【作者】 李琨琨；

【导师】 刘益清； 唐芙爱；

【作者基本信息】 郑州大学 ， 内科学， 2004， 硕士

【摘要】 背景与目的：大肠癌是最常见的恶性肿瘤之一，它的发生率和死亡率均占全部恶性肿瘤的前列。与其他恶性肿瘤一样，大肠癌在其发生和发展过程中也具有浸润和转移的特性，其具体机制不甚明了。近来越来越多的研究发现，转化生长因子-β（Transforming growth factor β，TGF-β）分泌紊乱及其转导通路的异常与肿瘤的发生发展关系密切。 TGF-β是一类多功能的多肽类生长因子，对细胞的增殖与分化，细胞外基质的产生，细胞凋亡及机体的免疫系统均起着重要的调节作用。活化的TGF-β与其Ⅱ型受体（transforming growth factor β receptorⅡ，TβRⅡ）结合从而形成TGF-β／TβRⅡ复合物并迅速导致Ⅰ型受体（transforming growth factor β receptor Ⅰ，TβRⅠ）的磷酸化。磷酸化的TβRⅠ可磷酸化受体调节Smads（Smad2，Smad3），并与共介导Smads（Smad4）结合形成异源复合物，转位到细胞核，调节靶基因的转录。TGF-β1是TGF-β家族的主要成员，多数肿瘤中可以观察到TGF-β₁的高表达，但其机制尚未完全阐明。Smad4是一种新近发现的抑癌基因，在TGF-β信号转导通路中具有重要作用，在人类多种肿瘤中表达水平降低。TGF-β信号转导通路的异常常见于一些来源于上皮细胞的肿瘤发展的晚期，并且与肿瘤的浸润及转移密切相关。 许多研究表明，肿瘤的生长是血管依赖性的，血管生成在肿瘤的浸润及转移中发挥了重要作用。肿瘤的血管生成受到许多癌基因，抑癌基因及生长因子的影响。文献报道，TGF-β1在多种肿瘤的血管生成中起重要作用，Smad4则可以抑制肿瘤的血管生成，但它们在大肠癌中的作用尚无定论。微血管密度（Microvesseldensity，MVD）是衡量血管生成的定量指标。CD34是特异性最强的血管内皮细胞郑州大学2004年硕士论文人大肠肿瘤TGF一阳和smad4的表达及其与血管生成的关系标记物，可用来检测MVD。本文通过检测大肠腺癌中TGF一pl，Smad4的表达及其相互关系，并探讨二者同MVD的关系，旨在阐明TGF一B信号转导通路障碍在大肠癌发生及浸润和转移中的可能机制，从而为大肠癌的诊断、治疗及预后判断提供一条新的途径。 材料与方法:（l）组织标本来源于郑州大学第一附属医院及郑州铁路局中心医院1999一2003年间手术切除标本。共选取64例大肠腺癌标本、24例大肠腺瘤标本、10例经病理证实为正常的癌旁组织标本，所有标本均经10%甲醛固定，常规脱水后石蜡包埋。（2）免疫组化sp法（streptavidin-peroxidase，sP）检测ToF一日l，Smad4及CD34在大肠正常粘膜、大肠腺瘤及大肠腺癌中的表达情况并计数MVD。（3）统计工具为sPsslo.0版软件包，统计方法采用xZ检验、spe^an等级相关分析、t检验以及方差分析，并以尸<0.05作为有统计学意义的判定标准。 结果:（l）TGF一m主要表达于细胞浆，偶见细胞核中也有表达，TGF一pl在正常大肠粘膜及腺瘤组织中着色较均匀，染色浅淡;在大肠腺癌组织中成棕黄色或棕褐色。Smad4的着色部位主要在细胞浆中，在细胞膜上也有着色，可见胞浆、胞膜中的棕黄色和棕褐色的颗粒状物质。CD34阳性反应物质主要位于血管内皮细胞的胞浆。TGF一州在大肠腺癌中的阳性表达率为70.31%，高于大肠腺瘤组的33.33%和癌旁正常大肠粘膜组的30.00%，差异均具有统计学意义（P<0 .05），而大肠腺瘤组与癌旁正常大肠粘膜组比较，TGF一印的阳性表达率虽然升高，但不具有统计学意义（P>0.05）。Smad4在大肠腺癌组中的阳性表达率为46.88%，与大肠腺瘤组的83.33%和癌旁正常大肠粘膜组的90.00%相比明显降低护<0.05)，大肠腺瘤与癌旁正常大肠粘膜组间比较无明显差异（外0.05）。大肠腺癌组MVD平均值为26.85士8.27，在大肠腺瘤组和癌旁正常大肠粘膜组MVD平均值分别为14.36士7.43和8.83士4.94，三组间两两相比均具有显著性差异（P<0.05），大肠腺癌组MVD显著高于大肠腺瘤组和癌旁正常大肠粘膜组，大肠腺瘤组的MVD也明显高于癌旁正常大肠粘膜组。 （2）根据大肠腺癌组织分化程度不同将大肠腺癌分为高/中分化组和低分化组，TGF一盯在高/中分化组和低分化组大肠腺癌的阳性表达率分别为69.05%、72.73%，两组间比较无显著性差异（P>0.05）。Smad4在高/中分化组和低分化组大肠腺癌的阳性表达率分别为50.00%、40.91%，呈一定的下降趋势，但两组间比郑州大学2004年硕士论文人大肠肿瘤TGF一娜和Smad4的表达及其与血管生成的关系较亦无显著性差异（P>0.05）。MVD平均值在高/中分化组和低分化组大肠腺癌分别为23.81士6.90和32.65士7.63，两组间比较有显著性差异（P<0.01）。 （3）根据癌细胞在肠壁内浸润深度的不同，可将大肠腺癌分为浸润未达外膜层组和浸润达到或超过外膜层组。TGF一邵在此两组内的阳性表达率分别为48.00%和84.62%，随浸润深度的增加呈上升趋势，两组间比较差异有显著性（P<0 .05）。Smad4在此两组内的阳性表达率分别为64.00%和35.90%，随浸润深度的增加而呈一定的下降趋势，两组间比较差异亦有显著性护<0.05)。两组的MVD平均值分别为19.93士5.01和31.26士6.73，随浸润深度的增加呈一定的上升趋势，两组间比较差异有显著性（尸<0.01）。 （4）根据大肠腺癌有?更多还原

