【作者】 黄维莉；

【导师】 迟宝荣；

【作者基本信息】 吉林大学 ， 内科学， 2014， 博士

【摘要】 胃癌（carcinoma of stomach）是我国最常见的恶性肿瘤之一，胃癌的死亡率高居各类恶性肿瘤第二位。胃癌被广泛的认为起源于胃壁最表层的粘膜上皮细胞，它常常可以侵犯胃壁的不同广度和深度，临床发现胃癌可发生于胃的各个部位（其中胃窦幽门区最多、胃底贲门区次之、胃体部略少）。早期胃癌是指癌灶仅仅局限在胃部粘膜内或粘膜下层，进展期胃癌是指癌灶侵犯肌层深度，或者有转移到胃以外区域。实际上胃癌有多种的形态和分类，最常见的有溃疡型、浸润型、表浅型、肿块型、溃疡癌（也有观点认为这属于慢性胃溃疡癌变）。通过显微镜观测，从组织学角度也可以将胃癌分为腺鳞癌、鳞状细胞癌、未分化癌、腺癌（占约90%，包括管状腺癌、粘液腺癌、乳头状腺癌、印戒细胞癌）以及类癌。即便显微镜下的组织学形态是一致的，胃癌细胞内部更细微的分子结构也有很多的不同，按照这种分法，还有很多独特的胃癌种类等待研究者们的进一步发现。凝集素是一类糖蛋白，它具有糖的专一性，并可以促进细胞的凝集。凝集素被发现可以可逆的与糖发生专一、非共价结合。动物细胞和植物细胞都可以自主的对凝集素进行合成和分泌，凝集素广泛存在于动物、植物等生物细胞膜、细胞质及细胞外基质中，其在细胞识别和粘着反应中起着重要作用。近年来，如核磁共振（NMR）、FAC、质谱（MS）等许多先进技术的应用使糖组学的研究取得了一定的进展，但是每种技术都有其各自的缺点和优点。事实上，各种技术对于样品准备和结果的分析都需要消耗大量的时间，且需要高昂的费用。而凝集素芯片技术则无需事先对样品的某些糖链进行修饰或移除，耗时短，相对费用也较低，从而能简便、快速、高通量地对蛋白组学及糖组学进行研究。寡糖、糖脂或糖蛋白均可以通过糖芯片固定在支持物上，以用于对蛋白质或者细胞与糖结合活性的检测。目前，各种基于凝集素的特异性的糖组学相关研究技术的高速发展，特别是基因芯片技术的逐步商业化，将有助于推动对糖复合物上的聚糖结构以及功能的研究，从而有助于了解其在生物过程中所扮演的重要角色。方法：本研究收集了来自北华大学附属医院的胃癌及胃溃疡患者活检组织，利用凝集素特异结合糖链的原理，建立了基于凝集素芯片技术检测糖蛋白的模型，分析了胃癌组织与胃溃疡组织中糖蛋白糖链结构的差异，并通过凝集素组织化学方法加以验证。结果：利用凝集素芯片分别对病人胃癌组织和胃溃疡组织的糖蛋白表达进行了检测及分析，结果显示：1.通过凝集素芯片分析发现，胃癌病人癌变组织中糖蛋白的表达种类多于胃溃疡组，且糖链分支数明显增加；2.通过凝集素芯片分析发现，癌变组织糖蛋白中的乙酰氨基半乳糖（GalNAc）较胃溃疡组相比明显增加；3.通过免疫组化研究进一步证实与MPL特异结合的含有GalNAc的糖蛋白，与VVA特异性结合的含有GalNAc和GalNAcα-Ser/Thr（Tn）的糖蛋白在胃癌组织中均高表达；4.与MPL及VVA特异结合的含有GalNAc的糖蛋白主要表达于细胞质，在癌组织中大部分着色为棕黄色和棕褐色，为中度或强染色。5.所有类型的胃癌组织均显示了较强的染色，与胃溃疡相比均有显著差异性。凝集素免疫组化结果与凝集素芯片结果一致。6.在不同类型的胃癌组织中，与MPL特异结合的含有GalNAc的糖蛋白的表达量从高到低依次为：粘膜内癌、印戒细胞癌、腺癌、淋巴转移腺癌、未分化癌、类癌；7.在不同类型的胃癌组织中，与VVA特异性结合的GalNAc和GalNAcα-Ser/Thr（Tn）的糖蛋白的表达量从高到低依次为：粘膜内癌、印戒细胞癌、类癌、未分化癌、腺癌、淋巴转移腺癌；8.凝集素芯片检测糖蛋白技术可为胃癌的早期诊断及治疗提供依据。更多还原

