胞浆溶解型儿茶酚胺氧位甲基转移酶（S-COMT）影响胰腺癌生物学行为的机制研究

发布时间：2018-04-25 13:50

本文选题：儿茶酚胺氧位甲基转移 + 胰腺癌　；参考：《北京协和医学院》2013年博士论文

【摘要】：研究背景胰腺导管腺癌(Pancreatic ductal adenocarcinoma, PDAC)是一种主要来源于胰腺导管上皮细胞的恶性肿瘤,其恶性程度极高,预后差。胰腺癌患者在确诊后的中位生存时间仅为4至6个月,超过12个月者仅占10%,5年总体生存率不超过5%。胰腺癌发病较为隐匿,缺乏典型的临床症状,因此早期诊断率低,绝大多数的患者在明确诊断时疾病已经处于中晚期。手术是目前临床上唯一可以根治胰腺癌、改善患者预后的方法,但根治性手术切除率仅为15-20%；化疗虽然是治疗胰腺癌的主要辅助手段,但肿瘤细胞对化疗药物产生的耐药性严重地影响了疗效,使胰腺癌成为当今医学领域最难治疗的癌症之一。因此,目前在胰腺癌不能早期诊断的情况下,寻找新的有效的治疗靶标已经成为国内外研究的热点。本实验室前期应用蛋白质组学的方法在胰腺癌患者血清中筛选出的儿茶酚胺氧位甲基转移酶(Catechol-O-Methyltransferase, COMT)可能为具有临床应用价值的胰腺癌相关免疫原性膜抗原。COMT蛋白在人体众多的组织器官中均有广泛表达,是雌激素分解代谢过程中的一个关键酶,它能迅速通过甲基化的方式灭活雌激素代谢中间产物。最近越来越多的研究显示,COMT不仅是中枢神经系统外多巴胺的主要降解酶,而且对于雌激素诱导的遗传毒性致癌作用具有保护作用。研究目的 1、验证COMT蛋白的两种同工酶MB-COMT、S-COMT在人胰腺癌组织及人胰腺癌细胞系中的表达情况。 2、探索COMT蛋白在体外及体内水平对胰腺癌细胞恶性表型的影响,在蛋白水平揭示该蛋白发挥作用的分子生物学机制,评估其作为胰腺癌新的治疗靶标的可能性。 3、分析COMT蛋白在肿瘤组织中的表达水平与胰腺癌患者临床病理特征及预后之间的关系,评价该蛋白作为胰腺癌诊断标记物及预后指标的临床应用价值。材料与方法 1、应用Western blotting法检测COMT蛋白在16例人胰腺癌组织、癌旁组织及9株常用人胰腺癌细胞系(SW1990, Su86.86, AsPC-1, BxPC-3, Capan-1, Panc-1, MIAPaCa-2, Colo357, T3M4)中的表达水平。 2、构建S-COMT基因的过表达质粒,并应用siRNA干扰技术瞬时过表达或抑制S-COMT蛋白在胰腺癌细胞系中的表达水平。应用CCK-8法、流式细胞仪分析法及Transwell法观察并对比干预前后胰腺癌细胞在增殖、凋亡和侵袭能力的变化。 3、应用Western blotting法检测过表达或抑制S-COMT蛋白后胰腺癌细胞系内与细胞增殖相关蛋白(如PI3K、p-Akt、GSK-3、PTEN)、细胞周期相关蛋白(如p53、p27、p21、CyclinD1、CDK4)、凋亡相关蛋白(如Bax、Bad、Bcl-2、 Bim)以及侵袭相关蛋白(E-cadherin)的表达水平,在蛋白水平揭示S-COMT影响胰腺癌细胞恶性表型的分子生物学机制。 4、以慢病毒为载体构建胰腺癌S-COMT蛋白稳定表达细胞系,应用裸鼠皮下成瘤实验进一步探讨S-COMT在体内水平对胰腺癌发生发展的影响作用,并验证体外实验结果。 5、收集我院2009年1月至2011年12月间收住院治疗并经过病理学检查确诊的胰腺癌标本53例,采用免疫组化方法检测S-COMT蛋白在癌组织与癌旁组织中的表达情况,应用Kaplan-Meier生存分析评价其与患者临床病理特征以及预后指标的相关性。结果 1、COMT蛋白两种同工酶(MB-COM、S-COMT)在胰腺癌肿瘤组织及癌旁组织中均有表达,其中S-COMT蛋白在癌组织内的表达水平显著的高于与其对应的癌旁组织(p=0.0129),而MB-COMT蛋白在肿瘤组织及癌旁中的表达差异无统计学意义(p=0.0987)。MB-COMT、S-COMT蛋白在9株人胰腺癌细胞系中均有不同程度的表达,其中S-COMT在Panc-1、Su86.86、Canpan-1细胞中呈低表达,在Colo357、MIAPaCa-2、T3M4中呈中等表达,在SW1990、AsPC-1、 BxPC-3中呈高表达。根据细胞系内表达水平,我们选择S-COMT蛋白分别呈低、中、高表达的三株胰腺癌细胞系Panc-1、MIAPaCa-2、SW1990作为本实验的研究对象。 2、在体外水平,S-COMT蛋白可显著影响胰腺癌细胞的生物学行为及恶性表型：过表达S-COMT蛋白后胰腺癌细胞可发生G1期阻滞,增殖能力减弱,侵袭能力下降,在吉西他滨的诱导下细胞凋亡水平增加；相反,抑制S-COMT蛋白后胰腺癌处于分裂期S期的细胞比例增加,增殖与侵袭能力增强而凋亡水平降低。裸鼠皮下成瘤实验结果也显示,S-COMT蛋白在体内水平同样可明显抑制肿瘤的形成及肿瘤增殖的速度。 3、S-COMT蛋白可影响胰腺癌细胞系内与细胞增殖相关蛋白(如PI3K、 p-Akt、GSK-3)、细胞周期相关蛋白(如p53、p27、p21、CyclinD1、CDK4)、凋亡相关蛋白(如Bax、Bad、Bcl-2、Bim)以及侵袭相关蛋白(E-cadherin)的表达水平。S-COMT蛋白可以通过调节与肿瘤细胞密切相关的重要信号传导通路PI3K/Akt及p53途径的活化程度来调节胰腺癌细胞的恶性表型。 4、免疫组化结果显示,S-COMT在胰腺癌组织中表达明显上调(P=0.0001),其表达水平与胰腺癌临床TNM分期及是否局部复发相关,而与患者的年龄、性别、肿瘤分化程度、肿瘤的部位、手术方式、手术切缘情况及是否存在远处转移无相关性。Kaplan-Meier生存分析结果显示胰腺癌患者S-COMT蛋白低表达提示预后不良,然而S-COMT蛋白的表达水平并不是影响手术患者预后的独立危险因素。结论 1、体外及体内实验结果显示,S-COMT可能通过调节细胞内重要的信号传导通路PI3K/Akt及p53途径的活化程度影响胰腺癌细胞的生物学行为,对胰腺癌的发生及演进具有显著的抑制作用,临床中通过调节其表达水平有望成为胰腺癌治疗的新策略。 2、S-COMT蛋白在胰腺癌组织中呈显著的高表达,其表达水平与肿瘤的临床TNM分期及是否局部复发相关；胰腺癌患者S-COMT蛋白低表达往往提示预后不良,然而S-COMT蛋白的表达水平并不是影响手术患者预后的独立危险因素。
