MGMT基因表达在肾细胞癌治疗中的作用研究

发布时间：2018-07-10 17:27

本文选题：MGMT + 肾透明细胞癌　；参考：《山东大学》2015年博士论文

【摘要】：肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)在所有肾细胞癌占75%-80%,是最常见的病理类型。化疗仍是目前临床上常用的方法,但治疗效果常因肿瘤本身具有耐药或治疗后出现耐药性而失败。目前为止,耐药机制主要有P-糖蛋白的过表达、DNA拓扑异构酶的低表达、抗凋亡基因bcl-2的过表达以及DNA修复蛋白的过表达等。烷化剂是一大类重要的临床抗肿瘤药物,主要造成DNA碱基的烷基化损伤,其中以形成O6-甲基鸟嘌呤对细胞的威胁最大,造成细胞突变与死亡。O6-甲基鸟嘌呤可以由O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)修复。MGMT对烷化剂损伤的修复使其在肿瘤化疗中发挥双重作用。一方面保护正常组织免于烷化剂的损伤和二次癌症的发生,另一方面引起肿瘤组织对烷化剂类化疗药物的耐药性。MGMT的表达在正常组织中要低于其肿瘤组织,而且不同肿瘤组织中呈现出不一样的模式。然而,在肾透明细胞癌中MGMT基因的表达目前仍不清楚。甲基化抑制剂5-Aza-CdR能够逆转DNA的异常甲基化、重新激活失活的抑癌基因的表达,因此其具有作为抗肿瘤药物的潜力。本课题拟从基因和蛋白水平研究MGMT基因在肾透明细胞癌中的表达,并阐明MGMT在耐药肾透明细胞癌治疗中的作用。同时我们将5-Aza-CdR与丝裂霉素联合作用于肾癌细胞,探讨应用5-Aza-CdR能否提高肾癌细胞对丝裂霉素的敏感性。第一部分MGMT基因表达对肾细胞癌的耐药性影响的研究一、MGMT在肾透明细胞癌组织和细胞中表达上调1、采用免疫组化的方法对60个组织标本中的MGMT表达情况进行了检测。免疫组化染色着色结果可以将60例病例分为MGMT不表达(-),低表达(+)和高表达(++,+++)三组,其中高表达的组占62%(37/60)。实时定量PCR结果显示,与正常组织相比,肿瘤组织中MGMT mRNA水平表达上调2倍以上的占85%(17/20)2、选取两株癌细胞ACHN和786-0以及正常肾细胞株HK-2为研究对象。结果表明两株癌细胞中MGMTmRNA水平远高于正常细胞。Western blot结果进一步验证了MGMT在肿瘤细胞中高表达。二、MGMT表达与肾透明细胞癌的阶段有关进一步分析了MGMT水平与癌症进程之间的关系。研究发现MGMT表达水平与肿瘤病理级别和临床阶段有着正相关。在G1,G2和G3/G4期的肾透明细胞癌中MGMT高表达的比例分别占40.0%,67.7%和77.3%(p=0.04)。在T1,T2和T3/T4阶段中MGMT高表达比例分别为39.1%,70.1%和80.0%(p=0.02)。三、下调MGMT表达可以增强ACHN和786-0细胞对烷化剂BCNU或TMZ的敏感性。1、首先通过western blot检测siRNA的作用,干扰siRNA作用48h后MGMT蛋白表达明显下降。2、MTT结果显示,下调MGMT表达可以使ACHN细胞对烷化剂BCNU和TMZ更加敏感,3、以肿瘤细胞株786-0为对象进行实验。下调MGMT同样显著增加786-0细胞对烷化剂BCNU和TMZ的敏感性。第二部分5-Aza-CdR对肾癌细胞株CAKI-1及786-0的化疗敏感性的影响一、MTT法检测丝裂霉素对CAKI-1细胞抑制作用我们用MTT法分别检测不同浓度丝裂霉素作用于CAKI-1细胞24 h的生长抑制率,发现丝裂霉素能抑制细胞的增值,并呈现剂量依赖性。二、初步结果显示甲基化抑制剂5-Aza-CdR能够增强肾癌细胞786-0对丝裂霉素的敏感性MTT检测发现单用5-Aza-CdR对人肾癌细胞株786-0增值呈现明显的抑制作用。不同浓度的5-Aza-CdR联合丝裂霉素对肿瘤细胞的抑制作用呈现剂量依赖性,随着5-Aza-CdR浓度的增加,肿瘤细胞的抑制率明显升高。第三部分结论及创新点一、结论1、研究证实了MGMT在肾透明细胞癌组织和细胞中的表达呈高表达；2、研究分析发现MGMT的表达水平与肾透明细胞癌的恶性程度呈正相关；3、MGMT参与肾透明细胞癌对烷化剂类化疗药物的耐药过程；4、甲基化抑制剂5-Aza-CdR能够增强肾癌细胞786-0对丝裂霉素的敏感性。二、创新点与不足之处1、本研究首次证实了MGMT在肾透明细胞癌组织和细胞中表达高表达,并且发现MGMT的表达水平与肾透明细胞癌的恶性程度呈正相关。本研究首次发现下调MGMT表达可以增强ACHN和786-0细胞对烷化齐BCNU或TMZ的敏感性。但MGMT是如何参与肾透明细胞癌的进展及对烷化剂耐受的机制尚不清楚。2、初步结果显示甲基化抑制剂5-Aza-CdR能够增强肾癌细胞786-0对丝裂霉素的敏感性。5-Aza-CdR是否是通过影响MGMT的表达或活性从而影响肾癌细胞对化疗药的敏感性的需要进一步研究。
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.11

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