發(fā)布時間：2018-02-03 08:42

  本文關鍵詞： RAD21 P53 DNA損傷修復 自噬 亞細胞定位 奧沙利鉑敏感性　出處：《南方醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：背景與目的:胃癌已成為全球癌癥死亡的第二大原因。在中國,胃癌在惡性疾病中排第三。由于癥狀模糊,多數(shù)胃癌患者在確診時已處于晚期。對于晚期胃癌患者,化療仍是最重要的治療方法。鉑類,尤其是第三代鉑類(奧沙利鉑等)已被廣泛應用于胃癌臨床的一線化療。然而,由于腫瘤固有或獲得性的鉑類耐藥機制的存在,使得鉑類藥物的臨床療效不佳。此外,自噬導致的DNA損傷修復是最重要的耐藥機制之一。RAD21編碼的蛋白是DNA雙鏈斷裂修復和核性蛋白質(zhì)。研究表明,RAD21與化療藥物的敏感性密切相關;RAD21可能成為一個新型的靶點,潛在地通過調(diào)節(jié)DNA損傷修復增強化療藥物的抗腫瘤效果。自噬參與了化療藥物介導的DNA損傷修復,并且自噬在促進腫瘤的化療抵抗中發(fā)揮了重要的作用。這些證據(jù)提示RAD21與自噬可能有著密切的關系。盡管大量研究已證實RAD21在許多腫瘤中表達上調(diào)并與化療藥物的敏感性密切相關,但在胃癌中,它與奧沙利鉑的藥物敏感性關系仍然沒有相關報道。本研究中,我們旨在探討胃癌中RAD21與奧沙利鉑藥物敏感性的關系,并闡述其具體的發(fā)生機制。材料與方法:1.運用PCR、WB檢測RAD21在胃癌細胞株和胃癌組織中的表達情況。2.運用MTT、流式及平板克隆檢測siRAD21與細胞增殖、凋亡及奧沙利鉑的敏感性關系。3.選擇47名胃癌患者(對奧沙利鉑敏感組)和9名胃癌患者(對奧沙利鉑不敏感組),用免疫組化分析胃癌患者RAD21漿表達或核漿表達的無進展生存。4.構建NLS-GV141-RAD21以及NES-GV141-RAD21載體,運用免疫熒光檢測RAD21不同的亞細胞定位與奧沙利鉑敏感性的關系。5.運用免疫共沉淀及彗星實驗探究P53、RAD21及自噬相關的化療藥物敏感性之間的關系。結果:1.各種胃癌細胞株及胃癌組織標本與對照組相比,RAD21表達水平明顯上調(diào)(p0.05)。2.干擾RAD21可增加胃癌細胞對奧沙利鉑的敏感性。3.RAD21的核轉位現(xiàn)象與胃癌患者的復發(fā)率、臨床分期及不良預后密切相關(p0.05)。4.RAD21的核轉入載體組與核轉出載體組相比,可明顯降低胃癌細胞對奧沙利鉑的敏感性。5.P53與RAD21呈現(xiàn)共定位并介導了 RAD21的亞細胞定位。6.干擾RAD21聯(lián)合自噬抑制劑可抑制細胞增殖并最大程度地促使DNA損傷現(xiàn)象的發(fā)生。在此過程中,ATG5與RAD21呈明顯的正相關(r=0.95,P0.01;r=0.96,P0.01)。結論:RAD21與奧沙利鉑的敏感性密切相關;深入機制探討,P53調(diào)控了 RAD21的亞細胞定位,并通過自噬降低了奧沙利鉑的敏感性,從而促進了胃癌的進展。綜上,這些結果提供了提高胃癌患者對奧沙利鉑敏感性的相關策略,使RAD21成為令人矚目的分子靶點。
[Abstract]:Background & objective: gastric cancer has become the second leading cause of cancer death in the world. Gastric cancer ranks third among malignant diseases in China. Most patients with gastric cancer are in advanced stage at the time of diagnosis. Chemotherapy is still the most important treatment for advanced gastric cancer patients. Especially the third generation platinum (oxaliplatin et al) has been widely used in first-line chemotherapy of gastric cancer. However due to the presence of intrinsic or acquired platinum resistance mechanisms. Make the clinical efficacy of platinum drugs poor. In addition. Repair of DNA damage induced by autophagy is one of the most important mechanisms of drug resistance. The protein encoded by RAD21 is DNA double strand break repair and nuclear protein. RAD21 was closely related to the sensitivity of chemotherapeutic drugs. RAD21 may become a novel target, potentially enhancing the anti-tumor effect of chemotherapeutic drugs by regulating DNA damage repair. Autophagy is involved in chemotherapy-mediated DNA damage repair. Autophagy also plays an important role in promoting chemotherapeutic resistance in tumors. These evidences suggest that RAD21 may be closely related to autophagy. Although numerous studies have confirmed the expression of RAD21 in many tumors. It was up-regulated and closely related to the sensitivity of chemotherapeutic agents. However, there is no correlation between oxaliplatin and oxaliplatin in gastric cancer. In this study, we aim to explore the relationship between RAD21 and oxaliplatin susceptibility in gastric cancer. Materials and methods: 1. The expression of RAD21 in gastric cancer cell line and gastric cancer tissue was detected by PCRX WB. 2. MTT was used. SiRAD21 and cell proliferation were detected by flow cytometry and plate cloning. Apoptosis and oxaliplatin sensitivity. 3. 47 gastric cancer patients (oxaliplatin sensitive group) and 9 gastric cancer patients (oxaliplatin insensitive group) were selected. The expression of RAD21 or nuclear cytoplasm in gastric cancer patients was analyzed by immunohistochemistry. 4. NLS-GV141-RAD21 and NES-GV141-RAD21 vectors were constructed. Using immunofluorescence to detect the relationship between subcellular localization of RAD21 and oxaliplatin sensitivity. 5. To explore p53 by immunoprecipitation and comet assay. The relationship between RAD21 and chemosensitivity of autophagy. Results: 1. All kinds of gastric cancer cell lines and gastric cancer tissue specimens were compared with the control group. The expression level of RAD21 was significantly up-regulated by p0.05. 2. Interfering with RAD21 could increase the sensitivity of gastric cancer cells to oxaliplatin. 3. Nuclear translocation of RAD21 and recurrence rate of gastric cancer patients. Clinical staging and poor prognosis were closely related to p0.05. 4. The nuclear transfer vector group was compared with the nuclear transfer vector group. It can significantly reduce the sensitivity of gastric cancer cells to oxaliplatin. 5.P53 is colocated with RAD21 and mediates the expression of oxaliplatin. The subcellular localization of RAD21. 6. Interfering with RAD21 combined with autophagy inhibitor can inhibit cell proliferation and promote DNA damage to the maximum extent. There was a significant positive correlation between ATG5 and RAD21. Conclusion: the sensitivity of oxaliplatin is closely related to the percentage of 1: RAD21, and the sensitivity of oxaliplatin to oxaliplatin is closely related to the sensitivity of oxaliplatin. To explore the mechanism that P53 regulates the subcellular localization of RAD21 and reduces the sensitivity of oxaliplatin through autophagy, thus promoting the progression of gastric cancer. These results provide strategies to improve the sensitivity of patients with gastric cancer to oxaliplatin and make RAD21 an attractive molecular target.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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本文編號：1486929