【作者】 才虹美；

【导师】 朱严冰；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:大肠癌是一种常见的消化道恶性肿瘤,是危害人类健康的主要恶性肿瘤之一。在西方发达国家,其发病率居恶性肿瘤的第二位。在我国,随着人民生活水平的提高和饮食习惯的改变,大肠癌的发病率有逐年上升的趋势。尽管治疗手段有了很大的进步,但五年生存率仍然很低。肿瘤的侵袭和转移是患者死亡的主要原因之一,因此,研究大肠癌转移的相关因素,对于揭示肿瘤转移的本质具有十分重要的科学意义。肿瘤转移是一个多步骤、多阶段的复杂过程,包括细胞脱落、迁徙、粘附、血管生成和逃逸机体免疫反应等。研究表明有多种蛋白参与肿瘤转移的各个过程,并对其有一定的调控和促进作用。Galectins(β-半乳糖苷结合蛋白)是生物体内广泛存在的一大类糖结合蛋白,属于凝集素家族。Galectins不仅存在于细胞质,也存在于细胞核内,具有多种生物功能。迄今,人们己经在生物体内发现了14个Galectins家族成员,其中Galectin-3广泛表达于上皮细胞和免疫细胞,参与多种生物学过程,如细胞生长、粘附、分化、血管生成和凋亡等。研究显示,Galectin-3与肿瘤侵袭和转移的某些过程相关,如血管生成、细胞基质相互作用、肿瘤细胞沿血道播散及逸出。因此,检测Galectin-3水平可能对了解一些肿瘤的转移及预后判断具有一定的价值。基质金属蛋白酶-2(matrixmetalloproteinase-2,MMP-2)是基质金属蛋白酶家族成员之一,能够降解细胞外基质(ECM)和基底膜(BM),促进肿瘤细胞向周围组织扩散,从而对肿瘤的侵袭和转移产生影响。MUC-1作为一种粘蛋白能加强肿瘤细胞与基底膜蛋白,如层粘连蛋白、纤维连接素和Ⅳ型胶原的粘附力,促进肿瘤的侵袭和转移。本研究采用免疫组织化学法,检测Galectin-3、MMP-2及MUC-1在大肠正常黏膜、癌旁组织、大肠癌组织中的表达情况,研究Galectin-3、MMP-2及MUC-1的表达与大肠癌的发生、发展、转移及其临床病理分期的关系,初步探讨它们在大肠癌发病和转移中的作用及它们之间的相关性,希望为阐明大肠癌转移的机制提供研究资料,为大肠癌的临床治疗以及判断预后提供有价值的参考指标。方法:本实验采用白求恩国际和平医院及河北医科大学第四附属医院2007年3月至5月间住院行外科手术治疗的大肠癌手术标本54例。其中男性16例,女性38例。年龄从45～72岁,平均年龄57岁。其中低分化26例,中高分化28例。Dukes分期中AB期29例,CD期25例。同时随机选取大肠癌旁组织12例,正常组织15例作为对照,癌旁组织在癌组织周围5cm以内进行取材,正常组织取材于病理证实无异常的组织块。所有患者术前均未接受化学药物治疗或放射线治疗。所有标本经10%中性福尔马林液中固定,常规石蜡包埋、4μm超薄切片,行免疫组化二步法染色,分别检测大肠癌组织、癌旁组织及正常组织中Galectin-3、MMP-2及MUC-1表达情况。结合临床资料对测定数据进行统计学分析,通过SPSS10.0统计软件进行四格表确切概率法、卡方检验、校正卡方检验、Spearman等级相关分析等统计学方法,以α=0.05为标准,P<0.05有统计学意义。结果:1 Galectin-3在大肠癌组织中阳性表达率为81.48%(44/54),癌旁组织Galectin-3阳性表达率为41.67%(5/12),正常组织中Galectin-3阳性表达率为40.00%(6/15)。Galectin-3在大肠癌组织中的表达明显高于相应的大肠正常黏膜组织及癌旁组织,有统计学意义(P<0.05 );在正常组织和癌旁组织中的表达无明显差异(P>0.05)。Galectin-3蛋白在大肠癌组织中的表达与患者年龄、性别、肿瘤大小无相关性(P>0.05);低分化大肠癌的表达比中高分化大肠癌表达明显增高(X2=5.402,P<0.05);侵及浆膜大肠癌的表达比未侵及浆膜大肠癌的表达明显增高(X2=7.364,P<0.05);有淋巴结转移大肠癌的表达比无淋巴结转移大肠癌的表达明显增高(X2=4.835,P<0.05);DukesCD期大肠癌的表达比DukesAB期大肠癌的表达明显增高(X2=4.835,P<0.05)。2 MMP-2在大肠癌组织中的阳性表达率为83.33%(45/54),癌旁组织MMP-2阳性表达率为50.00%(6/12),正常组织中MMP-2阳性表达率为40.00%(6/15)。MMP-2在大肠癌组织中的表达明显高于相应的癌旁正常黏膜组织及癌旁组织,有统计学意义(P<0.05);在正常组织和癌旁组织中的表达无明显差异(P>0.05)。MMP-2蛋白在大肠癌组织中的表达与患者年龄、性别、肿瘤大小无相关性(P>0.05);低分化大肠癌的表达比中高分化大肠癌表达明显增高(X2=4.287,P<0.05);侵及浆膜大肠癌的表达比未侵及浆膜大肠癌的表达明显增高(X2=5.134,P<0.05);有淋巴结转移大肠癌的表达比无淋巴结转移大肠癌的表达明显增高(X2=7.210,P<0.05);DukesCD期大肠癌的表达比DukesAB期大肠癌的表达明显增高(X2=7.210, P<0.05)。3 MUC-1在大肠癌组织中阳性表达率为51.85%(28/54)。在癌旁组织的阳性表达率为16.67%(2/12),在正常粘膜组织中阳性表达率为13.33%(2/15)。MUC-1在大肠癌组织中的表达明显高于相应的癌旁正常黏膜组织及癌旁组织,有统计学意义(P<0.05);在正常组织和癌旁组织中的表达无明显差异(P>0.05)。MUC-1蛋白在大肠癌组织中的表达与患者年龄、性别、肿瘤大小无相关性(P>0.05);低分化大肠癌的表达比中高分化大肠癌表达明显增高(X2=9.048, P<0.05);侵及浆膜大肠癌的表达比未侵及浆膜大肠癌的表达明显增高(X2=6.567,P<0.05);有淋巴结转移大肠癌的表达比无淋巴结转移大肠癌的表达明显增高(X2=4.862,P<0.05);DukesCD期大肠癌的表达比DukesAB期大肠癌的表达明显增高(X2=4.862,P<0.05)。4 Galectin-3和MMP-2在大肠癌组织中的表达呈正相关(r=0.298,P=0.028<0.05);Galectin-3和MUC-1在大肠癌组织中的表达呈正相关(r=0.304,P=0.025<0.05);而MMP-2和MUC-1在大肠癌中的表达无明显相关关系(r=0.166,P=0.231>0.05)。结论:1 Galectin-3在大肠癌组织中的表达量明显高于正常组织及癌旁组织,并且随着肿瘤分化程度的降低、侵犯深度的加深、淋巴结的转移以及Dukes分期的进展而升高,提示Galectin-3在大肠癌的发生、发展、侵袭和转移过程中发挥着重要的作用。2 MMP-2在大肠癌组织中的表达量明显高于正常组织及癌旁组织,同时随着肿瘤分化程度的降低、侵犯深度的加深、淋巴结的转移以及Dukes分期的进展,其表达水平有升高趋势,提示MMP-2也在大肠癌的发生、发展、侵袭和转移过程中发挥着重要的作用。3 MUC-1在大肠癌组织中的表达量高于正常组织及癌旁组织,随着肿瘤分化程度的降低、侵犯深度的加深、淋巴结的转移以及Dukes分期的进展,其表达水平有升高趋势,提示MUC-1同样在大肠癌的发生、发展、侵袭和转移过程中发挥着重要的作用。4 Galectin-3和MMP-2在大肠癌中的表达呈正相关。Galectin-3是MMP-2的底物,MMP-2的高表达使Galectin-3的表达上调,两者共同参与了大肠癌的侵袭和转移。Galectin-3和MUC-1在大肠癌中的表达也呈正相关。MUC-1作为Galectin-3的配体,通过上调Galectin-3的表达增加肿瘤细胞的粘附力和侵袭力,增大肿瘤的转移潜能。目前的研究结果尚不能表明MMP-2和MUC-1在大肠癌中的表达存在相关关系。总之,Galectin-3、MMP-2和MUC-1在大肠癌中高表达提示预后不良,三者同时检测可能对判断大肠癌的恶性程度及转移潜能具有重要的意义。更多还原

