The Research On The Mechanism Of Differentiation Of Barrett's Esophagus

Barrett's esophagus(BE) is an acquired condition that results from chronicgastro-oesophageal reflux.It is characterised by the metaplastic replacement of the normalsquamous epithelium of the lower oesophagus by columnar epithelium.In humans,it isclearly known that severe gastroesophageal reflux is main causes for BE,but it would be ofno importance were it not for its well-recognized association with esophagealadenocarcinoma.The rapid rise in the incidence of adenocarcinoma of the esophagus (ACE)has evoked greatly increased interest in BE,moreover,it is being seen as a growingpublic health problem in Western countries.Recent years,the prevalence of this disease isincreasing in China,partly because of increasing usage of endoscopy,and also because of atrue rise in the prevalence of the disease,mainly due to a concomitant rise ingastro-intestinal reflux disease(GERD).As more appropriately known,BE owes its importance to being a precursor lesion ofACE.The risk of cancerization is about 30～125 folds higher than general people.Researches have shown that specialized intestinal metaplasia (SIM) mucosa of Barrett'sesophagus (BE) predisposes to the development of esophageal adenocarcinoma and SIMhave always attracted scientists' interest.Earlier studies have demonstrated that SIMmucosa of BE had shown some similarities to normal intestinal epithelium on morphologyand histology.Then,it is reasonable that the signal pathways of forming intestinalepithelium may be involved in the formation of BE.Wnt-β-catenin/Tcf4 and Notch-Delta-Hes1-Math1 pathways maintain the proliferation and differentiation of normal intestinal epithelium.On normal condition ,assoon as Wnt pathways is activatied,β-catenin is generated and assembled inside of cell.Andβ-catenin combine with members of the Tcf/LEF family protein of DNA combinedprotein,which promoting the transcription ,activating cell proliferation.Notch pathwaydecide the direction of intestinal progenitor cell differentiation on early stage.The decisionis carried out by the reverse feedback regulation between Mathl and Hes1.Then thevarious intestinal cell are formed eventually.In this study ,the expression of key regulators of Wnt-β-catenin/Tcf4 andNotch-Delta-Hes1-Math1 pathways,Tcf4,Cdx2,Hes1 and Math1 were examined in biopsyspecimen of patients with BE.And human primary normal esophagus squamous epithelialcells were dealt with acid ,bile acid and the mixture of both ,the morphological changeswere observed,the expression of Tcf4,Cdx2,Hes1 and Math1 were examined in cells ofvarious groups.Our aimis to investigate the possible mechanism differentiation of BE.PartⅠExpression of Wnt and Notch signal pathways in Barrett'sesophagusAim:To investigate the expression of key regulators of Wnt and Notch signal pathwaysin Barrett's esophagus (BE).Methods:Patients were diagnosed by the Montreal definition and grouped by the Prague C& M criteria.Several samples were taken from BE and two paired normalesophagus samples were taken from at least 5cm above BE.The expression ofTcf4,Cdx2,Hes1,Math1 in 41 paired biopsy samples were measured byimmunohistochemistry staining and Real-time PCR ,the relationship betweenthese regulators was analyzed.Results:The mRNA and protein expression of Tcf4,Cdx2,Hes1,Mathl in the group ofintestinal metaplasia were greater than that of in the group of non-intestinal metaplasia (P＜0.05) ,and were greater in the group of C≥3Mn than other groups(P＜0.05) ,while the mRNA expression of these regulators in the group ofhigh-grade dysplasia was more than non- dysplasia group (P＜0.05) ,the value of△△CT of Tcf4,Cdx2,Hes1 and Math1 mRNA were correlated positively (P=0.000).Conclusion In BE,there is definitely the expression of key regulators of the Wnt andNotch signal pathways which mantaining intestinal proliferation anddifferentiation,related to intestinal metaplasia,dysplasia and the length of BEclosely.PartⅡIsolation,Identification and Subculture of human esophagueai squamousepithelial cellsAim:To seek the optimization for the culture of high purify and significant quantity ofnormal human esophageal squamous epithelial cells ;And to make base for furtherresearchMethods:Normal esophagus tissues obtained from the patients carried outoesophagectomy because of benign or malignant diseases.All tissues werejudged as normal esophagus tissues by pathologists.Cells were isolated fromthese tissues by dispaseⅡdigestion and trypsinization and then primarilycultured and subcultured in serumfree keratinocyte medium (K-SFM).Thebiological characteristics of the cells were investigated through directobservation ,living cells were accouting by Trypan blue staining Growthkinetic curves were drawn by accouting cell number.Cells were identifiedimmunohistochemically using antihuman monoclonal antibody of thecytokeratin 13/10 (CK13/10) and vimentin. Results:The cells displayed a cobblestone morphalogy,characteristic of epithelial cells andimmunocytochemistry stained positive for the epithelial cell markeranti-CK13/10,which indicated that the cells were human esophageal epithelialcells.The negative immunocytochemistry stained for anti-Vimentin whichwas interstitial cell marker showed that all cells were not contaminated byinterstitial cell .The cells' doubling day was 1.t 79d and population doubling time(PDT) was 49.42h,Conclusion:DispaseⅡdigestion and K-SFM are effective methods to obtain and culturehigh purify ,significant quantity of human normal esophageal epithelial cells invitro.PartⅢThe effects of acid exposure in normal human esophageal squamousepithelial cells in vitroAim:To observe the effects of acid exposure on cell morphological changes and theexpression of key regulators of Wnt and Notch signal pathways in normal humanesophageal squamous epithelial cells in vitor.Methods:Normal human esophageal squamous epithelial cells were grouped intofour ,treated by acidified medium (pH 4.0,pH 5.0,pH 6.0) for three times per day,10 min each time and the same treat for 7 days.The control group was not treatedby acidified medium.The morphological changes were observed through invertedphase contrast microscope,the mRNA and protein expression of Tcf4,Cdx2,Hes1,Math1 were detected by Real-time PCR and Western-blot,respectively.Results:①The cell proliferation of pH 4.0 group was lower than other groups and controlgroup (P＜0.05) on 5d and 7d.