本文選題：MGMT + 腎透明細(xì)胞癌　； 參考：《山東大學(xué)》2015年博士論文

【摘要】：腎透明細(xì)胞癌(clear cell renal cell carcinoma, ccRCC)在所有腎細(xì)胞癌占75%-80%,是最常見的病理類型�；熑允悄壳芭R床上常用的方法,但治療效果常因腫瘤本身具有耐藥或治療后出現(xiàn)耐藥性而失敗。目前為止,耐藥機(jī)制主要有P-糖蛋白的過表達(dá)、DNA拓?fù)洚悩?gòu)酶的低表達(dá)、抗凋亡基因bcl-2的過表達(dá)以及DNA修復(fù)蛋白的過表達(dá)等。烷化劑是一大類重要的臨床抗腫瘤藥物,主要造成DNA堿基的烷基化損傷,其中以形成O6-甲基鳥嘌呤對細(xì)胞的威脅最大,造成細(xì)胞突變與死亡。O6-甲基鳥嘌呤可以由O6-甲基鳥嘌呤-DNA甲基轉(zhuǎn)移酶(MGMT)修復(fù)。MGMT對烷化劑損傷的修復(fù)使其在腫瘤化療中發(fā)揮雙重作用。一方面保護(hù)正常組織免于烷化劑的損傷和二次癌癥的發(fā)生,另一方面引起腫瘤組織對烷化劑類化療藥物的耐藥性。MGMT的表達(dá)在正常組織中要低于其腫瘤組織,而且不同腫瘤組織中呈現(xiàn)出不一樣的模式。然而,在腎透明細(xì)胞癌中MGMT基因的表達(dá)目前仍不清楚。甲基化抑制劑5-Aza-CdR能夠逆轉(zhuǎn)DNA的異常甲基化、重新激活失活的抑癌基因的表達(dá),因此其具有作為抗腫瘤藥物的潛力。本課題擬從基因和蛋白水平研究MGMT基因在腎透明細(xì)胞癌中的表達(dá),并闡明MGMT在耐藥腎透明細(xì)胞癌治療中的作用。同時我們將5-Aza-CdR與絲裂霉素聯(lián)合作用于腎癌細(xì)胞,探討應(yīng)用5-Aza-CdR能否提高腎癌細(xì)胞對絲裂霉素的敏感性。第一部分MGMT基因表達(dá)對腎細(xì)胞癌的耐藥性影響的研究一、MGMT在腎透明細(xì)胞癌組織和細(xì)胞中表達(dá)上調(diào)1、采用免疫組化的方法對60個組織標(biāo)本中的MGMT表達(dá)情況進(jìn)行了檢測。免疫組化染色著色結(jié)果可以將60例病例分為MGMT不表達(dá)(-),低表達(dá)(+)和高表達(dá)(++,+++)三組,其中高表達(dá)的組占62%(37/60)。實(shí)時定量PCR結(jié)果顯示,與正常組織相比,腫瘤組織中MGMT mRNA水平表達(dá)上調(diào)2倍以上的占85%(17/20)2、選取兩株癌細(xì)胞ACHN和786-0以及正常腎細(xì)胞株HK-2為研究對象。結(jié)果表明兩株癌細(xì)胞中MGMTmRNA水平遠(yuǎn)高于正常細(xì)胞。Western blot結(jié)果進(jìn)一步驗(yàn)證了MGMT在腫瘤細(xì)胞中高表達(dá)。二、MGMT表達(dá)與腎透明細(xì)胞癌的階段有關(guān)進(jìn)一步分析了MGMT水平與癌癥進(jìn)程之間的關(guān)系。研究發(fā)現(xiàn)MGMT表達(dá)水平與腫瘤病理級別和臨床階段有著正相關(guān)。在G1,G2和G3/G4期的腎透明細(xì)胞癌中MGMT高表達(dá)的比例分別占40.0%,67.7%和77.3%(p=0.04)。在T1,T2和T3/T4階段中MGMT高表達(dá)比例分別為39.1%,70.1%和80.0%(p=0.02)。三、下調(diào)MGMT表達(dá)可以增強(qiáng)ACHN和786-0細(xì)胞對烷化劑BCNU或TMZ的敏感性。1、首先通過western blot檢測siRNA的作用,干擾siRNA作用48h后MGMT蛋白表達(dá)明顯下降。2、MTT結(jié)果顯示,下調(diào)MGMT表達(dá)可以使ACHN細(xì)胞對烷化劑BCNU和TMZ更加敏感,3、以腫瘤細(xì)胞株786-0為對象進(jìn)行實(shí)驗(yàn)。下調(diào)MGMT同樣顯著增加786-0細(xì)胞對烷化劑BCNU和TMZ的敏感性。第二部分5-Aza-CdR對腎癌細(xì)胞株CAKI-1及786-0的化療敏感性的影響一、MTT法檢測絲裂霉素對CAKI-1細(xì)胞抑制作用我們用MTT法分別檢測不同濃度絲裂霉素作用于CAKI-1細(xì)胞24 h的生長抑制率,發(fā)現(xiàn)絲裂霉素能抑制細(xì)胞的增值,并呈現(xiàn)劑量依賴性。二、初步結(jié)果顯示甲基化抑制劑5-Aza-CdR能夠增強(qiáng)腎癌細(xì)胞786-0對絲裂霉素的敏感性MTT檢測發(fā)現(xiàn)單用5-Aza-CdR對人腎癌細(xì)胞株786-0增值呈現(xiàn)明顯的抑制作用。不同濃度的5-Aza-CdR聯(lián)合絲裂霉素對腫瘤細(xì)胞的抑制作用呈現(xiàn)劑量依賴性,隨著5-Aza-CdR濃度的增加,腫瘤細(xì)胞的抑制率明顯升高。第三部分結(jié)論及創(chuàng)新點(diǎn)一、結(jié)論1、研究證實(shí)了MGMT在腎透明細(xì)胞癌組織和細(xì)胞中的表達(dá)呈高表達(dá)；2、研究分析發(fā)現(xiàn)MGMT的表達(dá)水平與腎透明細(xì)胞癌的惡性程度呈正相關(guān)；3、MGMT參與腎透明細(xì)胞癌對烷化劑類化療藥物的耐藥過程；4、甲基化抑制劑5-Aza-CdR能夠增強(qiáng)腎癌細(xì)胞786-0對絲裂霉素的敏感性。二、創(chuàng)新點(diǎn)與不足之處1、本研究首次證實(shí)了MGMT在腎透明細(xì)胞癌組織和細(xì)胞中表達(dá)高表達(dá),并且發(fā)現(xiàn)MGMT的表達(dá)水平與腎透明細(xì)胞癌的惡性程度呈正相關(guān)。本研究首次發(fā)現(xiàn)下調(diào)MGMT表達(dá)可以增強(qiáng)ACHN和786-0細(xì)胞對烷化齊BCNU或TMZ的敏感性。但MGMT是如何參與腎透明細(xì)胞癌的進(jìn)展及對烷化劑耐受的機(jī)制尚不清楚。2、初步結(jié)果顯示甲基化抑制劑5-Aza-CdR能夠增強(qiáng)腎癌細(xì)胞786-0對絲裂霉素的敏感性。5-Aza-CdR是否是通過影響MGMT的表達(dá)或活性從而影響腎癌細(xì)胞對化療藥的敏感性的需要進(jìn)一步研究。
