【作者】 曾伟伟；

【导师】 吴明利； 王士杰；

【作者基本信息】 河北医科大学 ， 内科学， 2011， 硕士

【摘要】 目的:近来研究发现,上皮细胞的微环境―间质在上皮肿瘤发生发展过程中不只是被动旁观者,还扮演着主动参与的作用,甚至是肿瘤的启动者,其中间质主要效应细胞―纤维母细胞因其重要作用而备受关注。目前大量研究证实肿瘤间质中存在一类活化的纤维母细胞,被称为肿瘤相关纤维母细胞(carcinoma-associated-fibroblasts, CAFs),通过分泌转化生长因子-β1(transforming growth factor-β1, TGF-β1)和肝细胞生长因子(hepatocyte growth factor, HGF)等因子促进肿瘤的发生发展,人原始造血细胞抗原(hunman hematopoietic progenitor cell antigen, CD34)阴性、平滑肌肌动蛋白-α(Smooth muscle actin-α,αSMA)阳性是其主要辨认标志。食管癌是我国最常见的恶性肿瘤之一,发病人数占世界50%以上,严重影响人民的生命健康,是我国重点防治的恶性肿瘤。长期大量的研究逐步明确了食管鳞状上皮癌变过程为正常→癌前病变→早期癌→进展期癌。对食管上皮癌变过程中肿瘤间质微环境的研究,将有助于解析食管鳞状上皮的癌变机制,有力推动食管癌的防治进程,但未见连续癌变过程中有关CAFs的报道。本研究观察食管间质纤维母细胞从正常→癌前病变→早期癌→进展期癌的癌变过程中α-SMA、CD34、TGF-β1及HGF的表达情况,探索间质CAFs及其相关因子TGF-β1和HGF在食管癌发生发展中的作用,并寻求潜在的具有临床意义的诊疗指标,从而为食管癌的病因、诊断、治疗和预后提供科学依据。方法:采用免疫组织化学(immunohistochemistry, IHC)链霉素亲生物素-过氧化酶法(S-P法)检测正常组、低级别上皮内瘤变组(中度不典型增生)、高级别上皮内瘤变组(重度不典型增生和原位癌)、早期癌组(粘膜癌)及进展期癌组共5组136例病例的食管组织中αSMA、CD34、TGF-β1及HGF表达情况,并计数各组的微血管密度(microvascular density, MVD),运用SPSS13.0统计软件包进行数据分析。结果:1αSMA在食管癌变过程中间质纤维细胞的表达αSMA在正常食管组织纤维细胞无表达(0/20),仅在肌细胞和血管壁平滑肌细胞中表达阳性。从低级别上皮内瘤变到癌的各组中表达逐渐升高,至进展期癌组达最高,表达率为95.7%(22/23)。阳性细胞呈梭形,主要存在于病变或癌巢周围间质中呈丝带样。结果显示食管鳞状上皮癌变过程中间质纤维母细胞αSMA表达呈上升趋势,其表达与患者性别、年龄无关,各组病例间质纤维母细胞αSMA表达的阳性率有显著性差异(χ~2 =56.423, P=0.000)。2 CD34在食管癌变过程中间质纤维细胞的表达CD34在正常食管间质纤维细胞中阳性表达率为95.0%(19/20),从低级别上皮内瘤变到癌的各组中表达逐渐降低,在进展期癌组中CD34仅在血管内皮细胞呈阳性表达,在癌周围间质纤维细胞中不表达(0/23)。结果显示食管鳞状上皮癌变过程中间质纤维母细胞CD34表达呈下降趋势,其表达与患者性别、年龄无关,各组病例CD34表达的阳性率有显著性差异(χ~2 =51.977, P=0.000)。3 TGF-β1在食管癌变过程中上皮细胞和间质纤维细胞的表达3.1 TGF-β1在上皮细胞中的表达TGF-β1在正常食管上皮细胞的表达率为5%(1/20);在低级别上皮内瘤变组、高级别上皮内瘤变组、早期癌组、进展期癌组的表达率分别为57.7%(15/26)、65.9%(29/44)、43.5%(10/23)、13.0%(3/23)。结果显示食管鳞状上皮癌变过程中上皮细胞的TGF-β1表达呈先升后降的趋势,其中在高级别上皮内瘤变组的表达达到峰值,其表达与患者性别、年龄无关,各组病例TGF-β1表达阳性率有显著差异(χ~2 =31.977, P=0.000)。3.2 TGF-β1在间质纤维母细胞中的表达TGF-β1在正常食管间质中不表达(0/20),在低级别上皮内瘤变组、高级别上皮内瘤变组、早期癌组、进展期癌组的表达率分别为3.8%(1/26)、20.5%(9/44)、34.8%(8/23)、60.9%(14/23)。结果显示癌变过程中食管间质TGF-β1表达呈上升趋势,其表达与患者性别、年龄无关,各组病例TGF-β1表达的阳性率有显著性差异(χ~2 =31.425, P=0.000)。4 HGF在食管癌变过程中间质纤维母细胞的表达HGF在正常食管间质中不表达(0/20),在低级别上皮内瘤变组、高级别上皮内瘤变组、早期癌组、进展期癌组的表达率分别为3.8%(1/26)、28.5%(13/44)、39.1%(9/23)、56.5%(13/23)。结果显示癌变过程中食管间质HGF表达呈上升趋势,其表达与患者性别、年龄无关,各组病例HGF表达的阳性率有显著性差异(χ2 =26.817, P=0.000)。5αSMA、CD34和TGF-β1在食管癌变过程间质纤维细胞表达的相关性分析αSMA和CD34在食管各级别病变间质纤维母细胞的阳性表达之间具有负相关性,相关系数R=-0.762,P=0.000。αSMA和TGF-β1在食管各级别病变间质纤维母细胞的阳性表达之间具有正相关性,相关系数R=0.419, P=0.000。6食管癌变过程各阶段的微血管密度(microvascular density, MVD)正常食管MVD值为12.33±1.68,低级别上皮内瘤变、高级别上皮内瘤变、早期癌、进展期癌的MVD值分别为15.72±1.97、20.91±2.20、26.42±1.96、30.01±2.35。结果显示癌变过程MVD值逐渐升高,5组的MVD值有显著性差异(P=0.000),且5组任意两者之间两两比较均有显著性差异(P=0.000)。MVD值大小与患者性别、年龄无关,但αSMA阳性组的MVD值明显高于阴性组,间质TGF-β1阳性组的MVD值也明显高于阴性组。结论:1在食管鳞状上皮从正常→癌前病变→癌的演变过程中,间质纤维母细胞向CAFs转化增加,提示间质CAFs可能参与上皮细胞恶性转化、促进癌进展。2推测CAFs通过分泌TGF-β1和HGF参与了食管鳞状细胞癌的发生、发展等一系列过程。3 CAFs可能在癌前病变阶段就促进微血管形成,并由此而促进食管的癌变和进展。4间质CAFs及其分泌因子TGF-β1和HGF可能是预测食管癌前病变转归的重要指标。更多还原

