發(fā)布時(shí)間：2018-04-25 13:50

  本文選題：兒茶酚胺氧位甲基轉(zhuǎn)移 + 胰腺癌�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文

【摘要】：研究背景 胰腺導(dǎo)管腺癌(Pancreatic ductal adenocarcinoma, PDAC)是一種主要來源于胰腺導(dǎo)管上皮細(xì)胞的惡性腫瘤,其惡性程度極高,預(yù)后差。胰腺癌患者在確診后的中位生存時(shí)間僅為4至6個(gè)月,超過12個(gè)月者僅占10%,5年總體生存率不超過5%。胰腺癌發(fā)病較為隱匿,缺乏典型的臨床癥狀,因此早期診斷率低,絕大多數(shù)的患者在明確診斷時(shí)疾病已經(jīng)處于中晚期。手術(shù)是目前臨床上唯一可以根治胰腺癌、改善患者預(yù)后的方法,但根治性手術(shù)切除率僅為15-20%；化療雖然是治療胰腺癌的主要輔助手段,但腫瘤細(xì)胞對(duì)化療藥物產(chǎn)生的耐藥性嚴(yán)重地影響了療效,使胰腺癌成為當(dāng)今醫(yī)學(xué)領(lǐng)域最難治療的癌癥之一。因此,目前在胰腺癌不能早期診斷的情況下,尋找新的有效的治療靶標(biāo)已經(jīng)成為國內(nèi)外研究的熱點(diǎn)。本實(shí)驗(yàn)室前期應(yīng)用蛋白質(zhì)組學(xué)的方法在胰腺癌患者血清中篩選出的兒茶酚胺氧位甲基轉(zhuǎn)移酶(Catechol-O-Methyltransferase, COMT)可能為具有臨床應(yīng)用價(jià)值的胰腺癌相關(guān)免疫原性膜抗原。COMT蛋白在人體眾多的組織器官中均有廣泛表達(dá),是雌激素分解代謝過程中的一個(gè)關(guān)鍵酶,它能迅速通過甲基化的方式滅活雌激素代謝中間產(chǎn)物。最近越來越多的研究顯示,COMT不僅是中樞神經(jīng)系統(tǒng)外多巴胺的主要降解酶,而且對(duì)于雌激素誘導(dǎo)的遺傳毒性致癌作用具有保護(hù)作用。 研究目的 1、驗(yàn)證COMT蛋白的兩種同工酶MB-COMT、S-COMT在人胰腺癌組織及人胰腺癌細(xì)胞系中的表達(dá)情況。 2、探索COMT蛋白在體外及體內(nèi)水平對(duì)胰腺癌細(xì)胞惡性表型的影響,在蛋白水平揭示該蛋白發(fā)揮作用的分子生物學(xué)機(jī)制,評(píng)估其作為胰腺癌新的治療靶標(biāo)的可能性。 3、分析COMT蛋白在腫瘤組織中的表達(dá)水平與胰腺癌患者臨床病理特征及預(yù)后之間的關(guān)系,評(píng)價(jià)該蛋白作為胰腺癌診斷標(biāo)記物及預(yù)后指標(biāo)的臨床應(yīng)用價(jià)值。 材料與方法 1、應(yīng)用Western blotting法檢測COMT蛋白在16例人胰腺癌組織、癌旁組織及9株常用人胰腺癌細(xì)胞系(SW1990, Su86.86, AsPC-1, BxPC-3, Capan-1, Panc-1, MIAPaCa-2, Colo357, T3M4)中的表達(dá)水平。 2、構(gòu)建S-COMT基因的過表達(dá)質(zhì)粒,并應(yīng)用siRNA干擾技術(shù)瞬時(shí)過表達(dá)或抑制S-COMT蛋白在胰腺癌細(xì)胞系中的表達(dá)水平。應(yīng)用CCK-8法、流式細(xì)胞儀分析法及Transwell法觀察并對(duì)比干預(yù)前后胰腺癌細(xì)胞在增殖、凋亡和侵襲能力的變化。 3、應(yīng)用Western blotting法檢測過表達(dá)或抑制S-COMT蛋白后胰腺癌細(xì)胞系內(nèi)與細(xì)胞增殖相關(guān)蛋白(如PI3K、p-Akt、GSK-3、PTEN)、細(xì)胞周期相關(guān)蛋白(如p53、p27、p21、CyclinD1、CDK4)、凋亡相關(guān)蛋白(如Bax、Bad、Bcl-2、 Bim)以及侵襲相關(guān)蛋白(E-cadherin)的表達(dá)水平,在蛋白水平揭示S-COMT影響胰腺癌細(xì)胞惡性表型的分子生物學(xué)機(jī)制。 4、以慢病毒為載體構(gòu)建胰腺癌S-COMT蛋白穩(wěn)定表達(dá)細(xì)胞系,應(yīng)用裸鼠皮下成瘤實(shí)驗(yàn)進(jìn)一步探討S-COMT在體內(nèi)水平對(duì)胰腺癌發(fā)生發(fā)展的影響作用,并驗(yàn)證體外實(shí)驗(yàn)結(jié)果。 