本文關(guān)鍵詞： 腎透明細胞癌 Nrf2 NQO1 免疫組化 RT-PCR　出處：《河北醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:腎細胞癌（renal cell carcinoma）是泌尿系統(tǒng)常見的惡性腫瘤，發(fā)病率及死亡率呈逐年上升趨勢，而其中60%-85%為透明細胞癌（clearcell renal cell carcinoma，ccRCC）。腎透明細胞癌惡性程度較高，容易發(fā)生遠處轉(zhuǎn)移，且對放化療均不敏感[2]。因此，探討腎透明細胞癌的發(fā)生和發(fā)展機制對其診斷、治療、預(yù)后等方面具有重要價值。 核因子E2相關(guān)因子2（nuclear factor E2-related factor，Nrf2）是CNC（cap-n-cohar，CNC）轉(zhuǎn)錄因子家族中調(diào)節(jié)抗氧化應(yīng)激反應(yīng)的重要轉(zhuǎn)錄因子，作為感受器感受氧化應(yīng)激，是細胞內(nèi)對抗氧化應(yīng)激最重要的機制。NQO1(NADPH quinine oxidoreductase)即醌氧化還原酶，是Nrf2/ARE抗氧化應(yīng)激系統(tǒng)中下游表達蛋白。大量的研究表明：Nrf2/NQO1信號通路在呼吸、消化、神經(jīng)、心血管系統(tǒng)等多種器官的氧化應(yīng)激反應(yīng)中發(fā)揮了重要作用，而Nrf2、NQO1表達水平及其活性變化與腫瘤的發(fā)生和發(fā)展均有密切關(guān)系。但Nrf2、NQO1在腎透明細胞癌中的表達目前尚無相關(guān)報道。本文主要研究Nrf2、NQO1的mRNA和蛋白在腎透明細胞癌組織和對應(yīng)癌旁組織中的表達情況，探討其在腎透明細胞癌發(fā)生、發(fā)展中的作用。 方法:收集2012年12月至2013年6月于河北醫(yī)科大學(xué)第二醫(yī)院泌尿外科住院手術(shù)病人的腎透明細胞癌標本52例，其中對照腎組織21例為癌旁4cm以外腎組織（病理學(xué)已證實）�；颊吣挲g37～82歲，平均年齡56（56.13±13.98）歲。所有患者術(shù)前均未行放療、化療及其他保守治療。所有腎細胞癌病例均已經(jīng)過病理證實為腎透明細胞癌。標本分為兩份，一份以10%福爾馬林固定，常規(guī)包埋、切片，另一份標本收集后立即以液氮進行快速冷凍并保存在㧟80℃冰箱中。根據(jù)世界衛(wèi)生組織（WHO）1997年Fuhrman三級分類標準進行病理分級，低級別組32例，高級別組20例；根據(jù)國際抗癌聯(lián)盟2009年(AJCC，2009) TNM分期標準進行臨床分期Ⅰ期15例、Ⅱ期16例、Ⅲ期14例和Ⅳ期7例。 1免疫組織化學(xué)方法檢測：檢測52例腎透明細胞癌及21例癌旁組織Nrf2及NQO1蛋白表達。運用SPSS17.0統(tǒng)計軟件處理數(shù)據(jù)：統(tǒng)計免疫組化染色陽性率數(shù)值，比較腎透明細胞癌組織和癌旁組織中Nrf2及NQO1蛋白的陽性表達率之間的差異，并分析與臨床分期、病理分級的關(guān)系，采用χ2檢驗或Fisher確切概率法檢驗；Nrf2與NQO1相關(guān)關(guān)系采用Spearman秩相關(guān)分析；認為P0.05差異有統(tǒng)計學(xué)意義。 2RT-PCR法檢測：檢測36例腎透明細胞癌及14例癌旁組織中Nrf2及NQO1mRNA的表達。按照Trizol試劑使用說明書提取冰凍組織中的RNA，檢測其濃度及純度，用PE基因擴增儀行逆轉(zhuǎn)錄并擴增，并行瓊脂糖凝膠電泳,以DNA Marker（DL2000）為標準片段作標記，應(yīng)用紫外透射儀觀察并用數(shù)碼相機照相電泳后條帶，并應(yīng)用Quantity One凝膠圖象分析軟件分析目的電泳條帶，參照相應(yīng)的內(nèi)參電泳條帶，結(jié)果以兩者的積分吸光度之比值統(tǒng)計,以x±s表示，，應(yīng)用t檢驗行計數(shù)資料組間比較；以P0.05差異有統(tǒng)計學(xué)意義。 結(jié)果: 1免疫組織化學(xué)染色結(jié)果顯示：在腎透明癌組織及其癌旁組織中均有Nrf2、NQO1蛋白表達。Nrf2蛋白在腎透明癌組織表達明顯高于癌旁組織中，兩種組織中的陽性率分別為76.9%和28.6%（χ2=15.005，P＜0.01），差異有統(tǒng)計學(xué)意義；NQO1蛋白在腎透明癌組織表達亦高于癌旁組織，兩種組織中的陽性表達率分別73.1%和23.8%（χ2=14.999，P0.01），差異有統(tǒng)計學(xué)意義；Nrf2與NQO1蛋白的表達與臨床分期和病理分級呈正相關(guān)P0.05；同時Nrf2與NQO1在腎透明細胞癌組織中表達呈正相關(guān)（r=0.594，P0.05）。 2RT-PCR結(jié)果:Nrf2和NQO1RT-PCR檢測結(jié)果：瓊脂糖凝膠電泳顯示：550bp為Nrf2目的條帶，488bp處為NQO1目的條帶，162bp處為內(nèi)參目的條帶。半定量分析結(jié)果顯示，腎透明癌組織中Nrf2和NQO1mRNA相對表達量分別為（0.767±0.125）和（1.025±0.148），而在癌旁組織中的相對表達量為分別為（0.299±0.025）和（0.181±0.051），即腎透明細胞癌組織中Nrf2和NQO1mRNA相對表達量顯著高于癌旁正常組織，差異有統(tǒng)計學(xué)意義(p＜0.05)。 結(jié)論: 1Nrf2和NQO1蛋白及其mRNA在腎透明細胞癌癌組織中的表達均高于癌旁組織中的表達。 2Nrf2與NQO1蛋白在腎透明細胞癌組織中的表達與臨床分期和病理分級均呈正相關(guān)。 3Nrf2和NQO1蛋白在腎透明細胞癌組織中的表達呈正相關(guān)。 4通過聯(lián)合檢測Nrf2和NQO1在ccRCC中的表達可能有助于判斷腫瘤的進展程度情況，同時Nrf2/NQO1信號通路有望成為治療腎透明細胞癌的新靶位，為臨床腎透明細胞癌的早期檢測和治療開辟新的道路。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.11

【參考文獻】

相關(guān)期刊論文 前1條

1 張海燕;孟欣;都鎮(zhèn)先;鄧娓娓;劉國良;王華芹;;Nrf2對蛋白酶體抑制劑誘導(dǎo)甲狀腺癌細胞凋亡影響的觀察[J];中華腫瘤防治雜志;2012年10期

本文編號：1476192