【作者】 吴文铭；

【导师】 赵玉沛； 吴元德；

【作者基本信息】 中国协和医科大学 ， 普通外科学， 2010， 博士

【摘要】 研究背景：在世界范围内,消化系统恶性肿瘤是一种威胁人类健康的重大疾病之一。其中多数都处于恶性肿瘤疾病谱发病率和死亡率的前几位。例如胰腺癌,其起病隐匿、进展快、预后极差,发病率与死亡率相当,被称为“二十一世纪的顽固医学堡垒”。再如结直肠癌,随着中国人生活水平的提高,结直肠癌的发病率近年来持续升高,已成为消化道恶性肿瘤中发病率最高的疾病。然而消化道恶性肿瘤的治疗效果却不乐观。疾病确诊时病程晚、手术切除率低、术后复发及转移等都是导致预后差的原因。根治性手术切除仍是消化道恶性肿瘤治疗的首选。因此提高预后的关键是如何提高手术切除率,而早期发现肿瘤无疑是最佳的解决方法。即使对于恶性程度极高的胰腺癌,早期病变经根治性手术切除后其1年存活率和生存期都可以得到明显的提高和延长。恶性肿瘤早期诊断的方法很多,然而效力却很有限,目前大部分恶性肿瘤都缺乏真正意义上的早期诊断方法。很多已在临床上使用的早期诊断方法其实仅仅是发现进展期病变中分期偏早的病人。因此临床上亟待出现实用的、方便价廉的、特异的早期诊断方法。血清肿瘤标志物检测是一种有效的肿瘤诊断方法,也是未来有可能应用在肿瘤早期诊断的方法之一。本实验室曾应用蛋白质组学的方法进行了胰腺癌相关膜抗原的筛查和鉴定工作,并在其中选定可能与胰腺癌发生、存在相关的膜抗原。其中COMT和CHCHD3在人胰腺癌细胞株SW1990,AsPc和P3中基因和蛋白水平均有表达；在与正常组织进行比较时,COMT蛋白在胰腺癌细胞株中的表达水平明显高于正常胰腺组织。因此有必要对这两种抗原蛋白展开进一步的研究,从组织层面明确它们在胰腺癌和正常胰腺组织中的表达,并扩大到其它消化系统恶性肿瘤,了解它们在消化系统肿瘤性疾病中的表达情况,为将来寻找和确定肿瘤标志物奠定实验基础。研究目的：从组织层面验证COMT和CHCHD3在胰腺癌、胰岛细胞瘤及正常胰腺中的表达差异；了解COMT和CHCHD3在其它消化系统恶性肿瘤中的表达情况。研究方法：首先针对COMT和CHCHD3进行生物信息学研究,了解其基因定位、功能、调控、转录等因素,了解两种蛋白在生物体中执行何种功能、受何影响。手术留取人胰腺癌、胰岛细胞瘤和癌旁正常胰腺标本,手术留取人肝癌及癌旁正常肝组织、胃癌及癌旁正常胃粘膜、结直肠癌及正常结直肠粘膜。提取上述标本的总RNA和总蛋白,分别进行逆转录后PCR和免疫印迹杂交,观察目的基因和蛋白在不同组织中的表达情况及差异；进行目的蛋白的组织芯片检测,采用免疫组化的方法了解蛋白在细胞中的定位、染色强度、染色阳性细胞比例和差异。结合生物信息学研究结果进行分析。实验结果：1.COMT基因具有单核苷酸多态性,其编码蛋白包括膜结合型蛋白和可溶性蛋白。COMT蛋白是多巴胺、肾上腺素及去甲肾上腺素的代谢酶,在儿茶酚雌激素的代谢中起重要作用。尤其是后者,可将雌激素代谢为具有抑癌作用的产物。2.CHCHD3基因目前功能不详,可能与PKA途径、氧化磷酸化及信号通路有关。3.RT-PCR实验结果表明COMT和CHCHD3基因在胰腺癌、胰岛细胞瘤及癌旁正常胰腺组织中均有表达,但表达无显著性差异。4.RT-PCR实验结果表明COMT和CHCHD3基因在肝癌和正常肝组织中均有表达,表达无显著性差异。5.RT-PCR实验结果表明COMT和CHCHD3基因在胃癌和正常胃粘膜中均有表达,但无显著性差异。6.RT-PCR实验结果表明COMT基因在结直肠癌和正常结直肠粘膜中均有表达,在结直肠癌中的表达强度显著高于在正常结直肠粘膜的表达水平,二者相比具有显著性差异(P＜0.05)。CHCHD3基因在结直肠癌和正常结直肠粘膜中均有表达,但无显著性差异。7.WB实验结果表明MB-COMT、S-COMT和CHCHD3蛋白在胰腺癌、胰岛细胞瘤及癌旁正常胰腺组织中均有表达,但表达无显著性差异。8.WB实验结果表明MB-COMT和S-COMT蛋白在肝癌和正常肝组织中均有表达,但表达无显著性差异。CHCHD3蛋白在肝组织中未见明显表达。9.WB实验结果表明MB-COMT和S-COMT蛋白胃癌和正常胃粘膜中均有表达,两者在正常胃粘膜中的表达强度均高于在胃癌中的表达强度,二组相比具有显著性差异(P＜0.05)。CHCHD3蛋白在胃组织中未见明显表达。10.WB实验结果表明MB-COMT和S-COMT蛋白在结直肠癌和正常结直肠粘膜中均有表达,S-COMT在结直肠癌中的表达强度显著高于在正常结直肠粘膜的表达水平,二者相比具有显著性差异(P＜0.05)。MB-COMT在两组间的表达水平无显著性差异。CHCHD3蛋白在结直肠组织中未见明显表达。11.免疫组化结果表明COMT蛋白在胰腺癌细胞浆和细胞核中的染色阳性细胞比例高于正常胰腺细胞浆和细胞核的阳性细胞比例,二者相比具有显著性差异(P＜0.05)。12.免疫组化结果显示COMT蛋白在肝癌和正常肝组织中均有染色,染色阳性细胞比例上两组无显著性差异(P＞0.05)。13.免疫组化结果显示COMT蛋白在正常胃粘膜细胞的染色阳性细胞比例高于胃癌细胞的染色阳性细胞比例,二者相比具有显著性差异(P＜0.05)。14.免疫组化结果显示COMT蛋白在结直肠癌细胞的染色阳性细胞比例高于正常结直肠细胞的染色阳性细胞比例,二者相比具有显著性差异(P＜0.05)。15.免疫组化结果显示CHCHD3在胰腺、肝脏、胃及结直肠肿瘤和正常组织中未见明显染色。实验结论：1.COMT在胰腺癌、胰岛细胞瘤及正常胰腺组织中均有表达；与正常胰腺组织相比,COMT在胰腺癌中呈高表达。2.COMT在肝脏、胃及结直肠肿瘤和正常组织中均有表达；与正常组织相比,COMT在结直肠癌组织中呈高表达,在胃癌组织中呈低表达。3.CHCHD3在胰腺癌、胰岛细胞瘤及正常胰腺组织中均有表达。更多还原

【Abstract】 BACKGROUND:The malignant neoplasms of digestive system are among the crucial diseases that threaten human’s health in the world. The incidence and mortality of those diseases rank top list in the malignant diseases. For example, pancreatic cancer is one of the most lethal human cancers. It is hard to diagnosed and progresses rapidly with worst prognosis. The incidence of pancreatic cancer is almost equal to the mortality, so it is called " the intractable fortress of 21st century". Make another example like colorectal cancer, the incidence grew fast in recent years with the development of people’s life standard so that it has become the most common cancer among the malignant tumors of digestive system. But the treatment remains not optimistic. The worse prognosis of digestive system tumors is partly because of the later