Nrf2及NQO1在肾透明细胞癌中的表达及意义

发布时间：2018-01-30 12:03

本文关键词： 肾透明细胞癌 Nrf2 NQO1 免疫组化 RT-PCR　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的:肾细胞癌（renal cell carcinoma）是泌尿系统常见的恶性肿瘤，发病率及死亡率呈逐年上升趋势，而其中60%-85%为透明细胞癌（clearcell renal cell carcinoma，ccRCC）。肾透明细胞癌恶性程度较高，容易发生远处转移，且对放化疗均不敏感[2]。因此，探讨肾透明细胞癌的发生和发展机制对其诊断、治疗、预后等方面具有重要价值。核因子E2相关因子2（nuclear factor E2-related factor，Nrf2）是CNC（cap-n-cohar，CNC）转录因子家族中调节抗氧化应激反应的重要转录因子，作为感受器感受氧化应激，是细胞内对抗氧化应激最重要的机制。NQO1(NADPH quinine oxidoreductase)即醌氧化还原酶，是Nrf2/ARE抗氧化应激系统中下游表达蛋白。大量的研究表明：Nrf2/NQO1信号通路在呼吸、消化、神经、心血管系统等多种器官的氧化应激反应中发挥了重要作用，而Nrf2、NQO1表达水平及其活性变化与肿瘤的发生和发展均有密切关系。但Nrf2、NQO1在肾透明细胞癌中的表达目前尚无相关报道。本文主要研究Nrf2、NQO1的mRNA和蛋白在肾透明细胞癌组织和对应癌旁组织中的表达情况，探讨其在肾透明细胞癌发生、发展中的作用。方法:收集2012年12月至2013年6月于河北医科大学第二医院泌尿外科住院手术病人的肾透明细胞癌标本52例，其中对照肾组织21例为癌旁4cm以外肾组织（病理学已证实）。患者年龄37～82岁，平均年龄56（56.13±13.98）岁。所有患者术前均未行放疗、化疗及其他保守治疗。所有肾细胞癌病例均已经过病理证实为肾透明细胞癌。标本分为两份，一份以10%福尔马林固定，常规包埋、切片，另一份标本收集后立即以液氮进行快速冷冻并保存在㧟80℃冰箱中。根据世界卫生组织（WHO）1997年Fuhrman三级分类标准进行病理分级，低级别组32例，高级别组20例；根据国际抗癌联盟2009年(AJCC，2009) TNM分期标准进行临床分期Ⅰ期15例、Ⅱ期16例、Ⅲ期14例和Ⅳ期7例。 1免疫组织化学方法检测：检测52例肾透明细胞癌及21例癌旁组织Nrf2及NQO1蛋白表达。运用SPSS17.0统计软件处理数据：统计免疫组化染色阳性率数值，比较肾透明细胞癌组织和癌旁组织中Nrf2及NQO1蛋白的阳性表达率之间的差异，并分析与临床分期、病理分级的关系，采用χ2检验或Fisher确切概率法检验；Nrf2与NQO1相关关系采用Spearman秩相关分析；认为P0.05差异有统计学意义。 2RT-PCR法检测：检测36例肾透明细胞癌及14例癌旁组织中Nrf2及NQO1mRNA的表达。按照Trizol试剂使用说明书提取冰冻组织中的RNA，检测其浓度及纯度，用PE基因扩增仪行逆转录并扩增，并行琼脂糖凝胶电泳,以DNA Marker（DL2000）为标准片段作标记，应用紫外透射仪观察并用数码相机照相电泳后条带，并应用Quantity One凝胶图象分析软件分析目的电泳条带，参照相应的内参电泳条带，结果以两者的积分吸光度之比值统计,以x±s表示，，应用t检验行计数资料组间比较；以P0.05差异有统计学意义。结果: 1免疫组织化学染色结果显示：在肾透明癌组织及其癌旁组织中均有Nrf2、NQO1蛋白表达。Nrf2蛋白在肾透明癌组织表达明显高于癌旁组织中，两种组织中的阳性率分别为76.9%和28.6%（χ2=15.005，P＜0.01），差异有统计学意义；NQO1蛋白在肾透明癌组织表达亦高于癌旁组织，两种组织中的阳性表达率分别73.1%和23.8%（χ2=14.999，P0.01），差异有统计学意义；Nrf2与NQO1蛋白的表达与临床分期和病理分级呈正相关P0.05；同时Nrf2与NQO1在肾透明细胞癌组织中表达呈正相关（r=0.594，P0.05）。 2RT-PCR结果:Nrf2和NQO1RT-PCR检测结果：琼脂糖凝胶电泳显示：550bp为Nrf2目的条带，488bp处为NQO1目的条带，162bp处为内参目的条带。半定量分析结果显示，肾透明癌组织中Nrf2和NQO1mRNA相对表达量分别为（0.767±0.125）和（1.025±0.148），而在癌旁组织中的相对表达量为分别为（0.299±0.025）和（0.181±0.051），即肾透明细胞癌组织中Nrf2和NQO1mRNA相对表达量显著高于癌旁正常组织，差异有统计学意义(p＜0.05)。结论: 1Nrf2和NQO1蛋白及其mRNA在肾透明细胞癌癌组织中的表达均高于癌旁组织中的表达。 2Nrf2与NQO1蛋白在肾透明细胞癌组织中的表达与临床分期和病理分级均呈正相关。 3Nrf2和NQO1蛋白在肾透明细胞癌组织中的表达呈正相关。 4通过联合检测Nrf2和NQO1在ccRCC中的表达可能有助于判断肿瘤的进展程度情况，同时Nrf2/NQO1信号通路有望成为治疗肾透明细胞癌的新靶位，为临床肾透明细胞癌的早期检测和治疗开辟新的道路。
