH and Dysplasia(DYS),one case had Carcinoma in situ(CIS) and DYS.3.1.2 Growth of fetal esophageal epitheliaThe esophageal epithelium at 3 months has been observed with stratified columnar epithelium with 1-4 layers of cells with similar cell morphology.For 4 to 8-month,the esophageal cell layers were up to 5-8;the basal cells in some parts were up to 3 layers with out-of-order arrangement and exhibited columnar and cubic morphology.The cytoplasm of these cells was strongly basophilic,and the superficial cells were quite different in shape,which exhibited cubic,columnar and squamous morphology,besides they always had cilium on free surface of cubic and columnar epithelium.For the esophageal epithelium with 9 months,the esophageal cell layers were up to 6-10;the basal cells were 1-2 layers with in-order arrangement,the superficial cells were mostly squamous cells and had less cilium.3.1.3 Genesis of fetal esophageal glandGenesis of esophageal glands in the submucosa:For the fetal esophagus with 4-month,esophageal gland ducts could be found in lamina propria of mucous membrane.For 6-month,esophageal gland ducts was found that became longer and perforated the muscularis mucosae downward,and in the submucosa we observed large numbers of mucous acini in each segment of the esophagus. Genesis of esophageal cardiac glands:Columnar epithelium islands appeared in the lower portion esophageal epithelium with 4 months.For 7-month,these columnar cells gradually sank into the lamina propria in lower portion esophagus,while some other same gestational age fetal esophagus had glands formed in the lamina propria of the lower portion.3.1.4 Contrast between fetal esophageal epithelium and ESCC3.1.4.1 Epithelial cells' hyperplasiaThe genesis of fetal esophageal epithelia has been proposed to develop from nascent simple columnar epithelia cell to stratified epithelium by Basic cell hyperplasia.The basal cells of fetal esophagus with 4 to 8 months showed hyperplasia in different extent,the basal cell layers were up to 2-4 in some sections.The basal cells exhibited columnar and cubic morphology;the cytoplasm of these cells was strongly basophilic.All those have big similarity with precancerous lesions of EC in morphology,such as BCH and DYS.3.1.4.2 Basement membraneThe basement membrane of fetal esophageal epithelia changed from none to exist and undulating protuberance by contraries happened in the process of the canceration of EC that changed from distinct to unconspicuous and disappearing.3.1.4.3 Apoptotic cellsApoptotic cells scattered in fetal esophageal epithelial cells as well as were familiar in EC-epithelia that proliferated fleetly.3.2 Immunohistochemical analysis3.2.1 S100A9 proteinThe positive rate was 65%(21/32) for S100A9 in fetal esophageal epithelia.The positive cell number for S100A9 increased with fetal aging,but the basal cells had no positive expression.The difference had the statistical significance(P＜0.05).The expression of S100A9 was highly positive in normal esophageal epithelial cells,but the positive cell number for S100A9 decreased in precancerous lesions and cancer. The positive rate was 73%(22/30) for S100A9 in ESCC.The difference had the statistics significance(P＜0.05).The level of moderately differentiated and poorly differentiated cells' expression was lower than that of well differentiated cells.The expression of S100A9 was concerned with the differentiation of ESCC(r=0.507).3.2.2 PRXⅥproteinThe positive rate for PRXⅥwas 72%(23/32) in fetal esophageal epithelia.The difference had the statistical significance(P＜0.05).The intensity of the positive expression and the positive rates increased with fetal development,and reached the climax at the age of 5-6 months,then decreased with fetal age.The positive rate was low(13%,4/30) for PRXⅥin ESCC.The difference had no statistics significance (P＞0.05).4 CONCLUSIONS4.1 The basal cells of fetal esophagus were quite similar to the process of the canceration of adult esophagus on the characteristic of form changing,such as state of active proliferation,formation of basement membrane,emergence of apoptotic cells.4.2 The positive cell number for S100A9 protein increased with fetal epithelia cells' differentiation,but gradually decreased from BCH to DYS and CIS,and from well differentiated SCC to moderately differentiated SCC and pooly differentiated SCC. The expression of S100A9 protein in fetal esophagus,precancerous lesions and EC suggests that the protein could be concerned with the differentiation level of cells.4.3 The expression of PRXⅥprotein in fetal esophagus and EC suggests that the protein could eliminate the peroxide that produced while metabolizing and protect the cells from injury as an antioxidase. |
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