【作者】 陈霞；

【导师】 侯晓华；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 Barrett食管(Barrett’s esophagus , BE)多由严重而长期的胃和十二指肠内容物反流刺激食管下段所致,正常的复层鳞状上皮被单层柱状上皮所取代的一种病理现象。随着认知和现代化的发展,我国胃食管反流病的发生愈来愈多,BE的发病率也逐年增加。BE被认为是一种癌前病变,与食管腺癌密切相关,其癌变的危险性较一般人群高出30～125倍。诸多研究已证实肠化生型BE上皮易于向食管腺癌演变,肠化生型BE是研究的热点,近年也逐渐引起国内学者的重视,但遗憾的是研究多着眼于BE的临床分析和BE演变为食管腺癌的机制,而极少涉及BE形成的机制研究。从早期的研究发现BE肠化生细胞的超微结构与正常小肠上皮类似,到近年利用小肠标志物来监测食管腺癌术后复发的研究,都提示肠化型BE与正常小肠上皮存在共性。Wnt-β-catenin/Tcf4及Notch-Delta-Hes 1-Math 1信号途径在维持正常状态下小肠上皮的增殖和分化中作用重要。Wnt-β-catenin/Tcf4信号途径维持正常状态下小肠上皮的增殖,Wnt途径被激活后,导致β-catenin在细胞浆内聚集并与DNA结合蛋白Tcf/LEF家族成员相互结合,促进转录,细胞增殖。Tcf-4基因敲除小鼠的模型,表现为小肠隐窝干细胞数目减少,失去功能,最终导致小鼠死亡。正常状态下,Notch-Delta-Hes1-Math1信号途径通过Hes1对Math1的负调控作用,来决定小肠早期祖细胞的分化方向,从而形成不同种类的小肠细胞。本研究检测了BE活检组织中Wnt及Notch信号途径中的关键分子的表达,并将人正常食管鳞状上皮细胞经酸、胆汁酸及两者的混合物处理。观察其形态变化,检测Wnt及Notch信号途径中的关键分子的表达,试图探讨BE发生的可能机制。第一部分Barrett食管Wnt及Notch信号途径的表达目的:研究BE组织中Wnt及Notch信号途径的关键分子Tcf4, Cdx2, Hes1,Math1的表达。方法:用免疫组织化学法测定41例BE组织(肠化18例,非肠化23例)和其配对的正常食管鳞状上皮组织中Tcf4, Cdx2, Hes1,Math1的蛋白表达,同时采用Real-time(荧光定量)PCR法测定mRNA的表达,计算△△C_T值,并分析指标间的相关性。结果:Tcf4, Cdx2, Hes1, Math1的表达在肠化组高于非肠化组(P<0.05),高度及低度异型增生组高于无异型增生组(P <0.05),在C≥3Mn组高于其他组(P <0.05,)。mRNA的表达Tcf4与Cdx2、Hes1、Math1正相关(P =0.000), Hes 1与Math 1、Cdx2正相关(P =0.000), Cdx2与Math1正相关(P =0.000)。结论:BE组织存在与小肠增殖分化相关的Wnt及Notch信号途径关键分子的表达,其表达与BE的肠化、异型增生及长度密切相关。第二部分人正常食管上皮原代细胞的提取分离、鉴定及培养目的:探讨人正常食管上皮原代细胞的提取分离、鉴定及培养的方法,以获得一定量的纯度高的细胞,为进一步研究奠定基础。方法:人食管上皮细胞来源于食管部分切除手术患者的正常食管组织,先后经DispaseⅡ和胰酶消化,从食管黏膜上分离上皮细胞,和波丝蛋白抗体免疫组织化学染色共同鉴定细胞,无血清角质细胞培养基(K-SFM)培养细胞。采用直接观察,台盼蓝染色进行活力鉴定及细胞计数法绘制细胞生长曲线。结果:细胞呈典型“铺路石”状排列,CK13抗体免疫组织化学染色阳性,波丝蛋白抗体免疫组织化学染色阴性,证明培养的细胞为人食管上皮细胞,无间质细胞污染。细胞倍增天数为1.179d,细胞群体倍增时间(population doubling time, PDT)为49.42h结论:DispaseⅡ消化分离细胞,K-SFM培养细胞可获得一定量的纯度高的人食管鳞状上皮细胞。第三部分酸对人正常食管上皮细胞的影响目的:探讨不同pH值的酸孵育后人正常食管鳞状上皮细胞的形态变化及Tcf4, Cdx2,Hes1,Math1的表达方法:体外培养人正常食管粘膜上皮细胞,分别在pH 4.0、pH 5.0、pH 6.0的培养液中每天3次,每次10 min孵育,共7天。并设对照组进行比较。采用倒置显微镜细胞大体形态变化,MTT法检测细胞增殖速率,电镜下观察细胞超微结构改变,CK13和CK20抗体荧光染色观察细胞角蛋白表达的变化,Real-time PCR方法检测Tcf4, Cdx2, Hes1,Math1 mRNA的表达,Western-blot方法检测Tcf4, Cdx2,Hes1,Math1蛋白的表达。