本文選題：鼻咽癌 + CXCR7-sh　； 參考：《成都醫(yī)學院》2017年碩士論文

【摘要】：論文工作分為兩部分:一、干擾CXCR7在抑制鼻咽癌轉移侵襲中的作用。二、白花丹醌通過ROS抑制鼻咽癌增殖的作用機制。一、干擾CXCR7在抑制鼻咽癌轉移侵襲中的作用鼻咽癌(Nasopharyngeal carcinoma,NPC)是我國最為常見的頭頸部惡性腫瘤之一,病理類型多屬分化差鱗癌,惡性程度較高,轉移傾向性強,嚴重危害患者的生命安全。雖然目前鼻咽癌的治療方法多樣,但各有利弊,伴有轉移的中晚期鼻咽癌的治療現(xiàn)狀更是不容樂觀。因此,闡明鼻咽癌侵襲、轉移的分子機制是開發(fā)鼻咽癌有效治療手段的重要因素之一。趨化因子受體7(Chemokine receptor 7,CXCR7)作為SDF-1近年來新發(fā)現(xiàn)的另一受體,不僅參與調控胚胎發(fā)育、趨化運動、干細胞歸巢等生理過程,還參與調控腫瘤的多種生物學行為。研究發(fā)現(xiàn)CXCR7在腫瘤組織中的表達普遍高于正常組織,下調CXCR7的表達,不僅可以抑制肝癌、乳腺癌細胞的生長,還能阻止卵巢癌細胞、結腸癌細胞的遷移或侵襲能力。但迄今為止CXCR7對鼻咽癌細胞的增殖、凋亡、遷移等生物學行為的研究報道較為罕見。在本研究中,我們擬搜集臨床標本,利用免疫組織化學方法證實CXCR7在鼻咽癌組織中的分布,分析其與鼻咽癌的臨床分期及預后相關性,探討CXCR7在調控鼻咽癌轉移侵襲中的作用。目的明確CXCR7對鼻咽癌轉移侵襲能力的影響并初步探討其作用機制,為今后開展以CXCR7為靶點的分子治療研究及轉化應用奠定前期基礎。方法1.應用免疫組織化學方法檢測鼻咽癌組織中CXCR7的表達情況,χ2檢驗分析CXCR7的表達與鼻咽癌患者的臨床分期和遠處轉移的關系。2.構建CXCR7-sh RNA慢病毒表達載體干擾人鼻咽癌細胞。將人鼻咽癌CNE2細胞分為實驗組、陰性對照及空白對照組,實驗組:轉染CXCR7-sh RNA慢病毒表達載體;對照對照組:轉染sh Control慢病毒載體;空白對照組:不做任何處理。慢病毒穩(wěn)定感染CNE2細胞后,通過Western blot檢測各組細胞中CXCR7蛋白的表達水平。3.分別利用MTT法和Transwell小室法考察CXCR7干擾對CNE2細胞增殖和侵襲能力的影響;同時通過Western blot檢測CXCR7干擾對AKT、P-AKT以及MMP7蛋白表達水平的變化。結果1.免疫組化結果顯示:CXCR7高表達于鼻咽癌組織;CXCR7陽性(+)表達的鼻咽癌患者的臨床分期和遠處轉移發(fā)生率遠高于CXCR7陰性(-)的鼻咽癌患者(P0.05)。2.轉染CXCR7-sh RNA慢病毒表達載體的實驗組CNE2細胞中CXCR7的蛋白表達水平顯著低于陰性對照組和空白對照組(P0.01)。3.MTT結果顯示:實驗組CNE2細胞的生長速度明顯低于陰性對照組和空白對照組(P0.05);Transwell小室結果顯示:實驗組中CNE2細胞穿過Matrigel的細胞數(shù)目明顯少于陰性對照和空白對照組(P0.01);此外,實驗組CNE2細胞中P-AKT以及MMP7蛋白表達水平顯著低于陰性對照組和空白對照(P0.01)。結論CXCR7的表達與鼻咽癌患者的臨床分期和遠處轉移程度密切相關;靶向抑制CXCR7基因表達能抑制鼻咽癌細胞的增殖與侵襲能力,其作用機制與CXCR7可以調控AKT活化以及MMP7的表達有關。二、白花丹醌通過ROS抑制鼻咽癌增殖的作用機制研究背景鼻咽癌是我國常見的頭頸部惡性腫瘤,對化療較為敏感。對于不能耐受放療、不宜進行手術的鼻咽癌患者,化療效果的優(yōu)劣直接決定其生存時間和生活質量,但當前常用的化療藥物并不十分理想。白花丹醌(Plumbgin,PLB)是我國民間常見中草藥白花丹的主要活性成分,具有多重藥理學功效。已有研究表明,白花丹醌對肺癌、肝癌、乳腺癌等多種腫瘤發(fā)揮良好的抗腫瘤效應,但其抗鼻咽癌的作用及具體分子機制并不完全清楚�；钚匝�(Reactive oxygen species,ROS)是細胞內關鍵的氧化還原信號分子,不僅在腫瘤的誘發(fā)中發(fā)揮作用,而且在抗腫瘤中也發(fā)揮著重要作用。正常情況下人體內的ROS維持在一個穩(wěn)定的范圍內,并在抗炎、抗菌等方面發(fā)揮積極作用。而當這種平衡被打破,ROS持續(xù)升高,將促使細胞發(fā)生轉化、導致惡性腫瘤的發(fā)生。近年來隨著對ROS生物學功能研究的深入,ROS對腫瘤細胞的殺傷效應逐漸被認知。目前許多臨床傳統(tǒng)化療藥物被發(fā)現(xiàn)其作用機制為誘導ROS積累產(chǎn)生抗腫瘤活性。但ROS在白花丹醌抗鼻咽癌治療中的作用罕有報道。目的研究白花丹醌對鼻咽癌增殖的影響;探討ROS在白花丹醌阻滯鼻咽癌細胞周期中的作用及機制,為白花丹醌抗鼻咽癌的臨床應用奠定良好的理論基礎。方法以人鼻咽癌6-10B細胞為模型,MTT法檢測不同濃度的白花丹醌對其增殖的影響;DCFH-DA探針法檢測ROS生成的變化;MTT法檢測活性氧清除劑NAC在白花丹醌抑制鼻咽癌細胞增殖中的作用;流式細胞術檢測白花丹醌、ROS對6-10B細胞周期的影響;Western blot檢測周期相關蛋白Aurora A、Confilin、P-Confilin(ser3)、P-GSK3β(ser9)、核內轉錄因子STAT3總蛋白及其磷酸化的表達變化。結果研究結果顯示白花丹醌能明顯抑制鼻咽癌的增殖并呈劑量依賴效應。ROS在白花丹醌處理6-10B細胞后顯著上升�；钚匝跚宄齽㎞AC可拮抗白花丹醌引起的6-10B細胞ROS的升高,并削弱了白花丹醌抑制鼻咽癌細胞增殖的作用。白花丹醌可導致6-10B細胞G2/M周期阻滯,并上調P-GSK3β(ser9),下調周期相關蛋白Aurora A、Confilin、P-Confilin(ser3)以及核內轉錄因子STAT3并抑制其磷酸化,而清除ROS可以阻斷白花丹醌可以引起G2/M周期阻滯、抑制白花丹醌對上述周期相關蛋白的下調作用。結論白花丹醌通過上調ROS抑制GSK3β/STAT3信號及下游周期相關蛋白,使細胞阻滯在G2/M期從而抑制鼻咽癌增殖。