[Abstract]:Clear cell renal cell carcinoma (ccRCC) is the most common pathological type in all renal cell carcinoma (RCC), which is the most common pathological type. Chemotherapy is still a common clinical method currently, but the therapeutic effect is often due to the resistance of the tumor itself or the emergence of drug resistance after treatment. So far, the mechanism of drug resistance is mainly the overexpression of P- glycoprotein. The low expression of DNA topoisomerase, the overexpression of the anti apoptotic gene Bcl-2 and the overexpression of the DNA repair protein. Alkylating agents are a major class of important clinical antitumor drugs, which mainly cause the alkylation of DNA base, among which the formation of O6- methyl guanine has the greatest threat to the cells, causing cell mutation and death of.O6- methyl guanine. Methotrexate can be repaired by O6- methyl guanine -DNA methyltransferase (MGMT) to repair the repair of.MGMT damage to alkylating agents to play a double role in tumor chemotherapy. On one hand, it protects normal tissues from alkylating agent damage and two cancers, and on the other hand, causes the expression of.MGMT in the tumor tissue against alkanochemotherapeutic drugs. However, the expression of MGMT gene in renal clear cell carcinoma is still unclear. Methylation inhibitor 5-Aza-CdR can reverse the abnormal methylation of DNA and reactivate the expression of the inactivated tumor suppressor gene, so it can be used as an antitumor. The potential of the drug is to study the expression of MGMT gene in the renal clear cell carcinoma from gene and protein level, and to elucidate the role of MGMT in the treatment of drug-resistant renal clear cell carcinoma. At the same time, we combine the role of 5-Aza-CdR and mitomycin in renal cell carcinoma cells to explore whether 5-Aza-CdR can improve the sensitivity of RCC cells to mitomycin. Part 1: Study on the effect of MGMT gene expression on the resistance of renal cell carcinoma 1. The expression of MGMT in the tissues and cells of renal clear cell carcinoma is up to 1. The expression of MGMT in 60 tissue specimens is detected by immunohistochemistry. The results of immunohistochemical staining can be divided into 60 cases of MGMT non expression (-), low table Three groups of high expression (+ + + + +), of which high expression group accounted for 62% (37/60). Real-time quantitative PCR results showed that the expression of MGMT mRNA in tumor tissues was up to 85% (17/20) 2, ACHN and 786-0, and normal renal cell line HK-2 in two cancer cells. The results showed that MGMT in two cancer cells. The mRNA level was far higher than the normal cell.Western blot results further verified the high expression of MGMT in the tumor cells. Two, the expression of MGMT and the stage of renal clear cell carcinoma further analyzed the relationship between the level of MGMT and the process of cancer. The study found that the level of MGMT expression was positively related to the pathological and clinical stages of the tumor. In G1, G2 The high expression of MGMT in renal clear cell carcinoma was 40%, 67.7% and 77.3% (p=0.04). The high expression of MGMT in T1, T2 and T3/T4 stages was 39.1%, 70.1% and 80% (p=0.02). Three, the downregulation of MGMT expression could enhance the sensitivity of ACHN and 786-0 cells to alkanating