發(fā)布時間：2018-07-03 02:24

  本文選題：腎癌 + Nrf2��； 參考：《河北醫(yī)科大學》2014年碩士論文

【摘要】：目的：腎癌是泌尿系腫瘤中常見的腫瘤，，70%-80%為透明細胞癌,近年發(fā)病有增加趨勢。尋找腎癌的新靶點對于腎癌的早期發(fā)現(xiàn)及預后并制定治療方案有著非常重要的意義。在腫瘤形成和細胞增殖過程中，氧化應激及炎癥反應與之密切相關，已成為國內外學者的研究熱點，與氧化應激相關途徑的有關基因有可能成為潛在的藥物治療腫瘤的作用靶點。目前研究發(fā)現(xiàn)很多藥物是通過調節(jié)Nrf2/HO-1信號通路來發(fā)揮抗氧化及抗炎作用的。為探討Nrf2和HO-1與腎癌發(fā)生、發(fā)展的關系，本實驗通過免疫組化SABC和RT-PCR法檢測Nrf2和HO-1在腎癌和癌旁正常組織中的表達情況，探討Nrf2和HO-1在腎癌發(fā)生、發(fā)展中的作用。 方法：選取河北醫(yī)科大學第二醫(yī)院外科手術切除的腎癌組織標本51例。選其中23例患者中的癌旁組織（經病理證實為正常腎組織）為對照組。所有病例術前均未進行過放療和（或）化療。所有標本分為兩份，一份經4%甲醛溶液固定，石蠟塊包埋，另一份保存于液氮之中。 免疫組織化學方法檢測：應用免疫組化技術，從蛋白水平檢測石蠟包埋組織中Nrf2和HO-1的表達，其中腎癌組織標本51例，正常腎組織標本23例。采用雙評分半定量法來進行評分，綜合統(tǒng)計染色陽性率及染色強度，運用SPSS17.0統(tǒng)計軟件處理數(shù)據，比較腎癌組織和正常腎組織中Nrf2及HO-1的陽性表達率之間的差異。采用χ2檢驗，把具有統(tǒng)計學意義顯著性差異的臨界值定為P0.05。Nrf2與HO-1關系采用Pearson相關分析。 RT-PCR方法檢測：根據Trizol試劑盒說明提取冰凍組織中的總RNA，加入RT反應體系管，進行逆轉錄反應合成cDNA，設立β-actin基因片段為Nrf2和HO-1基因表達的內參照，在RT-PCR儀上進行擴增，取每個標本的擴增產物行瓊脂糖凝膠電泳，以DNA Marker（DL2000）作為標準片段標記，電泳后用紫外透射儀觀察，應用Quantity One凝膠圖象分析軟件對目的電泳條帶進行分析，以β-actin電泳條帶作為參照，結果以兩者吸光度的比值表示。兩組間吸光度比值采用t檢驗進行統(tǒng)計分析。P0.05認為差異有統(tǒng)計學意義。 結果： 1免疫組織化學染色示：Nrf2主要表達于腫瘤細胞的細胞漿中，在腎癌組織中的陽性表達率為82.4%，在癌旁正常組織中表達的陽性率為34.8%，兩者差異有統(tǒng)計學意義（P=0.00，P0.05）。HO-1主要位于腫瘤細胞的胞漿中，其在腎癌組織中的陽性表達率為76.5%，在癌旁正常組織中的陽性表達率為39.1%，二者陽性表達率差異有統(tǒng)計學意義（P=0.02，P0.05）。Nrf2和HO-1蛋白的表達呈正相關（r=0.515P0.05）。 2RT-PCR結果顯示：Nrf2在腎癌和癌旁正常組織的表達量分別為0.747±0.101和0.374±0.071，二者差異有統(tǒng)計學意義（P0.05）。HO-1在腎癌和癌旁正常組織的表達量分別為0.853±0.078和0.410±0.085，二者差異有統(tǒng)計學意義（P0.05）。 結論： 1Nrf2和HO-1蛋白在腎癌組織中的表達高于癌旁正常組織中的表達。 2Nrf2、HO-1蛋白在腎癌組織中的表達呈正相關。 3Nrf2、HO-1蛋白可能參與了腎癌的發(fā)生、發(fā)展，并在演變過程中起到了重要作用。
[Abstract]:Objective: renal cancer is a common tumor in urological tumors. 70%-80% is a clear cell carcinoma. It has an increasing trend in recent years. The new target for searching for renal cancer is of great significance for the early detection and prognosis of renal cancer and the formulation of treatment scheme. In the process of tumor formation and cell proliferation, oxidative stress and inflammatory reaction are closely related. The relevant genes related to oxidative stress may become potential targets for the potential drug treatment of tumor. Many drugs have been found to play antioxidation and anti-inflammatory effects by regulating the Nrf2/HO-1 signaling pathway. To explore the relationship between the development of Nrf2 and HO-1 and the development of renal carcinoma, In this experiment, the expression of Nrf2 and HO-1 in renal carcinoma and adjacent normal tissues was detected by immunohistochemical SABC and RT-PCR method, and the role of Nrf2 and HO-1 in the development of renal carcinoma was discussed.
Methods: 51 cases of renal carcinoma tissue excised by surgical operation in Second Hospital of Hebei Medical University were selected and 23 of them were selected as control group. All cases were not treated with radiotherapy and / or chemotherapy before operation. All the specimens were divided into two parts, one with 4% Formaldehyde Solution and paraffin paraffin. Buried, the other in liquid nitrogen.