5、收集我院2009年1月至2011年12月間收住院治療并經(jīng)過病理學(xué)檢查確診的胰腺癌標(biāo)本53例,采用免疫組化方法檢測S-COMT蛋白在癌組織與癌旁組織中的表達(dá)情況,應(yīng)用Kaplan-Meier生存分析評(píng)價(jià)其與患者臨床病理特征以及預(yù)后指標(biāo)的相關(guān)性。 結(jié)果 1、COMT蛋白兩種同工酶(MB-COM、S-COMT)在胰腺癌腫瘤組織及癌旁組織中均有表達(dá),其中S-COMT蛋白在癌組織內(nèi)的表達(dá)水平顯著的高于與其對(duì)應(yīng)的癌旁組織(p=0.0129),而MB-COMT蛋白在腫瘤組織及癌旁中的表達(dá)差異無統(tǒng)計(jì)學(xué)意義(p=0.0987)。MB-COMT、S-COMT蛋白在9株人胰腺癌細(xì)胞系中均有不同程度的表達(dá),其中S-COMT在Panc-1、Su86.86、Canpan-1細(xì)胞中呈低表達(dá),在Colo357、MIAPaCa-2、T3M4中呈中等表達(dá),在SW1990、AsPC-1、 BxPC-3中呈高表達(dá)。根據(jù)細(xì)胞系內(nèi)表達(dá)水平,我們選擇S-COMT蛋白分別呈低、中、高表達(dá)的三株胰腺癌細(xì)胞系Panc-1、MIAPaCa-2、SW1990作為本實(shí)驗(yàn)的研究對(duì)象。 2、在體外水平,S-COMT蛋白可顯著影響胰腺癌細(xì)胞的生物學(xué)行為及惡性表型：過表達(dá)S-COMT蛋白后胰腺癌細(xì)胞可發(fā)生G1期阻滯,增殖能力減弱,侵襲能力下降,在吉西他濱的誘導(dǎo)下細(xì)胞凋亡水平增加；相反,抑制S-COMT蛋白后胰腺癌處于分裂期S期的細(xì)胞比例增加,增殖與侵襲能力增強(qiáng)而凋亡水平降低。裸鼠皮下成瘤實(shí)驗(yàn)結(jié)果也顯示,S-COMT蛋白在體內(nèi)水平同樣可明顯抑制腫瘤的形成及腫瘤增殖的速度。 3、S-COMT蛋白可影響胰腺癌細(xì)胞系內(nèi)與細(xì)胞增殖相關(guān)蛋白(如PI3K、 p-Akt、GSK-3)、細(xì)胞周期相關(guān)蛋白(如p53、p27、p21、CyclinD1、CDK4)、凋亡相關(guān)蛋白(如Bax、Bad、Bcl-2、Bim)以及侵襲相關(guān)蛋白(E-cadherin)的表達(dá)水平。S-COMT蛋白可以通過調(diào)節(jié)與腫瘤細(xì)胞密切相關(guān)的重要信號(hào)傳導(dǎo)通路PI3K/Akt及p53途徑的活化程度來調(diào)節(jié)胰腺癌細(xì)胞的惡性表型。 4、免疫組化結(jié)果顯示,S-COMT在胰腺癌組織中表達(dá)明顯上調(diào)(P=0.0001),其表達(dá)水平與胰腺癌臨床TNM分期及是否局部復(fù)發(fā)相關(guān),而與患者的年齡、性別、腫瘤分化程度、腫瘤的部位、手術(shù)方式、手術(shù)切緣情況及是否存在遠(yuǎn)處轉(zhuǎn)移無相關(guān)性。Kaplan-Meier生存分析結(jié)果顯示胰腺癌患者S-COMT蛋白低表達(dá)提示預(yù)后不良,然而S-COMT蛋白的表達(dá)水平并不是影響手術(shù)患者預(yù)后的獨(dú)立危險(xiǎn)因素。 結(jié)論 1、體外及體內(nèi)實(shí)驗(yàn)結(jié)果顯示,S-COMT可能通過調(diào)節(jié)細(xì)胞內(nèi)重要的信號(hào)傳導(dǎo)通路PI3K/Akt及p53途徑的活化程度影響胰腺癌細(xì)胞的生物學(xué)行為,對(duì)胰腺癌的發(fā)生及演進(jìn)具有顯著的抑制作用,臨床中通過調(diào)節(jié)其表達(dá)水平有望成為胰腺癌治療的新策略。 2、S-COMT蛋白在胰腺癌組織中呈顯著的高表達(dá),其表達(dá)水平與腫瘤的臨床TNM分期及是否局部復(fù)發(fā)相關(guān)；胰腺癌患者S-COMT蛋白低表達(dá)往往提示預(yù)后不良,然而S-COMT蛋白的表達(dá)水平并不是影響手術(shù)患者預(yù)后的獨(dú)立危險(xiǎn)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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R735.9

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