disease period, the lower respectability and the recurrence even the metastases. The surgical resection is still the primary choice for the treatment of malignant tumors of digestive system. So early detection and early diagnosis are the best way to solve the question that how to promote the radical resection rate. Even though the most malignant pancreatic cancer, the survival can be last for a longer time after the tumor has been resected in the early stage. We got many weapons to diagnose malignant tumors during their early stage, but less effective. Most methods used in the clinical practice such as CEA, CA19-9 are not for the early detection but just can screen the early phase in the advance stage. It is very urgent to develop some practical, cheap and sensitive early detection methods. The serum tumor biomarker test is a very effective tumor diagnostic method, and maybe become one of the early detection methods in the future. Our lab has finished a study about screening and identification of immunogenic membrane antigens in pancreatic cancer using proteomics. We chose some potential membrane antigens that may have some relationship with the genesis and existence of pancreatic cancer. Among them, COMT and CHCHD3 were found expressed in human pancreatic cancer cell lines SW1990, AsPc and P3 in the gene and protein level. Compared with normal pancreatic tissue, COMT were found more expressed in the pancreatic cancer cell lines. Further study is needed to prove the role of these two proteins in the pancreatic cancer, and expand the study to the tumors of other digestive system organs to understand the expression level. This is the basis for the further study of tumor marker.OBJECTIVE:In order to verify the expression of COMT and CHCHD3 in the tissue level of pancreatic cancer, insuloma and normal pancreatic tissue; To understand the expression of COMT and CHCHD3 in other malignant tumors of digestive system.METHODS:Make a deeper understand of COMT and CHCHD3 using bioinformation study. To know the genes’location, function, modulation and transcription, to explain the proteins’function in human body and what kind of influence they have on body. We kept samples of pancreatic cancer, insuloma, normal pancreatic tissue, hepatic cancer and normal hepatic tissue, gastric cancer and normal gastric mucosa, colorectal cancer and normal colorectal mucosa immediately after the samples were resected. The total RNA and total protein were extracted from the samples and then performed RT-PCR and western blot. Study the expression level and different expression between the tumors and normal tissues of all the groups. In tissue microarray, Using the immunohistochemistry to study the protein’s staining location in cells, staining intensity, staining rate. RESULTS:1. There are single nucleotide polymorphisms in COMT gene. The coding proteins include membrane-bound COMT and soluble COMT. COMT can catabolize dopamine, adnephrin and arterenol and play an important role in the metabolism of catechol estrogens whose metabolism products show an inhibitive activity against cancer.2. It is still unclear about the function of CHCHD3 gene. Maybe it relates to PKA pathway, oxidative phosphorylation and signal pathways.3. RT-PCR results showed both COMT and CHCHD3 expressed in pancreatic