【Abstract】 Objective: Recent studies have found that stromal microenvironment has increasing role in the initiation and progression of cancer, not bystander but a key player, or even a potential initiator. As the main effector, a type of activated fibroblasts have been been found in tumorous stromal, and so called Carcinoma-associated-fibroblasts(CAFs) with theαSMA(+) and CD34(-) characteristic can promote the initiation and progression of cancer through the secretions such as TGF-β1, HGF.Esophageal squmous carcinoma(ESC) is one of the most common malignancies in China, more than 50% ESC patients in the world are Chinese, So ESC is a focused malignant tumor to be prevented and treated in our country. Many studies have shown ESC undergoes a multistage process from normal, precancerosis, early cancer to cancer. The research of stromal microenvironment in the process will be able to promote the development of study and treatment on ESC.The experiment was made to observe the expressions ofαSMA, CD34, TGF-β1 and HGF in initiation and progression of esophageal cancer. To explore the role of stromal CAFs and related protein in tumorous initiation and progression. To seek the potential significant clinical indicators on diagnosis and treatment, so as to provide a scientific basis for the diagnosis, treatment and prognosis of esophageal cancer.Methods: The S-P methods of immunohistochemistry was employed to measure the expression ofαSMA, CD34, TGF-β1 and HGF in 136 specimens of esophageal tissue, in which have 20 specimens of normal esophageal tissue, 26 specimens of low level intraepithelial neoplasia, 44 specimens of high level intraepithelial neoplasia, 23 specimens of early cancer, 23 specimens of advanced cancer. And every group’s microvascular density(MVD) was counted. SPSS 13.0 was applied to analyze the results of experiment.Results:1 Expression ofαSMA in stromal fibroblasts of various esophageal lesionsThe normal esophageal stromal fibroblasts didn’t expressαSMA (0/20), but the smooth muscle tissue and small vascular smooth muscle cells expressedαSMA. At low level intraepithelial neoplasia the positive expression rate ofαSMA began to increase gradually, the expression rate of adanced cancer group was 95.7%(22/23). The spindle positive cells were looked like silk ribbons surrounding the lesion tissue or tumorous nests. Results showed a ascendant trend inαSMA expression of stromal fibroblasts in the process of esophageal epithelial canceration. The expression was not obviously correlated with patient’s gender,age. Significant difference has been found in all groups about the positive expression ofαSMA(χ~2=56.423, P=0.000).2 Expression of CD34 in stromal fibroblasts of various esophageal lesionsThe CD34 positive expression rate in normal esophageal stromal fibroblasts was 95.0%(19/20). At low level intraepithelial neoplasia the positive expression rate of CD34 began to decrease gradually,in adanced cancer group stromal fibroblasts didn’t express CD34, Only endothelial cells expressed CD34. Results showed a descendant trend in CD34 expression of stromal fibroblasts in the process of canceration. The expression was not obviously correlated with patient’s gender,age. Significant difference has been found in all groups about the positive expression of CD34(χ~2=51.977, P=0.000).3 Expression of TGF-β1 in epithelium and stromal fibroblasts of various esophageal lesions3.1 Expression of TGF-β1 in epithelium of various esophageal lesions The TGF-β1 positive expression rate in normal esophageal epithelium was 