【Abstract】 Objective: Colorectal carcinoma is one of the most common digestive tract malignant tumors, which is one of the main tumor which harm human health too. It was the second tumor killer in western countries. In our country, the incidence rate of colorectal carcinoma is increasing with the development of people’s life and the alteration of the dietary. Although therapy methods have been improved, survival rate of five years is still very low. Tumor invasion and metastasis are the major cause of death in patients with colorectal carcinoma. It is important to study the metastasis associated factors so as to reveal the essence of tumor metastasis.The invasion and metastasis of malignancy is a complicated process including cell defluxion, metabasis, adhesion, angiogenesis and escaping immunoreactions. Recent studies have showed that many kinds of protein are involved in the process of metastasis, regulating or promoting the metastatic ability of the cancer cell. Galectins is a family of animal lectins defined byβ-galactoside affinity widely seen in mammalian. To date, 14 mammalian members in galectin family have been sequenced. Galectin-3 expresses generally in endothelial cell and immunological cell participates in many biological courses. The studies indicate that Galectin-3 is also involved in cell-matrix interaction, angiogenesis, the process of invasion and metastasis in the dissemination and effusion of tumor cells. To detect the level of Galectin-3 is very important for understanding the metastasis of tumor and evaluating prognosis. Matrixmetalloproteinase-2(MMP-2) is one of the matrixmetalloproteinases(MMPs), which can degrade extracellular matrix(ECM) and basement membrane(BM). MMP-2 can regulate the biological behavior of tumor cells, such as invasion, metastasis and so on. MUC-1 is a kind of mucin, which can strengthen the adhesion of tumor cells and basement membrane, such as laminin, fibronectin and typeⅣcollagen, and promote the invasion and metastasis of tumor. The immunohistochemistry was used to evaluate the expressions of Galectin-3, MMP-2 and MUC-1 in normal colonic epithelium、borderline tissues and colorectal carcinoma, and the correlation between expressions of Galectin-3, MMP-2 and MUC-1 in the carcinomatous tissues of colorectal carcinoma and clinical pathologic factors of the cancer, and to explore the role of Galectin-3, MMP-2 and MUC-1 in pathogenesis and metastasis of colorectal carcinoma so that some fundamental data might be supplied to the study of metastasis-associated factors.Methods: Collect tissues from 54 colorectal adenoca patients by surgical operation, 12 with borderline tissues and 15 with normal intestine tissues. 16 male, 38 women. Age from 45-72 years old, the average is 57 years old. 26 poorly-differentiated, 28 well-differentiated. 