【作者】 徐志彬；

【导师】 王士杰； 吴明利；

【作者基本信息】 河北医科大学 ， 外科学， 2014， 博士

【摘要】 食管鳞状细胞癌（Esophageal squamous cell carcinoma，ESCC）是我国常见的恶性肿瘤之一，死亡率高，5年生存率低，浸润和转移是影响患者死亡及预后的重要因素。食管癌具体发生机制尚不明确，以往关于食管癌的研究多专注于恶变的鳞状上皮细胞，而很少关注上皮细胞周围的间质微环境，近年来，肿瘤间质微环境的研究逐渐成为研究热点。活化的癌相关纤维母细胞(Cancer-associated fibroblasts CAFs)是肿瘤间质微环境内的主要效应细胞，研究证实，包括食管癌在内的多种肿瘤组织内的癌相关纤维母细胞，与正常成纤维细胞相比，在形态结构、生长方式、增殖活性、运动能力等发面均发生了显著变化，并且能够分泌大量的促进肿瘤细胞增殖、血管新生的信号分子以介导上皮-间质之间复杂的相互作用。目的：食管癌的发展是一个渐进的过程，从正常鳞状上皮发展到鳞状细胞癌的过程中存在中间过渡阶段—不典型增生鳞状上皮或异型增生鳞状上皮，此种上皮与正常鳞状上皮和癌上皮细胞有着不同的结构与功能，在癌前病变间质组织中，是否也存在正常成纤维细胞向癌相关纤维母细胞转化的过渡阶段-不典型增生间质成纤维细胞尚不知晓，与正常成纤维细胞和癌相关纤维母细胞相比，其结构和功能特性又会有何不同，在食管癌变进展中起到何种作用，具体作用机制如何，均有待研究证实。食管间质成纤维细胞在食管癌变进程中扮演何种角色，深入研究其具体作用机制可能有助于食管癌及食管癌前病变的早期诊断，早期治疗，为食管癌及食管癌前病变的药物治疗提供作用靶点。方法：研究食管癌变过程中间质成纤维细胞的表型转变规律及间质成纤维细胞相关蛋白的表达变化，采用免疫组织化学方法检测食管正常组、低级别上皮内瘤变组、高级别上皮内瘤变组、早期癌组及进展期癌组间质和上皮组织中α-SMA、TGFβ1、smad4、HGF、c-met、MMP2、MMP9及VEGF表达变化，计数各组微血管密度，统计分析各组指标与病变的相关性，统计分析间质表达α-SMA与TGFβ1、HGF的相关性，统计分析间质TGFβ1与上皮smad4，间质HGF与上皮c-met的表达相关性；体外分离培养人食管不同病变阶段组织中的间质成纤维细胞，包括食管正常成纤维细胞（normal fibroblasts, NFs）、食管不典型增生成纤维细(atypical hyperplasiafibroblasts, AFs）、食管癌相关纤维母细胞（cancer-associated fibroblasts，CAFs），观察三种间质成纤维细胞的形态学特征，检测各自的免疫表型和生物学特性，细胞内相关基因的表达变化,组织块贴壁法和酶消化法体外培养三种食管间质成纤维细胞，采用倒置相差显微镜、透射电子显微镜观察细胞大体形态及内部结构，瑞氏-吉姆萨染色方法，观察细胞着色情况，细胞免疫组织化学技术，检测细胞表面蛋白，包括角蛋白（cytokeratin，CK）、波形蛋白（Vimentin，Vim）、α-平滑肌肌动蛋白（α-smooth muscleactin，α-SMA）的表达情况，采用四氮唑蓝比色法绘制NFs，AFs及CAFs7天生长曲线，流式细胞术检测NFs，AFs及CAFs细胞周期，RT-PCR技术检测细胞内α-SMA、TGFβ1、HGF、MMP2、MMP9及VEGF-A的基因表达，Transwell技术检测癌细胞分别与三种间质成纤维细胞间接接触前后，4种细胞内基因的表达变化，同时检测癌细胞对间质成纤维细胞侵袭力的影响；体外模拟微环境分析三种间质成纤维细胞与癌细胞体外间接接触作用机制，建立动物模型，体内研究三种间质成纤维细胞与癌细胞直接接触作用机制。将癌细胞株分别与三种食管间质成纤维细胞混合注射于裸鼠皮下，构建实验动物模型，观察成瘤时间、成瘤率以及肿瘤增长速度，免疫组化检测间质成纤维细胞的表型变化，PCNA蛋白染色检测食管癌细胞的增殖能力变化，CD34标记血管内皮细胞以检测肿瘤间质微血管密度。结果：第一部分：随食管癌变进展，食管间质成纤维细胞的表型发生了转变，由α-SMA阴性表达转变为阳性表达，且呈逐渐增高趋势；间质和上皮中TGFβ1、HGF、MMP2、MMP9蛋白表达均逐渐上升，上皮中c-met蛋白表达亦呈逐渐上升趋势，而smad4蛋白表达呈下降趋势，间质中VEGF蛋白表达逐渐升高。在间质成纤维细胞中α-SMA与TGFβ1、HGF表达呈正相关。随食管癌变进展，MVD值逐渐增高，MVD值与TGFβ1、HGF和VEGF表达呈正相关。第二部分：体外成功培养出食管正常成纤维细胞、不典型增生成纤维细胞和癌相关纤维母细胞，纯化后的细胞纯度达到95%。光镜下观察，三种间质成纤维细胞呈扁平长梭形，大小基本一致，突触较多，食管正常成纤维细胞间存在密度抑制和接触抑制，而不典型增生成纤维细胞和癌相关纤维母细胞抑制和接触抑制消失，细胞排列紊乱，局部出现重叠生长的现象；电镜下观察，食管三种成纤维细胞富含粗面内质网、核糖体、高尔基体，食管正常成纤维细胞内可见微丝，不典型增生成纤维细胞和癌相关纤维母细胞内可见大量微丝，局部形成微丝束，甚至成团形成致密斑。三种间质成纤维细胞角蛋白染色均为阴性，波形蛋白染色均为阳性，食管正常成纤维细胞的α-SMA染色为阴性，食管不典型增生成纤维细胞和癌相关纤维母细胞α-SMA表达均为阳性。NFs、AFs和CAFs生长曲线均为S形，3-5d为对数生长期，细胞生长旺盛，CAFs是活化的成纤维细胞，增殖能力明显强于NFs和AFs，AFs增殖能力则介于NFs和CAFs之间，接近于CAFs。NFs，AFs，CAFs组处于S期细胞百分比分别为23.7，32.8，33.4。第三部分：从NFs→AFs→CAFs，Vimentin mRNA的表达无变化，α-SMAmRNA的表达逐渐增强，TGFβ1，HGF, MMP2，MMP9mRNA在NFs、AFs、CAFs中表达水平依次增高。 VEGF-A在NFs未见表达，在AFs、CAFs中高表达。三种食管间质成纤维细胞分别与食管癌细胞ECa109间接共培养后，TGFβ1，HGF, MMP2，MMP9均表达增强。NFs对ECa109无明显促进增殖作用,而AFs、CAFs刺激食管癌细胞株ECa109细胞内PCNA表达量显著增加，促进ECa109的增殖。侵袭实验发现，空白对照组中仅有少量食管癌细胞穿透Matrigel，到达滤膜下方的细胞数为(18.0±5.4)个，NFs组到达滤膜下方的细胞数为(35.0±4)个，AFs和CAFs作用下滤膜下方的细胞数分别为(125.2±7.1)、(135.5±8)个。动物模型结果显示，食管间质成纤维细胞与癌细胞株混合注射可明显缩短成瘤时间，提高成瘤率，肿瘤内微血管密度增加，从而加速肿瘤的生长。结论：1在食管癌变进程中，食管正常间质成纤维细胞与癌相关纤维母细胞间存在中间过渡阶段-食管不典型增生成纤维细胞。从正常成纤维细胞→不典型增生成纤维细胞→癌相关纤维母细胞，其免疫表型逐步发生了改变。2体外可成功培养三种食管间质成纤维细胞。从正常成纤维细胞→不典型增生成纤维细胞→癌相关纤维母细胞，细胞功能越发活跃，生物学特性发生了改变，处于增殖期细胞比例增高。3癌细胞可通过旁分泌细胞因子，间接刺激食管间质成纤维细胞，使其功能发生改变。食管间质成纤维细胞亦可通过旁分泌细胞因子，间接刺激癌细胞的增殖，同时可增强癌细胞的侵袭力。4动物模型显示：正常食管间质成纤维细胞在与癌细胞直接接触后，可使其表型向癌相关纤维母细胞发生转化。活化的食管间质成纤维细胞在与癌细胞直接接触后，可加速肿瘤的生长。更多还原

