【作者】 冯雪萍；

【导师】 肖志强；

【作者基本信息】 中南大学 ， 病理与病理生理学， 2010， 博士

【摘要】 鼻咽癌(nasopharyngeal carcinoma, NPC)是我国南方常见的恶性肿瘤,其发病率和死亡率均居世界首位,严重威胁我国人民的生命和健康。放疗是NPC的主要治疗手段。虽然放疗设备和方法不断改进,但NPC疗效无明显改善。放疗失败的主要原因是NPC组织中存在一定比例的放疗抗拒细胞,放疗后残留的这些肿瘤细胞不仅放射抗拒性增强,而且更具侵袭性、容易发生淋巴结和远处转移。因此,放疗抗拒仍然是制约NPC疗效的瓶颈。但NPC放射抗拒的分子机制不太清楚。寻找NPC放射抗拒相关的蛋白质,不仅有助于揭示NPC放疗抗拒的机制,而且能为NPC放疗敏感性预测以及NPC的靶向和个体化治疗提供科学依据,对提高NPC的疗效、改善NPC预后具有重要作用。为筛选NPC放疗抗拒相关的蛋白质,我们采用亚致死剂量放射线反复照射法建立了放射抗拒的NPC细胞株CNE2-IR,该细胞系来源于NPC细胞株CNE2,为接受5次亚致死剂量放射线(11Gy)照射后存活的亚克隆细胞。然后采用蛋白质组学技术比较放射抗拒CNE2-IR细胞与放射敏感CNE2细胞蛋白质表达谱的差异,筛选NPC的放疗抗拒相关蛋白质。即应用二维凝胶电泳(Two-dimensional gel electrophoresis,2-DE)分离两株细胞的总蛋白质,PDQuest软件分析识别差异表达蛋白质点,基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF-MS)和电喷雾电离串联质谱(ESI-Q-TOF MS／MS)鉴定差异表达的蛋白质。Western blot对蛋白质组学筛选到的差异蛋白质进行验证。建立了高质量的放射抗拒CNE2-IR细胞与放射敏感CNE2细胞的2-DE图谱,质谱共鉴定出34种差异表达的蛋白质。其中14-3-3σ和Maspin表达在放射抗拒的CNE2-IR细胞中明显下调,而GRP78和Mn-SOD表达明显上调。应用Western blot验证了4种蛋白质(14-3-3σ、Maspin、GRP78和Mn-SOD)在CNE2-IR和CNE2细胞株中的差异表达水平。为了探讨差异表达蛋白质能否作为预测NPC放疗敏感性的分子标志,采用免疫组化检测差异蛋白14-3-3σ、Maspin、GRP78和Mn-SOD在39例放射抗拒和51例放射敏感NPC组织中的表达,采用ROC(Receiver Operating Characteristic, ROC)曲线分析4个蛋白质预测NPC放疗敏感性的价值。结果显示,与放疗敏感的NPC组织比较,14-3-3σ和Maspin表达在放射抗拒NPC组织中明显下调,而GRP78和Mn-SOD表达明显上调。14-3-3σ和Maspin表达下调,以及GRP78和Mn-SOD表达上调与NPC放射抗拒性有明显的相关性；4个蛋白质组合判别放射敏感与放射抗拒NPC的敏感性为90%、特异性为88%。为进一步证实4个差异蛋白质在NPC放射抗拒产生中的作用,我们选择差异蛋白质14-3-3σ进行功能分析。既采用基因转染技术上调14-3-3σ在CNE2-IR细胞中的表达,采用克隆存活实验检测14-3-3σ表达上调对其放疗敏感性的影响。结果显示,14-3-3σ表达上调可部分逆转CNE2-IR细胞的放射抗拒性,这提示14-3-3σ在NPC放射抗拒产生中具有重要的作用。本文研究结果表明,14-3-3σ、Maspin、GRP78和Mn-SOD蛋白是预测NPC放疗敏感性的潜在生物标志物,它们表达失调可能参与NPC放射抗拒性的产生。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China with the highest incidence and mortality rate in the world. It poses one of the most serious public health problems in this area. Radiotherapy is the primary treatment for NPC. Although the radiotherapeutic equipments and technique are improved continuously, the radiotherapeutic efficacy of NPC remains unfavorable. The main reason for the failure of NPC radiotherapy is that there are a certain percentage of radioresistant cells in the NPC tissues, which not only have higher radioresistance, but also are more aggressive after radiotherapy, prone to lymph node and distant metastasis. Therefore, the radioresistance remains a serious obstacle to successful treatment in some NPC cases.However, the molecular mechanism underlying radioresistance of NPC has not been elucidated yet. The finding of radioresistant proteins will be useful for revealing the mechanism of NPC radioresistance, and providing the scientific basis for predicting NPC sensitivity as well as targets and personalized therapy, which plays an important role in improving efficacy and prognosis of NPC.To identify proteins involved in NPC resistance, we established a radioresistant subclone cell line (CNE2-IR) derived from NPC cell line CNE2 by treating the cells with five rounds of sublethal ionizing radiation (11Gy). Proteomics was then performed to compare protein profiles of CNE2-IR and CNE2 cells. Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of CNE2-IR and CNE2 cells, PDQuest software was applied to analyze the differential protein spots between the CNE2-IR and CNE2 cell lines, these differential spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS,and the differential proteins identified by proteomic approach were confirmed by Western blot.Highly reproductive and resolvable 2-DE maps of CNE2-IR and CNE2 cells were established. A total of 34 differential proteins was identified by MALDI-TOF MS and ESI-Q-TOF MS.Among them,14-3-3σand Maspin was significantly downregulated, and GRP78 and Mn-SOD was significantly upregulated in the radioresistant CNE2-IR as compared with control CNE2. The differential expression levels of the four proteins (14-3-3σ, Maspin, GRP78 and Mn-SOD) between the CNE2-IR and CNE2 cell lines were confirmed by Western blot.To investigate whether the differentially expressed proteins serve as predictive molecular markers for radiosensitivity of NPC, immunohistochemistry was performed to detect the expression levels of 14-3-3σ, Maspin, GRP78 and Mn-SOD proteins in the 39 radioresistant and 51 radiosensitive NPC tissues. The ability of 14-3-3σ, Maspin, GRP78 and Mn-SOD in distinguishing the radiosensitive and radioresistant NPC tissues was evaluated by the ROC (receiver operating characteristic) analysis. The results showed that 14-3-3σand Maspin was significantly downregulated, and GRP78 and Mn-SOD was significantly upregulated in the radioresistant NPC tissues as compared with radiosensitive NPC tissues.The downregulation of 14-3-3σand Maspin and upregulation of Mn-SOD and GRP78 were significantly correlated with NPC radioresistance.As a panel, the four proteins achieved a sensitivity of 90% and a specificity of 88% in discriminating radiosensitive from radioresistant NPC.To address the question whether the expression levels of the four proteins may associate with the development of NPC radioresistance, we selected 14-3-3σ, one of the four differential proteins to be further functionally studied. pcDNA3-14-3-3σplasmid was transfected into the CNE-2-IR cells to upregulate the expression of 14-3-3σ, and the effect of 14-3-3σ-upregulation on CNE-2-IR radiosensitivity was measured by using a clonogenic survival assay. The results indicated that the radioresistance can be partially reversed by overexpression of 14-3-3σin the CNE2-IR cells.Our data showed that 14-3-3σ, Maspin, GRP78 and Mn-SOD proteins are potential biomarkers for predicting NPC radiosensitivity.Their dysregulation may be involved in the development of NPC radioresistance.更多还原