[Abstract]:Objective: renal cell carcinoma (renal cell carcinoma) is a common malignant tumor in urinary system, disease incidence rate and the mortality rate is rising year by year, and the 60%-85% was clear cell carcinoma (ClearCell renal cell carcinoma, ccRCC). The malignant degree of renal cell carcinoma is high, prone to distant metastasis, and not sensitive to radiotherapy and chemotherapy of [2]. therefore, to explore the treatment mechanism of occurrence and development of renal cell carcinoma for the diagnosis, prognosis and other aspects, has important value.
Nuclear factor E2 related factor 2 (nuclear factor E2-related factor, Nrf2) CNC (cap-n-cohar, CNC) is an important transcription factor regulating oxidative stress response transcription factor family, as the receptor mechanism of oxidative stress,.NQO1 cells against oxidative stress is the most important (NADPH quinine oxidoreductase): quinone oxidoreductase, expression the downstream protein oxidative stress in Nrf2/ARE. A large number of studies show that Nrf2/NQO1 signaling pathway in respiratory, digestive, nervous, play an important role in oxidative stress in various organs in the cardiovascular system and the occurrence and development of Nrf2, there was a close correlation between NQO1 expression and activity and tumor. But Nrf2, expression of NQO1 in kidney clear cell carcinoma has not yet been reported. This paper mainly studies the Nrf2, mRNA and NQO1 protein in renal cell carcinoma tissues and corresponding adjacent tissues To explore its role in the development of renal clear cell carcinoma.
Methods: from December 2012 to June 2013 in clear cell renal cell carcinoma surgical specimens of hospitalized patients in Department of Urology Second Hospital of Hebei Medical University in 52 cases, the control kidney tissues of 21 cases of renal carcinoma (except 4cm pathology confirmed). Patients 37~82 years of age, the average age of 56 (56.13 + 13.98) years old. All the patients were not radiotherapy, chemotherapy and other conservative treatment. All renal cell carcinoma cases have been confirmed by pathology of renal cell carcinoma. The specimens were divided into two parts, one in 10% formalin fixed, conventional embedding, slicing, another specimen collected immediately after the liquid nitrogen was frozen and stored in the refrigerator at 80? WHO (WHO). According to the pathological grading of 1997 Fuhrman three classification standards, low grade group 32 cases, 20 cases of high grade group; according to UICC 2009 (AJCC, 2009) TNM staging criteria for clinical stage I There were 15 cases, 16 cases in stage II, 14 in stage III and 7 in stage IV.