结果:①pH 4.0组5d和7d细胞增殖速率低于其他实验组和空白对照组(P<0.05)。(②电镜下细胞无明显超微结构的改变。③CK13和CK20抗体荧光染色观察细胞角蛋白表达无明显变化;④pH 4.0组细胞Tcf4, Cdx2 mRNA的表达于5d和7d时高于其他组(P<0.05)。⑤pH 4.0组细胞Tcf4, Cdx2蛋白的表达于5d和7d时高于其他组(P<0.05)。结论:pH 4.0条件下人正常食管黏膜上皮细胞增殖速率降低,Tcf4, Cdx2mRNA及蛋白表达水平升高。第四部分胆汁酸对人正常食管上皮细胞的作用目的:探讨不同浓度的胆汁酸孵育后人正常食管鳞状上皮细胞的形态变化及Tcf4,Cdx2, Hes1,Math1的表达方法:体外培养人正常食管粘膜上皮细胞,分别在含有浓度分别为250umol、400 umol和500 umol胆汁酸的培养液中每天3次,每次10 min孵育,共7天。并设未加胆汁酸的空白对照组进行比较。采用倒置显微镜细胞大体形态变化,MTT法检测细胞增殖速率,电镜下观察细胞超微结构改变,CK13和CK20抗体荧光染色观察细胞角蛋白表达的变化,Real-time PCR方法检测Tcf4, Cdx2, Hes1,Math1mRNA的表达,Western-blot方法检测Tcf4, Cdx2, Hes 1,Math 1蛋白的表达。结果:①5d和7d时各实验组细胞增殖速率低于对照组。②电镜下400 umol组细胞7d后可见张力纤维减少,少数细胞见类似于腺体的超微结构变化。③荧光染色见400 umol组细胞7d CK20蛋白表达;④400 umol孵育组的人正常食管黏膜上皮细胞Tcf4, Cdx2, Hes1,Math1mRNA的表达于7d时高于其他组(P<0.05)。⑤400umol孵育组的人正常食管黏膜上皮细胞Tcf4, Cdx2, Hes1,Math1蛋白的表达于7d时高于其他组(P<0.05)。结论:不同浓度的胆汁酸降低人正常食管上皮细胞增殖速率;含有浓度为400 umol胆汁液的培养液中孵育的人正常食管上皮细胞部分可出现超微结构的改变,角蛋白表达发生变化,Tcf4, Cdx2, Hes1,Math1mRNA及蛋白表达水平显著升高。第五部分酸及胆汁酸的混合物对人正常食管上皮细胞的影响目的:探讨酸及胆汁酸的混合物孵育后人正常食管鳞状上皮细胞的形态变化及Tcf4,Cdx2, Hes1,Math1的表达方法:体外培养人正常食管粘膜上皮细胞,在pH值为4.0、胆汁酸浓度为400uml的培养液中每天3次,每次10 min孵育,共7天。设未加酸及胆汁酸混合物的空白对照组、pH4.0组及含400 umol浓度胆汁酸组3组进行比较。采用倒置显微镜细胞大体形态变化,MTT法检测细胞增殖速率,电镜下观察细胞超微结构改变,CK13和CK20抗体荧光染色观察细胞角蛋白表达的变化, Real-time PCR方法检测Tcf4, Cdx2, Hes1,Math1 mRNA的表达,Western-blot方法检测Tcf4,Cdx2, Hes1,Math1蛋白的表达。结果:①7d时各实验组细胞增殖速率低于对照组,混合组细胞增殖速率低于400umol组。②电镜下细胞无明显超微结构的改变。③7d时混合组细胞可见微弱的CK20的表达。④7d时混合物组Tcf4, Cdx2 mRNA的表达高于未加空白对照组(P<0.05),低于pH4.0组及400 umol浓组(P<0.05) ; 7d时混合物组Hes1,Math1mRNA的表达高于对照组和pH4.0组(P<0.05),低于400 umol组(P<0.05)。⑤7d时混合物组Tcf4, Cdx2蛋白的表达高于未加空白对照组(P<0.05),低于pH4.0组及400 umol组(P<0.05) ; 7d时混合物组Hes 1,Math 1蛋白的表达高于对照组和pH4.0组(P<0.05),低于400 umol组(P<0.05)。结论:酸及胆汁酸联合作用降低人正常食管上皮细胞增殖速率,对细胞增殖速率的影响强于胆汁酸。酸及胆汁酸混合组Tcf4, Cdx2, Hes1,Math1rnRNA及蛋白表达水平升高,但表达低于胆汁酸。本研究结果提示BE组织中存在Wnt及Notch信号途径的关键分子Tcf4,Cdx2, Hes1,Math1的异常表达。人食管正常鳞状上皮细胞在pH4.0的条件下Tcf4, Cdx2呈高表达。人食管正常鳞状上皮细胞在含有浓度为400 umol胆汁液的培养液中孵育,Tcf4, Cdx2, Hes1,Math1的表达升高。更多还原

【Abstract】 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.更多还原