[Abstract]:The work is divided into two parts: one, the role of interfering CXCR7 in inhibiting the metastasis and invasion of nasopharyngeal carcinoma. Two, the mechanism of inhibiting the proliferation of nasopharyngeal carcinoma by ROS. 1, the role of CXCR7 in inhibiting the metastasis and invasion of nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is one of the most common head and neck malignant tumors in China, and the disease is one of the most common diseases in China. Although various types of differential squamous cell carcinoma are differentiated, the malignant degree is high and the metastatic tendency is strong, it is serious harm to the life safety of the patients. Although there are various treatment methods for nasopharyngeal carcinoma at present, each has advantages and disadvantages, and the treatment status of the middle and Late Nasopharyngeal Carcinoma with metastasis is not optimistic. Therefore, the molecular mechanism of nasopharyngeal carcinoma is to develop nasopharyngeal carcinoma. One of the important factors of effective therapy. Chemokine receptor 7 (Chemokine receptor 7, CXCR7) is a new receptor found in SDF-1 in recent years. It not only participates in the physiological processes of regulating embryonic development, chemotaxis, and stem cell homing, but also participates in the regulation of various biological behavior of tumor. The study found that the expression of CXCR7 in the tumor tissues has been found. The expression of CXCR7, which is generally higher than normal tissue, can not only inhibit the growth of liver cancer and breast cancer cells, but also prevent the migration and invasion of ovarian cancer cells and colon cancer cells. But so far, CXCR7 has been rare in the study of biological lines such as proliferation, apoptosis and migration of nasopharyngeal carcinoma cells. In this study, we intend to search The distribution of CXCR7 in nasopharyngeal carcinoma tissue was confirmed by immunohistochemical method, and the correlation between the clinical stage and prognosis of nasopharyngeal carcinoma was analyzed. The role of CXCR7 in regulating the metastasis and invasion of nasopharyngeal carcinoma was discussed. Objective to clarify the effect of CXCR7 on the metastasis and invasion ability of nasopharyngeal carcinoma and to explore its mechanism for future development. CXCR7 as the target of molecular therapy research and transformation application lay the preliminary basis. Method 1. the expression of CXCR7 in nasopharyngeal carcinoma tissues was detected by immunohistochemistry. The relationship between the expression of CXCR7 and the clinical stage and distant metastasis of nasopharyngeal carcinoma patients was analyzed by x 2 test,.2. construction of CXCR7-sh RNA Lentivirus Expression Vector interfered with human nasopharynx. Human nasopharyngeal carcinoma CNE2 cells were divided into experimental group, negative control and blank control group, experimental group: transfected CXCR7-sh RNA Lentivirus Expression Vector; control control group: transfected sh Control lentivirus vector; blank control group: no treatment. Lentivirus stable infection CNE2 cells, Western blot detection of CXCR7 eggs in each group of cells in each group of CXCR7 eggs