【作者】 刘琴；

【导师】 来茂德； 徐芳英；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2016， 硕士

【摘要】 结直肠癌是一类常见的消化系统恶性肿瘤,据世界癌症协会2014年的研究报道,结直肠癌的致病率和致死率依然高居第三位。近年来,关于结直肠癌的研究已进展较快,但肿瘤复发、转移、耐药性仍是难以克服的问题。因此,深入研究结直肠癌发生发展的调节因素和机制,寻找有效的治疗靶点对于结直肠癌的治疗至关重要。促泌素是2000年由Wagner等采用免疫筛选技术从人胰腺β细胞cDNA文库中筛选出来的一个新的基因,该基因定位于染色体6p22.1-22.3,编码一个由276个氨基酸组成,分子量为32KDa的蛋白质。通过对该蛋白氨基酸序列分析发现,其含有6个EF-手型钙离子结合环,属于S100家族蛋白。人体内促泌素基因在胰腺及垂体中表达最高,在其他组织中也有表达,如脑、肾上腺、甲状腺及胃肠道等。在我们实验室早期研究中,促泌素(secretagogin, SCGN)是我们首次筛选到的一个在结直肠癌中表达下调的蛋白,并经免疫印迹及免疫组织化学反馈性验证。有研究表明促泌素蛋白有控制细胞生长和分化的作用。近来有报导显示,促泌素的过表达会通过抑制小细胞肺癌细胞的凋亡从而诱导细胞的化学耐药。在肾透明细胞癌中,促泌素的高表达与高转移有联系。我们实验室的免疫组织化学结果表明,促泌素主要表达于正常结直肠黏膜隐窝的基底层,细胞形态呈锥形,与肠道神经内分泌细胞极为相似。已证明促泌素蛋白是一个很好的神经内分泌标记物。神经内分泌分化(neuroendocrine differentiation, NED)现象广泛存在于一般的非内分泌组织、良性肿瘤及恶性肿瘤组织中,且恶性肿瘤中以腺癌多见,神经内分泌细胞作为腺癌组织的一种伴随成分以单个细胞或细胞巢的形式分散存在。目前已在多种非神经内分泌器官腺癌包括食管癌、胃癌、结直肠癌、前列腺癌、乳腺癌等中发现了神经内分泌细胞分化(neuroendocrine cell differentiation, NED)的现象,这种现象被称为腺癌伴神经内分泌分化。本课题主要是探究促泌素在结直肠癌发展及预后中的意义。腺癌中,关于神经内分泌分化表达的预后价值莫衷一是。有研究报道,腺癌伴发神经内分泌分化的临床意义与肿瘤原发部位相关。有人认为,胰腺癌伴发神经内分泌分化预后较好,前列腺癌伴神经内分泌分化则提示着较差的预后。同时又有报道称神经内分泌分化对于乳腺癌的预后并无确切影响。虽然大部分研究认为伴神经内分泌分化提示了一个较差的预后,关于结直肠腺癌中神经内分泌分化对预后的影响仍持有争议。Grabowski等研究发现神经内分泌细胞分化能够被用来作为预测晚期(Ⅲ、Ⅳ期)结直肠癌患者的独立预后指标,此外,神经内分泌细胞分化相较于中或高分化腺癌而言更易出现在低分化腺癌。在结直肠癌中,促泌素的表达是否与结直肠癌的预后有相关性呢?为证实以上假设,我们首先检测了417例结直肠癌病人组织标本中促泌素的表达。然后在结直肠癌细胞中构建了过表达促泌素的细胞株,探索促泌素对结直肠癌细胞耐药的影响。结果表明,在417例结直肠癌病人组织标本中,促泌素的表达不影响预后,P=0.230。在经过化疗的结直肠癌病人标本中,促泌素的表达是病人预后的有利因素,P=0.004。此外,在这批组织标本中,促泌素的表达与膜E-Cadherin的表达均呈显著正相关,P值为0.018。在构建的过表达促泌素结直肠癌细胞株中,检测到细胞对5-FU的敏感性增强。此外,检测了过表达促泌素细胞株的侵袭、迁移能力均下降,上皮标志物E-Cadherin表达显著升高。通过研究,我们得出以下结论：1.在进行术后化疗的结直肠癌患者中,促泌素表达阳性者预后较好,促泌素表达可增强结直肠癌细胞对5-氟尿嘧啶的敏感性；2.促泌素抑制结直肠癌细胞迁移侵袭能力。更多还原