1 immunohistochemistry: the expression was detected in 52 cases of renal clear cell carcinoma and 21 cases of adjacent tissue Nrf2 and NQO1 protein. Using SPSS17.0 statistical software to deal with data statistics: immunohistochemical staining positive rate of numerical difference between the positive expression of Nrf2 and NQO1 protein in renal cell carcinoma tissues and adjacent tissues of the rate then, analysis and clinical stage, the relationship between pathology classification, using 2 test or Fisher exact test; Nrf2 and NQO1 correlation with Spearman rank correlation analysis; there was significant difference in P0.05.
2RT-PCR detection: the expression of Nrf2 and NQO1mRNA were detected in 36 cases of renal clear cell carcinoma and 14 cases of adjacent tissues. In accordance with the instructions on the use of Trizol reagent extraction in frozen tissue RNA, detect its purity and concentration, PE gene amplification instrument for reverse transcription and amplification, parallel agarose gel electrophoresis, DNA Marker (DL2000) marking for the standard application of UV transilluminators fragment, and observed after electrophoresis bands with a digital camera, and using Quantity One gel image analysis software to analyze the internal reference bands, electrophoresis corresponding zone, results in both the ratio of integral absorbance statistics, with x + s said, using t test line count data between two groups; P0.05 difference was statistically significant.
Result:
1 immunohistochemical staining results showed that both in renal carcinoma and the adjacent tissue Nrf2, NQO1 protein expression of.Nrf2 protein in renal carcinoma tissues was significantly higher than that in paracancerous tissues, the positive rate of two kinds of tissues were 76.9% and 28.6% (2=15.005, P < 0.01), the difference was statistically significant NQO1; protein in renal carcinoma tissue expression was also higher than that of the adjacent tissues, the positive expression rate of two tissues were 73.1% and 23.8% (2=14.999, P0.01), the difference was statistically significant; the Nrf2 and NQO1 protein expression and clinical stage and pathologic grade was P0.05; while Nrf2 and NQO1 expression in renal cell cancer tissues were positively correlated (r=0.594, P0.05).
Results: 2RT-PCR Nrf2 and NQO1RT-PCR test results: agarose gel electrophoresis showed: 550bp Nrf2 band, 488bp NQO1 band, 162bp band as a reference. Semi quantitative analysis showed that the renal carcinoma tissue Nrf2 and NQO1mRNA expression levels were (0.767 + 0.125) and (1.025 + 0.148), while in the adjacent tissues relative expression respectively (0.299 + 0.025) and (0.181 + 0.051), namely renal clear cell carcinoma Nrf2 and NQO1mRNA expression was significantly higher than that in normal tissues, the difference was statistically significant (P < 0.05).
Conclusion:
The expression of 1Nrf2 and NQO1 protein and their mRNA in the tissues of renal clear cell carcinoma are higher than those in the para cancerous tissue.
The expression of 2Nrf2 and NQO1 protein in the tissue of renal clear cell carcinoma is positively correlated with the clinical stage and pathological grade.
The expression of 3Nrf2 and NQO1 protein in the tissues of renal clear cell carcinoma is positively correlated.
4, the joint detection of Nrf2 and NQO1 expression in ccRCC may help to predict the progression of tumor. Meanwhile, Nrf2/NQO1 signaling pathway is expected to become a new target for the treatment of renal clear cell carcinoma, and will open up a new way for the early detection and treatment of renal clear cell carcinoma.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

【参考文献】