detected by Western blot The effect of CXCR7 interference on the proliferation and invasion of CNE2 cells was investigated by MTT and Transwell cell method, and the expression of CXCR7 interference on AKT, P-AKT and MMP7 protein was detected by Western blot. Results 1. immunohistochemical results showed that CXCR7 was highly expressed in nasopharyngeal carcinoma and positive (+) expression. The clinical stage and distant metastasis rate of nasopharyngeal carcinoma patients were much higher than that of CXCR7 negative (P0.05) nasopharyngeal carcinoma (P0.05).2. transfected with CXCR7-sh RNA Lentivirus Expression Vector, the protein expression level of CXCR7 in the experimental group was significantly lower than that of the negative control group and the blank control group (P0.01).3.MTT results: the growth of the CNE2 cells in the experimental group. The speed was significantly lower than that of the negative control group and the blank control group (P0.05), and the Transwell cell results showed that the number of CNE2 cells passing through Matrigel in the experimental group was significantly less than that of the negative control and the blank control group (P0.01). In addition, the expression level of P-AKT and MMP7 protein in the CNE2 cells in the experimental group was significantly lower than that of the negative control group and the blank control (P0.01). Conclusion the expression of CXCR7 is closely related to the clinical staging of nasopharyngeal carcinoma and the degree of distant metastasis. Targeting inhibition of CXCR7 gene expression can inhibit the proliferation and invasion of nasopharyngeal carcinoma cells. The mechanism of its action is related to CXCR7 regulation of AKT activation and the expression of MMP7. Two, the mechanism of white anthraquinone to inhibit the proliferation of nasopharyngeal carcinoma by ROS Background nasopharyngeal carcinoma is a common head and neck cancer in China and is more sensitive to chemotherapy. For nasopharyngeal carcinoma patients who are not tolerant to radiotherapy and should not be operated on, the effect of chemotherapy is directly determined by the survival time and quality of life, but the current commonly used chemotherapeutic drugs are not very ideal. Plumbgin (PLB) is the folk of our country. The main active components of the common Chinese herbal medicine, white flower, have multiple pharmacological effects. It has been shown that white anthraquinone has a good antitumor effect on a variety of tumors, such as lung cancer, liver cancer, and breast cancer, but the role and specific molecular mechanism of the anti nasopharyngeal carcinoma are not completely clear. Reactive oxygen species (ROS) is a cell Neiguan. The redox signalling molecules of the bond play an important role not only in the induction of tumor, but also in the antitumor. Under normal circumstances, the ROS in the human body is maintained in a stable range and