[Abstract]:Research background
Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor derived mainly from pancreatic ductal epithelial cells. Its malignant degree is very high and the prognosis is poor. The median survival time of the patients with pancreatic cancer is only 4 to 6 months after diagnosis, and only 10% for more than 12 months. The overall survival rate of 5 years is not more than that of 5%. pancreatic cancer. Occult, lack of typical clinical symptoms, so the early diagnosis rate is low, most of the patients are in the middle and advanced stages when the diagnosis is clear. The operation is the only method to cure pancreatic cancer and improve the prognosis of the patients, but the radical resection rate is only 15-20%; chemotherapy is the main auxiliary hand for the treatment of pancreatic cancer. But the drug resistance of tumor cells seriously affects the efficacy of chemotherapeutic drugs, making pancreatic cancer one of the most difficult to treat in the medical field. Therefore, it has become a hot spot at home and abroad to find new effective therapeutic targets in the case of early diagnosis of pancreatic cancer. The screening of Catechol-O-Methyltransferase (COMT) in the serum of patients with pancreatic cancer (COMT) may be a clinical application of pancreatic cancer related immunogenic membrane antigen (.COMT), which is widely expressed in many tissues and organs in human body, and is the process of estrogen catabolism. A key enzyme that can quickly inactivate intermediate products of estrogen metabolism through methylation. More and more recent studies have shown that COMT is not only the main degradation enzyme of dopamine outside the central nervous system, but also has a protective effect on estrogen induced genotoxic carcinogenesis.
research objective
1, verify the expression of two isozymes MB-COMT and S-COMT of COMT protein in human pancreatic cancer tissues and human pancreatic cancer cell lines.
2, to explore the effects of COMT protein on the malignant phenotype of pancreatic cancer cells in vitro and in vivo, and to reveal the molecular biological mechanism of the protein at the protein level, and to assess the possibility of the protein as a new therapeutic target for pancreatic cancer.
3, the relationship between the expression level of COMT protein in tumor tissue and the clinicopathological features and prognosis of pancreatic cancer patients was analyzed, and the clinical value of the protein as a diagnostic marker and prognostic indicator of pancreatic cancer was evaluated.