【Abstract】 Squamous cell carcinoma of the esophagus is one of the most commonmalignant tumors in China and the cancer patients have higher mortality ratesand5-year survival rate is lower. Invasion and metastasis is an importantfactor affecting mortality and prognosis in patients.Precise mechanism ofesophageal cancer remains unclear. Previous studies on esophageal cancermainly paid much attention to malignant transformation of squamousepithelial cells, but paid little attention to changes in the stromamicroenvironment surrounding epithelial cells. Tumor microenvironmentcontained matrix protein, immune cells, activated fibroblasts and adjacentunique vasculature. Activated cancer-associated fibroblasts are the maineffecting cells. Compared with normal fibroblasts, activated cancer-associatedfibroblasts has undergone significant changes on morphological structure,growth pattern,athletic ability and proliferation activity. They can also secretemore cytokines to promote tumor cell proliferation. Paracrine signalings andextracellular matrix components could mediate epithelial-mesenchymalinteractions.Objective：The process of esophageal carcinoma is moderately advanced. Atypicalhyperplasia squamous epithelia is intermediate stage between normalsquamous epithelia and carcinoma epithelia. Atypical hyperplasia squamousepithelia is different from normal squamous epithelia and carcinoma epitheliain structure and function. We don’t know if there is atypical hyperplasiafibroblasts in stroma of precancerous lesion which is intermediate stagebetween normal fibroblasts and cancer-associated fibroblast. Compared withnormal fibroblasts and cancer-associated fibroblasts, what features is itsstructure and function? What is atypical hyperplasia fibroblasts’role and its mechanism in esophageal carcinogenesis? Studies on the different stagefibroblasts may be helpful for early diagnosis and treatment for esophagealprecancerous lesion and provide targets of drug action.Method:In this study,we study phenotypic transformation of fibroblast andchanges of expression of proteins(including α-SMA、TGFβ1、HGF、MMP2、MMP9and VEGF)secreted by interstitial fibroblasts and changes ofexpression of proteins(including c-met and smad4) in the epithelia in theesophageal carcinogenesis process. Detection of α-SMA, TGF-β1andSMAD4, HGF, HGF, VEGF and MMP2and MMP9expression changes inesophageal normal group and low grade intraepithelial neoplasia group, highgrade intraepithelial neoplasia group, early in the cancer and advanced cancergroups via immunohistochemical staining. Counting microvessel density