②Distinct ultrastructure changs were not observedby electron microscope.③The expression spectrum of cytokeratin did notchange ,which investigated by immunofluorescence staining for anti-CK13/10 andanti-CK20.④The mRNA expression of Tcf4,Cdx2 in the group of the pH 4.0 were higher than other groups on 5d and 7d (P＜0.05).(5)The protein expression of Tcf4,Cdx2 in the group of the pH 4.0 were higher than other groups on 5d and 7d(P＜0.05).Conclusion:In vitro,the proliferation of normal human esophageal squamous epithelialcells treated by acidified medium (pH 4.0) was lower,but the mRNA and proteinexpression ofTcf4,Cdx2 in the cells treated by acid (pH 4.0) were higher.PartⅣThe effects of bile acid exposure in normal human esophageal squamousepithelial cells in vitroAim:To observe the effects of bile acid exposure on cell morphologicalchanges and the expression of key regulators of Wnt and Notch signal pathways innormal human esophageal squamous epithelial cells in vitor.Methods:Normal human esophageal squamous epithelial cells were grouped intofour ,treated by medium contained various concentration bile acid (250umol,400umol and 500 umol)for three times per day,10 min each time and the same treat for7 days.The control group was not treated by bile acid.The morphological changeswere observed through inverted phase contrast microscope,the mRNA and proteinexpression of Tcf4,Cdx2,Hes1,Math1 were detected by Real-time PCR andWestern-blot,respectively.Results:①The cell proliferation of all groups treated by bile acid was lower than controlgroup (P＜0.05) on 5d and 7d.②Distinct ultrastructure changs could be observedby electron microscope in the cells of 400 umol group on 7d.③The expression(red) were detected by immunofluorescence staining for anti-CK13/10,also theexpression (green) were detected for anti-CK20 which was a special bio-markerof BE in the 400 umol group on 7d.④The mRNA expression of Tcf4,Cdx2 ,Hes1,Math1 in the 400 umol group were higher than other groups on 7d(P＜0.05).⑤The protein expression of Tcf4,Cdx2 ,Hes1,Math1 in the 400 umol group were higher than other groups on 7d (P＜0.05).Conclusion:In vitro,the proliferation of normal human esophageal squamous epithelialcells treated by medium contained various concentration bile acid was lower;themRNA and protein expression of Tcf4,Cdx2 ,Hes1,Math1 in the cells treated bymedium contained 400 umol bile acid were higher.PartⅤThe effects of mixture of acid and bile acid exposure in normal humanesophageal squamous epithelial cells in vitroAim:To observe the effects of mixture of acid and bile acid exposure on cellmorphological changes and the expression of key regulators of Wnt and Notchsignal pathways in normal human esophageal squamous epithelial cells in vitor.Methods:Normal human esophageal squamous epithelial cells were treated by acidified atpH4.0 and contained 400 umol bile acid medium (mixture group)for three times perday,10 min each time and the same treat for 7 days.Other group (pH4.0 acidifiedgroup ,400 umol bile acid contained group and untreated group) were ascontrol.The morphological changes were observed through inverted phase contrastmicroscope,the mRNA and protein expression of Tcf4,Cdx2,Hes1,Math1 weredetected by Real-time PCR and Western-blot,respectively.Results:①The cell proliferation of all treated groups was lower than untreated group(P＜0.05) on 7d,and that of mixture group were lower than 400 umol group.②Distinct ultrastructure changs were not observed by electron microscope.③Theweak protein expression of CK20 were investigated by immunofluorescencestaining on 7d.④On 7d,the mRNA expression of Tcf4,Cdx2 in the mixture groupwere higher than the un-teated group on 7d (P＜0.05),but less than the 400umolgroup and the pH4.0 group;the mRNA expression of Hes1,Math1 in the mixturegroup were higher than the un-teated group and the pH4.0 group (P＜0.05),but less than the 400umol group .⑤On 7d,the protein expression of Tcf4,Cdx2 in themixture group were higher than the untreated group on 7d (P＜0.05),but less than the400umol group and the pH4.0 group;the mRNA expression of Hes1,Math1 in themixture group were higher than the un-teated group and the pH4.0 group(P＜0.05),but less than the 400umol group.Conclusion:In vitro,the proliferation of normal human esophageal squamous epithelialcells treated by mixture of acid and bile acid was lower,which has stronger effectthan bile acid alone.The mRNA and protein expression of Tcf4,Cdx2,Hes1,Math1in the cells treated by mixture of acid and bile acid were higher,but still less than thatof the cells treated by bile acid alone.In summary,we observed that there was definitely abnormal expression of keyregulators of the Wnt and Notch signal pathways in BE,and in vitro,the mRNA andprotein expression of Tcf4,Cdx2 in normal human esophageal squamous epithelialcells treated by acid (pH 4.0) were higher,the mRNA and protein expression of Tcf4,Cdx2 ,Hes1,Math1 in the cells treated by medium contained 400 umol bile acid werehigher.