【Abstract】 Background and objectives: Colorectal carcinoma is one of the common malignant tumors that its incidence rate and death rate both rank the forefront of all the malignant tumors. As other tumors, colorectal carcinoma also has characteristics such as invasion and metastasis in process of its occurrence and development, but the mechanism is not fully understood. Recent studies have showed that secretive disorders and signaling pathway disturbance of transforming growth factor (TGF- ) are closely related to the progression and aggressiveness of malignant tumors.TGF- signal transduction is involved in fundamental biological processes, such as cell growth regulation, differentiation, adhension, the formation of extracellullar matrix (ECM), immune regulation and cell apoptosis. Recent progress has led to the elucidation of a general TGF- B signal pathway: Firstly, TGF- ligand adheres to transforming growth factor B receptor I , II (T B R I ,T R II) and T R I is phosphorylated by T B RII. Then, Smad2 and Smad3 are activated by T B R I and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. TGF- B 1 is a major member of TGF- B family and it expresses a high level in many tumor tissues, but its mechanism is not clear. Smad4, a new anti-oncogene, plays a key role in TGF- B signal transduction and express a low level in many human tumor tissues. The disorders of TGF- B signal transduction are always closely to the invasion and metastasis of tumors.Researches have indicated that the growth of tumor is dependent on angiogenesis.Angiogenesis of tumors plays an important role in tumor cells’ invasion and metastasis. Microvessel density (MVD) is quantitative indicator of angiogenesis. In addition, angiogenesis is also mediated by proto-oncogene, anti-oncogene and some growth factors. Studies have showed that TGF- P 1 plays an important role in angiogenesis of many tumors while Smad4 is an inhibitor of angiogenesis, but there exist lots of differences in colorectal carcinoma. In order to investigate the possible mechanism of colorectal carcinoma , an immunohistochemical streptavidin-peroxidase (SP) method was used to exmine the expression of TGF- P 1 , Smad4 and MVD in colorectal carcinoma and the correlation between these three proteins.Materials and methods: (1) 64 surgically resected carcinoma of colorectal carcinoma samples, 24 adenoma samples and 1 0 normal mocosa samples of colorectal carcinoma which were adjacent to carcinoma mucosa, were confirmed pathologically. All the tissues were fixed in 10% neutral formalin and embedden in paraffin. (2) SP immunohistochemical staining technique was used to exmine the expression of TGF- P 1 , Smad4 and CD34 in normal mucosa, adenoma and colorectal carcinoma. (3) The datawere analyzed by software SPSS 10.0. x -test or t-test was used to analysize the difference between different groups. P value < 0.05 was considered as statistically significant.Results: (1) TGF- P 1 protein was stained mainly in the cytoplasm of cells .Normal epithelial cells and adenoma were homogeneously stained by TGF- P 1 , whereas the positive tumor cells were heterogeneously distributed within colorectal carcinoma. Smad4 protein was stained mainly in the cytoplasm of cells, and occasionally evident in membrane of cells. CD34 protein was stained mainly in the cytoplasm of vascular endothelial cells. The positive rates of TGF- P 1 in colorectal carcinoma group, colorectal adenoma group and normal mocosa group were 70.31%, 33.33% and 30% respectively. There were significant differences (P<0.05) between normal mocosa group or adenoma group and carcinoma group, but no differences were observed between normal mocosa group and adenoma group. The positive rates of Smad4 in colorectal carcinoma group, colorectal adenoma group and normal mocosa group were 46.88%, 83.33% and 90% respectively. There were significant differences (P<0.05) betweennormal mocosa group or adenoma group and carcinoma group, but no significance differ更多还原