【Abstract】 Gastric cancer is the most common epithelial cancer worldwide and the second leadingcause of cancer death, with an incidence of18.9/100,000/year. In china, these are400,000new cases of gastric cancer and300,000deaths annually, making gastric cancer China’s thirdmost common type of cancer. Surgery is the only known cure, so new prognostic indicators ortumor markers are necessary to increase patient survival, treatment outcome, and facilitateearly diagnosis.The relation between gastric ulcer and cancer has long been disput ed ever sinceCruveilhier in1839first distinguished clearly between chronic ulcer and cancer bothclinically and pathologically, but there is accumulating evidence that gastric ulcer disease ispositively associated with the risk of developing stomach cancer. Discovery of a tool fordistinguishing early gastric cancer from ulcer is necessary.Protein glycosylation, the attachment of a saccharide moiety to a protein, is amodification that occurs posttranslationally. The difference in tissue distribution and substratespecificity toward peptides are related to the diverse functions of these different subtypes ofGalNAc-Tases. GalNAc-Tase expression pattern have been reported in several types ofcancers, including gastric cancer, which suggested that GalNAc played a key role in gastriccancer genesis.Lectin microarray is a sensitive tool with the potential to allow high-throught analysis ofcancer-associated changes in glycosylation, and it has been used for biomarker discovery forcancer.Methods：Human gastric ulcer and gastric cancer tissueswere obtained from AffiliateHospital of Beihua University (Jilin, China) as frozen tissues, and they were proved bypathologists.A lectin microarray was produced by using37lectins with different binding preferences covering N-and O-linked glycans. Each sample was observed consistently by three repeatedslides and the normalized medians of each lectin from9repeated blocks were averaged and itsSD was counted.Data are presented as means±SD for the indicated number of independent experiments.Statistical differences between groups were calculated by using Student’s two-tailed t-test.Differences were considered statistically significant for values.Result：Lectin histochemistry indicated that more positive cells and intensive stainingcould be visible in gastric cancer when compared with gastic ulcer using the selected twolectins (MPL and VVA), which was consistent with the results of lectin microarray, suggestingthat lectins-MPL and VVA could be used for distinguishing gastric cancer from ulcer.1. The types of glycoprotein expression and the numbers of sugar chain branch in gastriccancer tissue were increased compared with in gastric ulcer tissue through the lectinmicroarray analysis.2. The GalNAc′s content of glycoprotein in gastric cancer tissue was increasedcompared with in gastric ulcer tissue through the lectin microarray analysis.3. The GalNAc′s content combined with MPL specific and GalNAcα-Ser/Thr (Tn)′scontent combined with VAA specific in gastric ulcer tissue were high expression byimmunohistochemical study.4. The glycoprotein contains GalNAc combined with MPL or VAA specific was mainlyexpressed in the cytoplasm, and most color was tan and brown of moderate or strong stainingin gastric cancer tissue.5. All types of gastric cancer group showed strong staining, were significantly differencecompared with gastric ulcer. The results of lectins immunohistochemical were consistent withthe results of lectin chip.6. In different types of gastric cancer tissues, MPL specific glycoprotein containingGalNAc expression quantity order from high to low was: intra-mucosal carcinoma, signet ringcell carcinoma, adenocarcinoma, lymphatic metastatic carcinoid, undifferentiated carcinoma,adenocarcinoma;7. In different types of gastric cancer tissues, VAA specific glycoprotein containing GalNAc and GalNAcα-Ser/Thr（Tn）expression quantity order from high to low was:intra-mucosal carcinoma, signet ring cell carcinoma, carcinoma and undifferentiatedcarcinoma, adenocarcinoma, lymphatic metastasis adenocarcinoma.8. Lectins chip detection glycoprotein technology could provide the basis for earlydiagnosis and treatment of gastric cancer.更多还原