Immunohistochemical method detection: using immunohistochemical technique, the expression of Nrf2 and HO-1 in paraffin embedded tissue was detected from protein level, including 51 cases of renal carcinoma tissue and 23 normal renal tissue specimens. The double score and half quantitative method was used to score, and the positive rate and intensity of dyeing were statistically analyzed, and the number of SPSS17.0 statistical software was used. The difference between the positive expression rates of Nrf2 and HO-1 in renal carcinoma and normal renal tissue was compared. The critical value of statistically significant difference was determined to be the relationship between P0.05.Nrf2 and HO-1 by Pearson correlation analysis by the x 2 test.
RT-PCR method detection: according to the Trizol kit, the total RNA in the frozen tissue was extracted, and the RT reaction system tube was added, and cDNA was synthesized by reverse transcriptase. The gene fragment of the beta -actin was set up as the internal reference for the expression of Nrf2 and HO-1 gene, and the amplified product of each specimen was amplified by the agarose gel electrophoresis with DNA Marker (DL). 2000) as a standard fragment mark, after electrophoretic ultraviolet transmittance, Quantity One gel image analysis software was used to analyze the target bands. The ratio of the absorbance between the two groups was expressed with the ratio of the absorbance between the two groups. The two groups of absorbance ratio using t test were statistically analyzed and.P0.05 thought the difference was statistically significant. Learning meaning.
Result:
1 immunohistochemical staining showed that Nrf2 was mainly expressed in the cytoplasm of tumor cells, the positive expression rate in renal carcinoma tissue was 82.4%, and the positive rate was 34.8% in normal tissues adjacent to the cancer. The difference was statistically significant (P=0.00, P0.05).HO-1 was mainly located in the cytoplasm of tumor cells, and the positive expression rate in the renal carcinoma tissue For 76.5%, the positive expression rate in normal tissues adjacent to cancer was 39.1%, and the difference of positive expression rate between the two groups was statistically significant (P=0.02, P0.05) and the expression of.Nrf2 and HO-1 protein was positively correlated (r=0.515P0.05).
The results of 2RT-PCR showed that the expression of Nrf2 in renal carcinoma and adjacent normal tissues was 0.747 + 0.101 and 0.374 + 0.071 respectively. The difference between the two groups was statistically significant (P0.05).HO-1 was 0.853 + 0.078 and 0.410 + 0.085 in the normal tissues of renal carcinoma and adjacent to cancer, and the two differences were statistically significant (P0.05).
Conclusion:
The expression of 1Nrf2 and HO-1 protein in renal cell carcinoma was higher than that in adjacent normal tissues.
The expression of 2Nrf2 and HO-1 protein was positively correlated with renal cell carcinoma.
3Nrf2 and HO-1 proteins may play an important role in the development and progression of renal cell carcinoma.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.11

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