5.0%(1/20), the low level intraepithelial neoplasia group was 57.7%(15/26), the high level intraepithelial neoplasia group was 65.9%(29/44), the early cancer group was 43.5%(10/23) and the adanced cancer group was 13.0%(3/23). Results showed a fist ascendant and then descendant trend in TGF-β1 expression of epithelium in the process of canceration. The expression was not obviously correlated with patient’s gender, age. Significant difference has been found in all groups about the positive expression of TGF-β1(χ~2=31.977, P=0.000).3.2 Expression of TGF-β1 in stromal fibroblasts of various esophageal lesionsThe normal esophageal stromal fibroblasts didn’t express TGF-β1(0/20). At low level intraepithelial neoplasia the positive expression rate of TGF-β1 began to increase gradually, the expression rate of adanced cancer group was 60.9%(14/23). Results showed a ascendant trend in TGF-β1 expression of stromal fibroblasts in the process of canceration. The expression was not obviously correlated with patient’s gender,age. Significant difference has been found in all groups about the positive expression of TGF-β1(χ~2=31.425, P=0.000).4 Expression of HGF in stromal fibroblasts of various esophageal lesionsThe normal esophageal stromal fibroblasts didn’t express HGF (0/20). At low level intraepithelial neoplasia the positive expression rate of HGF began to increase gradually, the expression rate of adanced cancer group was 56.5%(13/23). Results showed a ascendant trend in HGF expression of stromal fibroblasts in the process of canceration. The expression was not obviously correlated with patient’s gender,age. Significant difference has been found in all groups about the positive expression of HGF(χ~2=26.817,p=0.000).5 Expression correlation betweenαSMA and CD34, TGF-β1 in stromal fibroblasts of various esophageal lesionsThe expression ofαSMA in stromal fibroblasts of various esophageal lesions was inversely correlated with CD34(R=-0.762, P=0.000). The expression ofαSMA in stromal fibroblasts of various esophageal lesions was correlated with TGF-β1(R=0.419, P=0.000).6 The MVD of the various esophageal lesionsThe MVD value of normal esophagus was 12.33±1.68, The MVD of low level intraepithelial neoplasia was 15.72±1.97, The MVD of high level intraepithelial neoplasia was 20.91±2.20, The MVD of early cancer was 26.42±1.96, The MVD of adanced cancer was 30.01±2.35. The MVD was not obviously correlated with patient’s gender, age. Significant difference has been found in all groups about The MVD value(P=0.000), and also in any two of all groups(P=0.000).Conclusions:1 With the process of esophageal squamous epithelial canceration, there were more stromal fibroblasts transforming into CAFs. Suggests that CAFs can induce esophageal squamous epithelial transforming and further developing.2 CAFs can contribute to the initiation and progression of ESC through factors such as TGF-β1, HGF.3 CAFs may promote microvascular formation in precancerous stage, and thus promote the initiation and progression of esophageal cancer.4 Indicates CAFs and the related factors such as TGF-β1, HGF in esophageal precancerosis probably are good prognosis indicators.更多还原