BCNU or TMZ. The expression of MGMT protein decreased significantly after the interference of siRNA 48h. MTT results showed that the down-regulation of MGMT expression could make ACHN cells more sensitive to BCNU and TMZ of alkylating agents, 3, with tumor cell line 786-0 as the object. The down regulation MGMT also significantly increased the sensitivity of 786-0 cells to alkanating BCNU and TMZ. Second part of the kidney. The effect of CAKI-1 and 786-0 on the chemosensitivity of cancer cell strain one. MTT assay was used to detect the inhibitory effect of mitomycin on CAKI-1 cells. We detected the growth inhibition rate of mitomycin at 24 h in CAKI-1 cells by MTT method. It was found that mitomycin could inhibit the proliferation of cells and showed a dose dependence. Two, preliminary results showed methyl methylation The inhibitor 5-Aza-CdR can enhance the sensitivity of renal cell carcinoma cell 786-0 to mitomycin MTT detection. The inhibitory effect of 5-Aza-CdR on human renal cell carcinoma cell line 786-0 is obviously inhibited. The inhibitory effect of 5-Aza-CdR combined with mitomycin on tumor cells at different concentrations is dependent on the dose dependent manner. With the increase of 5-Aza-CdR concentration, the tumor is increased. The inhibitory rate of cells increased significantly. Third conclusions and innovation point 1. Conclusion 1. The study confirmed that the expression of MGMT in the tissues and cells of renal clear cell carcinoma was highly expressed. 2, the study found that the expression level of MGMT was positively correlated with the malignant degree of renal clear cell carcinoma; 3, MGMT was involved in the chemotherapeutic drugs of renal clear cell carcinoma to alkylating agents. 4, methylation inhibitor 5-Aza-CdR can enhance the sensitivity of renal cell carcinoma cell 786-0 to mitomycin. Two, innovation and deficiency 1. This study first confirmed the high expression of MGMT in the tissues and cells of renal clear cell carcinoma, and found that the expression level of MGMT was positively correlated with the malignancy of renal clear cell carcinoma. This study for the first time found that down-regulation of MGMT expression could enhance the sensitivity of ACHN and 786-0 cells to alkylated BCNU or TMZ. However, the mechanism of MGMT to participate in the progression of renal cell carcinoma and the mechanism for the tolerance to alkylating agents is not clear.2. Preliminary results show that the methylation inhibitor 5-Aza-CdR can enhance the sensitivity of mitomycin 786-0 to renal cell carcinoma cells. It is necessary to further study whether 5-Aza-CdR affects the sensitivity of renal cell carcinoma cells to chemotherapeutic drugs by affecting the expression or activity of MGMT.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Gen Tamura;;Alterations of tumor suppressor and tumor-related genes in the development and progression of gastric cancer[J];World Journal of Gastroenterology;2006年02期

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本文編號：2114090