ineach group and statistically analyze the correlations between indicators andlesions. Analyze correlations between α-SMA and TGFβ1， correlationsbetween α-SMA and HGF. Analyze correlations between TGFβ1and smad4，correlations between HGF and c-met;In vitro, interstitial fibroblasts successfully were isolated and culturedfrom different stages of lesion of esophagus, including NFs, AFs and CAFs.Observation of morphological characteristics of three types of stromalfibroblasts and detection of immunological and biological characteristics andexpression of related genes in them were carried out. To analyze and comparethe results of interaction of three kinds of stromal fibroblasts and cancer cells.Comprehensively analyze esophageal stromal fibroblasts function in thesetting and progression of esophageal squamous cell carcinoma.Threeesophageal interstitial fibroblasts were cultured by explants adherent methodand enzymatic digestion method in vitro. To observe cell morphology andinternal structure using inverted microscope, transmission electronmicroscope.Using immuno-cell staing method to detect the expression ofα-SMA, CK and Vimentin. Using tetrazolium blue colorimetric method drawsa7days growth curve of NFs, AFs and CAFs. Detection of NFs, AFs and CAFs cell cycle using flow cytometry. detecting intracellular level of α-SMA、TGFβ1、HGF、MMP2、MMP9and VEGF-A mRNA using RT-PCR method.Using transwell technique to detect gene expression changes of each cell aftercancer cells indirectly contacted with three types of stromalfibroblasts.Detection of cancer cells impact on invasiveness of stromalfibroblasts. Cancer cell lines were mixed with three kinds of esophagealstromal fibroblasts respectively when injected into nude mice subcutaneouslyand observe the growth of the tumors. Immunohistochemical detection ofproliferation of cancer cells and tumor microvessel formation was carried out.Result:The first part: In esophageal carcinogenesis, esophageal stromalfibroblasts changed from α-SMA-phenotype to α-SMA+phenotype.Expression of TGFβ1、HGF、c-met、MMP2、MMP9and VEGF proteinincreased. Inversely, smad4protein decreased. Alpha-SMA correlated withTGFβ1and HGF positively. MVD increased with progression of esophagealcarcinogenesis. MVD correlated with TGF-beta1, HGF and VEGF expressionpositively.The second part: NFs, AFs and CAFs could be cultured in vitrosuccessfully with purity approaching to95%. In light microscope three kindsof stromal fibroblasts show to be long flat spindle. No differences in size. TheSynapse was abundant. There was synaptic inhibition and contact inhibition inNFs, otherwise in AFs and CAFs. Under electron microscopy, three kinds ofesophageal fibroblasts were rich in rough endoplasmic reticulum, Ribosome,Golgi. Microwires were visible in esophageal normal fibroblasts, abundant inAFs and CAFs. Local micro-filaments formed dense patches. Negativeexpression of CK and