plays an active role in anti-inflammatory, antibacterial and other aspects. When the balance is broken and the ROS continues to rise, the cell will be transformed. It leads to the occurrence of malignant tumor. In recent years, with the in-depth study of the biological function of ROS, the killing effect of ROS on tumor cells is gradually recognized. At present, many clinical traditional chemotherapeutic drugs have been found to induce the anti tumor activity of ROS accumulation. But the role of ROS in the treatment of nasopharyngeal carcinoma with white anthraquinone is rare. To study the effect of white anthraquinone on the proliferation of nasopharyngeal carcinoma and to explore the role and mechanism of ROS in the cell cycle of nasopharyngeal carcinoma block by white anthraquinone, to lay a good theoretical basis for the clinical application of white anthraquinone against nasopharyngeal carcinoma. Methods the 6-10B cell of human nasopharyngeal carcinoma was used as the model, and the MTT method was used to detect the effect of the different concentration of white anthraquinone on the proliferation of nasopharyngeal carcinoma; DCFH -DA probe was used to detect the changes of ROS formation; MTT assay was used to detect the effect of reactive oxygen scavenger NAC on the proliferation of nasopharyngeal carcinoma cells; flow cytometry was used to detect the effect of white anthraquinone and ROS on the cell cycle of 6-10B; Western blot detection cycle related protein Aurora A, Confilin, P-Confilin, beta, and internal transcription factors T3 total protein and its phosphorylation changes. Results the results showed that white anthraquinone could significantly inhibit the proliferation of nasopharyngeal carcinoma and significantly increase the dose dependent effect of.ROS after white anthraquinone treatment of 6-10B cells. Reactive oxygen scavenger NAC could antagonize the increase of ROS in 6-10B cells caused by white anthraquinone and weaken the inhibitory effect of white anthraquinone on nasopharynx. The effect of cancer cell proliferation. White anthraquinone can lead to 6-10B cell G2/M cycle block, and up regulation of P-GSK3 beta (ser9), down regulation of cycle related protein Aurora A, Confilin, P-Confilin (ser3), and nuclear factor STAT3 and inhibit its phosphorylation. Conclusion white anthraquinone inhibits the proliferation of nasopharyngeal carcinoma by inhibiting the GSK3 beta /STAT3 signal and the downstream cycle related proteins by up regulation of ROS, so that the cells block at G2/M phase.

【學位授予單位】：成都醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.63

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9 李爍;鼻咽癌組織中磷酸果糖激酶1的表達及RNA干擾靶向沉默磷酸果糖激酶1對鼻咽癌細胞株CNE2生物學特性的影響[D];華中科技大學;2014年

10 譚遙;新疆維吾爾、漢族鼻咽癌患者HLA基因多態(tài)性、LMP2A特異性T細胞免疫應答及預后差異性研究[D];新疆醫(yī)科大學;2016年

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6 馬，

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