干扰CXCR7抑制鼻咽癌转移侵袭及白花丹醌通过ROS抑制鼻咽癌增殖的作用机制

发布时间：2018-05-02 14:21

本文选题：鼻咽癌 + CXCR7-sh　；参考：《成都医学院》2017年硕士论文

【摘要】：论文工作分为两部分:一、干扰CXCR7在抑制鼻咽癌转移侵袭中的作用。二、白花丹醌通过ROS抑制鼻咽癌增殖的作用机制。一、干扰CXCR7在抑制鼻咽癌转移侵袭中的作用鼻咽癌(Nasopharyngeal carcinoma,NPC)是我国最为常见的头颈部恶性肿瘤之一,病理类型多属分化差鳞癌,恶性程度较高,转移倾向性强,严重危害患者的生命安全。虽然目前鼻咽癌的治疗方法多样,但各有利弊,伴有转移的中晚期鼻咽癌的治疗现状更是不容乐观。因此,阐明鼻咽癌侵袭、转移的分子机制是开发鼻咽癌有效治疗手段的重要因素之一。趋化因子受体7(Chemokine receptor 7,CXCR7)作为SDF-1近年来新发现的另一受体,不仅参与调控胚胎发育、趋化运动、干细胞归巢等生理过程,还参与调控肿瘤的多种生物学行为。研究发现CXCR7在肿瘤组织中的表达普遍高于正常组织,下调CXCR7的表达,不仅可以抑制肝癌、乳腺癌细胞的生长,还能阻止卵巢癌细胞、结肠癌细胞的迁移或侵袭能力。但迄今为止CXCR7对鼻咽癌细胞的增殖、凋亡、迁移等生物学行为的研究报道较为罕见。在本研究中,我们拟搜集临床标本,利用免疫组织化学方法证实CXCR7在鼻咽癌组织中的分布,分析其与鼻咽癌的临床分期及预后相关性,探讨CXCR7在调控鼻咽癌转移侵袭中的作用。目的明确CXCR7对鼻咽癌转移侵袭能力的影响并初步探讨其作用机制,为今后开展以CXCR7为靶点的分子治疗研究及转化应用奠定前期基础。方法1.应用免疫组织化学方法检测鼻咽癌组织中CXCR7的表达情况,χ2检验分析CXCR7的表达与鼻咽癌患者的临床分期和远处转移的关系。2.构建CXCR7-sh RNA慢病毒表达载体干扰人鼻咽癌细胞。将人鼻咽癌CNE2细胞分为实验组、阴性对照及空白对照组,实验组:转染CXCR7-sh RNA慢病毒表达载体;对照对照组:转染sh Control慢病毒载体;空白对照组:不做任何处理。慢病毒稳定感染CNE2细胞后,通过Western blot检测各组细胞中CXCR7蛋白的表达水平。3.分别利用MTT法和Transwell小室法考察CXCR7干扰对CNE2细胞增殖和侵袭能力的影响;同时通过Western blot检测CXCR7干扰对AKT、P-AKT以及MMP7蛋白表达水平的变化。结果1.免疫组化结果显示:CXCR7高表达于鼻咽癌组织;CXCR7阳性(+)表达的鼻咽癌患者的临床分期和远处转移发生率远高于CXCR7阴性(-)的鼻咽癌患者(P0.05)。2.转染CXCR7-sh RNA慢病毒表达载体的实验组CNE2细胞中CXCR7的蛋白表达水平显著低于阴性对照组和空白对照组(P0.01)。3.MTT结果显示:实验组CNE2细胞的生长速度明显低于阴性对照组和空白对照组(P0.05);Transwell小室结果显示:实验组中CNE2细胞穿过Matrigel的细胞数目明显少于阴性对照和空白对照组(P0.01);此外,实验组CNE2细胞中P-AKT以及MMP7蛋白表达水平显著低于阴性对照组和空白对照(P0.01)。结论CXCR7的表达与鼻咽癌患者的临床分期和远处转移程度密切相关;靶向抑制CXCR7基因表达能抑制鼻咽癌细胞的增殖与侵袭能力,其作用机制与CXCR7可以调控AKT活化以及MMP7的表达有关。二、白花丹醌通过ROS抑制鼻咽癌增殖的作用机制研究背景鼻咽癌是我国常见的头颈部恶性肿瘤,对化疗较为敏感。对于不能耐受放疗、不宜进行手术的鼻咽癌患者,化疗效果的优劣直接决定其生存时间和生活质量,但当前常用的化疗药物并不十分理想。白花丹醌(Plumbgin,PLB)是我国民间常见中草药白花丹的主要活性成分,具有多重药理学功效。已有研究表明,白花丹醌对肺癌、肝癌、乳腺癌等多种肿瘤发挥良好的抗肿瘤效应,但其抗鼻咽癌的作用及具体分子机制并不完全清楚。活性氧(Reactive oxygen species,ROS)是细胞内关键的氧化还原信号分子,不仅在肿瘤的诱发中发挥作用,而且在抗肿瘤中也发挥着重要作用。正常情况下人体内的ROS维持在一个稳定的范围内,并在抗炎、抗菌等方面发挥积极作用。而当这种平衡被打破,ROS持续升高,将促使细胞发生转化、导致恶性肿瘤的发生。近年来随着对ROS生物学功能研究的深入,ROS对肿瘤细胞的杀伤效应逐渐被认知。目前许多临床传统化疗药物被发现其作用机制为诱导ROS积累产生抗肿瘤活性。但ROS在白花丹醌抗鼻咽癌治疗中的作用罕有报道。目的研究白花丹醌对鼻咽癌增殖的影响;探讨ROS在白花丹醌阻滞鼻咽癌细胞周期中的作用及机制,为白花丹醌抗鼻咽癌的临床应用奠定良好的理论基础。方法以人鼻咽癌6-10B细胞为模型,MTT法检测不同浓度的白花丹醌对其增殖的影响;DCFH-DA探针法检测ROS生成的变化;MTT法检测活性氧清除剂NAC在白花丹醌抑制鼻咽癌细胞增殖中的作用;流式细胞术检测白花丹醌、ROS对6-10B细胞周期的影响;Western blot检测周期相关蛋白Aurora A、Confilin、P-Confilin(ser3)、P-GSK3β(ser9)、核内转录因子STAT3总蛋白及其磷酸化的表达变化。结果研究结果显示白花丹醌能明显抑制鼻咽癌的增殖并呈剂量依赖效应。ROS在白花丹醌处理6-10B细胞后显著上升。活性氧清除剂NAC可拮抗白花丹醌引起的6-10B细胞ROS的升高,并削弱了白花丹醌抑制鼻咽癌细胞增殖的作用。白花丹醌可导致6-10B细胞G2/M周期阻滞,并上调P-GSK3β(ser9),下调周期相关蛋白Aurora A、Confilin、P-Confilin(ser3)以及核内转录因子STAT3并抑制其磷酸化,而清除ROS可以阻断白花丹醌可以引起G2/M周期阻滞、抑制白花丹醌对上述周期相关蛋白的下调作用。结论白花丹醌通过上调ROS抑制GSK3β/STAT3信号及下游周期相关蛋白,使细胞阻滞在G2/M期从而抑制鼻咽癌增殖。
[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.

【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.63

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