positive expression of vimentin could be observed inthem. Alpha-SMA was negative expression in NFs, but positive in AFs andCAFs. NFs, AFs and CAFs growth curves were s-shaped. Logarithmic growthstage was from3rd to5th day. CAFs were activated fibroblast. Its proliferationcapacity is significantly stronger than NFs and AFs. The ability of AFsproliferation is between NFs and CAFs. Percentage of s-phase cells in NFs, AFs and CAFs group is23.7%,32.8%,33.4%respectively.The third part: From NFs→AFs→CAFs, there were no difference in theexpression of vimentin mRNA, whereas the the expression of α-SMA mRNAincreased in three kinds of fibroblasts. The expression of VEGF-A, TGFβ1,HGF, MMP2and MMP9mRNA was higher in CAFs than that in NFs and AFs.VEGF-A、MMP2、MMP9didn’t expressed in NFs and AFs, but expressedprominently in CAFs.Weak expression of TGFβ1and HGF was observed inAFs. Overexpression of TGFβ1and HGF was in CAFs. After esophagealcarcinoma cells ECa109indirectly was co-cultured with three kinds ofesophageal stromal fibroblasts, TGFβ1and HGF expression enchanced in NFs.VEGF-A,TGFβ1,HGF, MMP2and MMP9expression enchanced in AFs andCAFs. NFs didn’t obviously promote proliferation of ECa109line. AFs andCAFs will significantly promote the proliferation of ECa109line. PCNAexpression in esophageal cancer cell line ECa109increased significantly.Invasive experiments found that only a small amount of esophageal cancercells in the control group penetrate through Matrigel with cell number for(18.0±5.4), NFs group with cell number for (35.0±4), AFs group with cellnumber for (125.2±7.1), CAFs group with cell number for (135.5±8). Animalmodels showed that esophageal stromal fibroblasts and cancer cell strain ofmixed injection can shortened the time for tumor formation, accelerate thegrowth of tumors. it is not confirmed that they can stimulate the proliferationof tumor cells, but can increase tumor microvascular visibly.Conclusions:1In the course of esophageal carcinogenesis, immunophenotype ofesophageal stromal fibroblasts transformed and interstitial fibroblasts wereprogressively activated.there are intermediate stages.With their activation,esophageal stromal fibroblasts’ structures and biological characteristicsgradually changed, secretory function enhanced.2Three esophageal interstitial fibroblasts could be successfully culturedin vitro.3Cancer cells can indirectly stimulate esophageal stromal fibroblasts through paracrine cytokine and change their function. Esophageal stromalfibroblasts can indirectly stimulate the proliferation and invasiveness of cancercells through cytokine secretion.4After Esophageal normal stromal fibroblasts direct contact with cancercells,its phenotype transformed to the phenotype of cancer-associatedcarcinomas。 Activated esophageal stromal fibroblasts cancer cells’ directcontact with cancer cells may accelerate the building and growth of tumors.更多还原