29 Dukes classification AB, 25 Dukes classification CD. And None had accepted the treatment of any anti-tumor before operation. All of samples were fixed by neutrality formalin solutions, embedded by paraffin, sliced in to 4μm slice, using immunohistochemistry two-step method, examining Galectin-3, MMP-2 and MUC-1 expression in the tissue. The 3 proteins (Galectin-3, MMP-2 and MUC-1) were detected and the correlation among them were studied using involved statistic methods such as the chi-square test and Spearman correlation of ranks test, etc. And P value less than 0.05 is considered to significantly different.Result:1 The positive rate of Galectin-3 is 81.48%(44/54) in the colorectal carcinoma, 41.67%(5/12) in the borderline tissues, and 40.00%(6/15) in the normal intestine tissues. The expression of the Galectin-3 in colorectal carcinoma was significantly higher than normal colonic epithelium and borderline tissues, which has statistical significance (p<0.05); and the positive rate of Galectin-3 between the normal intestine tissues and the borderline tissues has no statistical significance (p>0.05). The expression of the Galectin-3 in colorectal carcinoma has no relation with some clinical parameters including age, sex, size(p>0.05); The expression of the Galectin-3 in colorectal carcinomas with poorly differentiated was significantly higher than that of the well differentiated one(p<0.05); The expression of the Galectin-3 in colorectal carcinomas with invasion of the adventitia was significantly higher than that without invasion of the adventitia one (p<0.05); The expression of the Galectin-3 in colorectal carcinomas with lymph node metastasis was significantly higher than that without lymph node metastasis one (p<0.05); The expression of the Galectin-3 in colorectal carcinomas of Dukes AB stage was significantly higher than that of Dukes CD stage one(p<0.05).2 The positive rate of MMP-2 is 83.33%(45/54) in the colorectal carcinoma, 50.00%(6/12) in the borderline tissues, and 40.00%(6/15) in the normal intestine tissues. The expression of the MMP-2 in colorectal carcinoma was significantly higher than normal colonic epithelium and borderline tissues, which has statistical significance (p<0.05); and the positive rate of MMP-2 between the normal intestine tissues and the borderline tissues has no statistical significance (p>0.05). The expression of the MMP-2 in colorectal carcinoma has no relation with some clinical parameters including age, sex, size(p>0.05);The expression of the MMP-2 in colorectal carcinomas with poorly differentiated was significantly higher than that of the well differentiated one(p<0.05); The expression of the MMP-2 in colorectal carcinomas with invasion of the adventitia was significantly higher than that without invasion of the adventitia one (p<0.05); The expression of the MMP-2 in colorectal carcinomas with lymph node metastasis was significantly higher than that without lymph node metastasis one(p<0.05); The expression of the MMP-2 in colorectal carcinomas of Dukes AB stage was significantly higher than that of Dukes CD stage