Materials and methods
1, the expression level of COMT protein in 16 human pancreatic cancer tissues, para cancerous tissue and 9 common human pancreatic cancer cell lines (SW1990, Su86.86, AsPC-1, BxPC-3, Capan-1, Panc-1, MIAPaCa-2, Colo357, T3M4) was detected by Western blotting.
2, the overexpression plasmid of S-COMT gene was constructed, and the expression level of S-COMT protein in pancreatic cancer cell lines was temporarily overexpressed or inhibited by siRNA interference technique. CCK-8, flow cytometry and Transwell were used to observe and compare the proliferation, apoptosis and invasion ability of pancreatic cancer cells before and after intervention.
3, the expression level of cell cycle related proteins (such as p53, p27, p21, CyclinD1, CDK4) and the expression level of cell proliferation related proteins (such as p53, p27, p21, CyclinD1, PTEN) in the pancreatic cancer cell lines after the expression or inhibition of S-COMT protein were detected by the Western blotting method. The level reveals the molecular biological mechanism of S-COMT affecting the malignant phenotype of pancreatic cancer cells.
4, using lentivirus as the carrier to construct a stable expression cell line of S-COMT protein in pancreatic cancer. The effect of S-COMT on the development of pancreatic cancer in vivo was further investigated by subcutaneous tumor formation experiment in nude mice, and the experimental results were verified in vitro.
5, 53 cases of pancreatic cancer were collected from January 2009 to December 2011 in our hospital and confirmed by pathological examination. Immunohistochemical method was used to detect the expression of S-COMT protein in cancer tissues and para cancerous tissues. The correlation between the clinicopathological features and prognosis of the patients was evaluated by Kaplan-Meier survival analysis.
Result
1, two COMT protein isozymes (MB-COM, S-COMT) were expressed in tumor tissues and adjacent tissues of pancreatic cancer, and the expression level of S-COMT protein in cancer tissues was significantly higher than that of its corresponding para cancerous tissue (p=0.0129), but the expression of MB-COMT protein in tumor tissues and adjacent to cancer was not statistically significant (p=0.0987).MB-COMT, S-COMT Protein expression was expressed in 9 human pancreatic cancer cell lines, and S-COMT expressed low expression in Panc-1, Su86.86 and Canpan-1 cells. It expressed moderately in Colo357, MIAPaCa-2, T3M4, and expressed high in SW1990, AsPC-1, BxPC-3. According to the level of expression in the cell line, we chose three strains of low, medium and high expression respectively. Pancreatic cancer cell lines Panc-1, MIAPaCa-2 and SW1990 were used as the subjects of this experiment.
2, in vitro, S-COMT protein can significantly affect the biological behavior and malignant phenotype of pancreatic cancer cells: after the expression of S-COMT protein, pancreatic cancer cells can have G1 phase block, the proliferation ability is weakened, the invasion ability is decreased, and the apoptosis level of the cells is increased under the guidance of gemcitabine. On the contrary, the pancreatic cancer is divided into division after the inhibition of S-COMT protein. The proportion of cells in phase S increased, the proliferation and invasion ability increased and the apoptosis level decreased. The results of subcutaneous tumorigenesis in nude mice also showed that S-COMT protein could also inhibit the formation of tumor and the rate of tumor proliferation in the body level.
3, S-COMT protein can affect cell proliferation related proteins (such as PI3K, p-Akt, GSK-3), cell cycle related proteins (such as p53, p27, p21, CyclinD1, CDK4), and the expression level of apoptosis related proteins, such as Bax, Bad, Bcl-2, and invasion related proteins, can be closely related to tumor cells. The activation of PI3K/Akt and p53 pathways in important signal transduction pathways regulate the malignant phenotype of pancreatic cancer cells.
4, the immunohistochemical results showed that the expression of S-COMT was significantly up-regulated in the pancreatic cancer tissues (P=0.0001). The expression level was related to the clinical TNM staging of pancreatic cancer and the local recurrence, while the age, sex, the degree of differentiation of the tumor, the site of the tumor, the mode of operation, the cutting edge of the hand, and the existence of distant metastasis were not related to.Kaplan-Meie. The results of R survival analysis showed that the low expression of S-COMT protein in pancreatic cancer patients suggests poor prognosis, but the expression level of S-COMT protein is not an independent risk factor affecting the prognosis of the patients.
conclusion
1, in vitro and in vivo experimental results show that S-COMT may affect the biological behavior of pancreatic cancer cells by regulating the activation of PI3K/Akt and p53 pathway in the important signal transduction pathway in the cell, and has a significant inhibitory effect on the occurrence and evolution of pancreatic cancer. In clinical, the expression level of the node is expected to be a new treatment for pancreatic cancer. Strategy.
2, S-COMT protein is highly expressed in pancreatic cancer tissue. Its expression level is related to the clinical TNM stage of the tumor and the local recurrence. The low expression of S-COMT protein in pancreatic cancer patients often indicates poor prognosis. However, the expression level of S-COMT protein is not an independent risk factor affecting the prognosis of the patients.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R735.9

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