【Abstract】 Colorectal cancer (CRC) is a general vicious digestive system carcinoma, the World Cancer Association in 2014 showed that the morbidity and mortality of colorectal cancer is still ranked third.Secretagogin was identified in 2000 by Wagner using immune screening technology in human pancreatic βcells cDNA library. The gene is located on chromosome 6 p22.1-22.3, encoding a 32KDa protein containing 276 amino acids. The protein contains six EF-hand calcium rings according to the protein amino acid sequence analysis, belonging to the S100 protein family. The secretagogin gene is expressed in abundance in pancreas, pituitary, brain, adrenal gland, thyroid gland and the gastrointestinal tract in human. Secretagogin is the first screened protein that is down-regulation in colorectal cancer in our laboratory and has been verified by western blotting and immunohistochemistry. At present the function of the protein is not very clear. Studies have shown that the secretagogin protein has an influence on cell growth and differentiation. Immunohistochemical results show that secretagogin is mainly expressed in base layer of normal colorectal mucosa fossae, a cone-shaped cell morphology, and is very similar to gut neuroendocrine cells. It has been verified that secretagogin is a very good neuroendocrine marker.Neuroendocrine cell differentiation (neuroendocrine cell differentiation, NED) phenomenon has been confirmed in a variety of adenocarcinoma of non-neuroendocrine organs, and differentiated neuroendocrine cells exist in the form of a single cell or cell nests dispersed, as a composition coupling with cancers. So it is called adenocarcinoma accompanied by neuroendocrine differentiation. It is reported that this kind of malignant tumor includes esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, breast cancer, etc. Here we mainly explore the significance of secretagogin in the progress and prognosis of colorectal cancer.Recent reports show that secretagogin will induce chemical resistance by the inhibition of the apoptosis in small cell lung cancer cell. In renal clear cell carcinoma, the high expression of secretagogin is associated with high metastasis. As opinions vary, the relationship between the neuroendocrine differentiation with prognosis in adenocarcinoma is not clear. Studies showed that the clinical significance of adenocarcinoma accompanied by endocrine differentiation patients is associated with primary sites.Some argue that endocrine differentiation would indicate a good prognosis in the patients with pancreatic cancer, while a bad prognosis would be indicated in prostate cancer with neuroendocrine differentiation. At the same time, there have been reports that neuroendocrine differentiation takes no exact influence on the prognosis of breast cancer. In colorectal cancer, researches about the relationship between neuroendocrine differentiation and prognosis still hold controversial, but most of the researches indicated neuroendocrine differentiation suggests a poor prognosis. Grabowski found that differentiation of neuroendocrine cell can be used as predicting the late phase (III, IV) as independent prognostic indicators in patients with colorectal cancer. NE cell differentiation would be more likely to appear in the poorly differentiated adenocarcinoma compared to the medium or well differentiated adenocarcinoma. So in colorectal cancer, is SCGN related with the chemotherapy drug resistance or high metastasis?To confirm the above assumptions, we first tested the expression of SCGN in 417 tissue samples of colorectal cancer patients. Then we constructed an SCGN-overexpressed cell line and explored whether SCGN takes effects on the drug resistance in colorectal cancer cell or not. Results show that expression of SCGN in 417 cases of tissue samples does not influence prognosis, P=0.230. Interestingly, expression of SCGN was significantly positively related with prognosis in patients after chemotherapy, P=0.004. In addition, in these samples, SCGN is significantly positively related with the expression of membrane E-Cadherin, P values were 0.018. In the SCGN-overexpressed cell lines which we construced, SCGN enhanced the drug sensitivity. In addition, the overexpression of SCGN promoted invasion, migration capacity in CRC cells. At the protein level and mRNA level, expression of epithelial markers such as E-Cadherin increased significantly.Based on the above data, we draw the following conclusions:1. Expression of secretagogin suggests a good prognosis in CRC patients with chemotherapy. Secretagogin may enhance the sensitivity of colorectal cancer cells to 5-fluorouracil.2. Secretagogin may inhibit colorectal cancer cell migration and invasion ability.更多还原