one(p<0.05).3 The positive rate of MUC-1 is 51.85%(28/54) in the colorectal carcinoma, 16.67%(2/12) in the borderline tissues, and 13.33%(2/15) in the normal intestine tissues. The expression of the MUC-1 in colorectal carcinoma was significantly higher than normal colonic epithelium and borderline tissues, which has statistical significance (p<0.05); and the positive rate of MUC-1 between the normal intestine tissues and the borderline tissues has no statistical significance (p>0.05). The expression of the MUC-1 in colorectal carcinoma has no relation with some clinical parameters including age, sex, size(p>0.05); The expression of the MUC-1 in colorectal carcinomas with poor differentiated was significantly higher than that of the well differentiated one(p<0.05); The expression of the MUC-1 in colorectal carcinomas with invasion of the adventitia was significantly higher than that without invasion of the adventitia one (p<0.05); The expression of the MUC-1 in colorectal carcinomas with lymph node metastasis was significantly higher than that without lymph node metastasis one(p<0.05); The expression of the MUC-1 in colorectal carcinomas of Dukes AB stage was significantly higher than that of Dukes CD stage one(p<0.05).4 There are significanly positive corrleation between Galectin-3 and MMP-2 (r=0.298, P=0.028<0.05); significanly positive correlation between Galectin-3 and MUC-1 (r=0.304, P=0.025<0.05); and no correlation between MMP-2 and MUC-1(r=0.166,P=0.231 >0.05).Conclusion:1 The expression of Galectin-3 in colorectal carcinoma was significantly higher than normal intestine tissues and borderline tissues, and the higher expressions of them was closely correlated with the pathogenesis、differentiation、invasion and metastasis of colorectal carcinoma.2 The expression of MMP-2 in colorectal carcinoma was significantly higher than normal intestine tissues and borderline tissues, and the higher expressions of them was closely correlated with the pathogenesis、differentiation、invasion and metastasis of colorectal carcinoma.3 The expression of MUC-1 in colorectal carcinoma was significantly higher than normal intestine tissues and borderline tissues, and the higher expressions of them was closely correlated with the pathogenesis、differentiation、invasion and metastasis of colorectal carcinoma.4 The expression of Galectin-3 and MMP-2 in colorectal carcinoma was positive correlation. Galectin-3 is the substrate of MMP-2, and the higher expression of MMP up-regulate the expression of Galectin-3.The two protein promote the invasion and metastasis of colorectal carcinoma. The expression of Galectin-3 and MUC-1 was also positive correlation. MUC-1 is a natural ligand for Galectin-3, which promotes the tumor cell adhesion and invasion by up-regulating Galectin-3.There was no correlation between MMP-2 and MUC-1.In a word, the higher expressions of Galectin-3、MMP-2 and MUC-1 might play an important role in the evaluation of prognosis. Detections of them have important signification in estimating the colorectal carcinoma progression and potentiality of lympho node metastasis.更多还原