cancer, insuloma and normal pancreatic tissue, but there was no significantly different between the groups.4. RT-PCR results showed both COMT and CHCHD3 expressed in hepatic cancer and normal hepatic tissue, but there was no significantly different between the groups.5. RT-PCR results showed both COMT and CHCHD3 expressed in gastric cancer and normal gastric mucosa, but there was no significantly different between the groups.6. RT-PCR results showed COMT expressed in colorectal cancer and normal colorectal mucosa, and was significantly over-expressed in the colorectal cancer compared with the normal colorectal mucosa (P<0.05). CHCHD3 expressed in colorectal cancer and normal colorectatl mucosa, but there was no significantly different between the groups.7. Western blot results showed MB-COMT、S-COMT and CHCHD3 protein expressed in pancreatic cancer, insuloma and normal pancreatic tissue, but there was no significantly different between the groups.8. Western blot results showed MB-COMT、S-COMT protein expressed in hepatic cancer and normal hepatic tissue, but there was no significantly different between the groups. CHCHD3 protein didn’t express in the hepatic tissues.9. Western blot results showed MB-COMT and S-COMT protein expressed in gastric cancer and normal gastric mucosa, and was significantly down-expressed in the gastric cancer compared with the normal gastric mucosa (P<0.05). CHCHD3 protein didn’t express in the gastric tissues.10.Western blot results showed MB-COMT and S-COMT protein expressed in colorectal cancer and normal colorectal mucosa, and S-COMT was significantly over-expressed in the colorectal cancer compared with the normal colorectal mucosa (P<0.05). MB-COMT was not significantly different between the groups. CHCHD3 protein didn’t express in the colorectal tissues.11.Immunohistochemistry results showed COMT protein mostly stained in the cytoplasm and nuclear of the pancreatic cancer and normal pancreatic tissue. The staining rate of pancreatic cancer is significantly higher than the normal pancreatic tissue (P<0.05)12.Immunohistochemistry results showed COMT protein stained in the hepatic cancer and normal hepatic tissue, the staining rate showed no significantly different.13.Immunohistochemistry results showed COMT protein stained in the gastric cancer and normal gastric mucosa. The staining rate of normal gastric mucosa is significantly higher than the gastric cancer (P<0.05).14.Immunohistochemistry results showed COMT protein stained in the colorectal cancer and normal colorectal mucosa. The staining rate of the gastric cancer is significantly higher than the normal gastric mucosa (P<0.05)15.The CHCHD3 protein immunohistochemistry results showed there are no obviously stain in the pancreatic tissue, hepatic tissue, gastric tissue and colorectal tissue in the CHCHD3.CONCLUSIONS:1. COMT expresses in the pancreatic cancer, insuloma and normal pancreatic tissue, and is over-expressed in the pancreatic cancer compared with the normal pancreatic tissue.2. COMT expresses in the liver, stomach and colorectal tissue, and is over-expressed in the colorectal cancer and down-expressed in the gastric cancer compared with the normal tissue.3. CHCHD3 expresses expresses in the pancreatic cancer, insuloma and normal pancreatic tissue.更多还原