| 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. |
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