鼻咽癌细胞放射抗拒的差异蛋白质组学分析

发布时间：2018-05-11 22:12

本文选题：鼻咽癌 + 放射抗拒　；参考：《广西医科大学》2011年硕士论文

【摘要】：【研究背景及目的】鼻咽癌(nasopharyngeal carcinoma,NPC)是我国常见的恶性肿瘤之一,以南方地区尤为高发,放疗被认为是首选的治疗手段。然而,临床上部分病人放疗后出现局部残留与局部复发,肿瘤细胞的放射抗拒性是重要原因之一。目前国内外在基因水平上对肿瘤放射抗拒机制进行了大量的研究,发现与细胞周期调控、细胞凋亡及DNA损伤修复等相关基因及蛋白有关,但尚未发现在鼻咽癌表达中能良好预测肿瘤放射抗拒性和预后的基因或蛋白,据推测主要原因可能为mRNA的表达丰度与其相应的蛋白质表达水平的不一致性造成的。由此得知,鼻咽癌放射抗拒机制的发生并非由单一基因或蛋白改变引起,而是多基因或蛋白构成的分子网络相互作用的结果。因此,应用蛋白质组学技术为从蛋白质表达水平进一步探讨鼻咽癌放射抗拒机制提供可能性。目前,鼻咽癌的蛋白质组学研究主要集中于鼻咽癌的早期诊断和筛选肿瘤标志物等方面。以体外培养的肿瘤细胞为研究对象,可以避免临床组织样本引起的组织异质性、影响因素复杂、阳性结果易丢失等诸多缺点。因此,从体外培养的肿瘤细胞系开始逐步过渡到临床成为当今研究放射抗拒机制的主要方法之一。本研究旨在应用蛋白质组学技术寻找与鼻咽癌放射抗拒发生相关的候选蛋白,通过建立和优化细胞蛋白质样品的制备方法,再对具有稳定放射抗拒性的鼻咽癌细胞系及其亲本细胞系进行比较蛋白质组学分析,从而找到与放射抗拒相关的差异表达蛋白。拟在蛋白质表达水平初步阐明鼻咽癌放射抗拒的分子机制,为后续候选蛋白的生物学、功能验证及动物实验奠定基础,为今后临床预测鼻咽癌病人个体放射敏感性并实施个体化放疗提供新的思路。【研究方法】 1、分别采用三种方法对人鼻咽癌细胞CNE-2进行总蛋白质的提取和双向电泳分析,建立稳定性与重复性俱佳的细胞蛋白质样品制备方法。 2、体外培养具有稳定放射抗拒性细胞株CNE-2R及其亲本细胞株CNE2,分别提取处于对数生长期的CNE-2R、CNE2细胞总蛋白。采用双向凝胶电泳(2-DE)即第一向固相pH梯度电泳、第二向SDS-聚丙烯酰胺凝胶电泳。分离细胞CNE-2R、CNE-2的总蛋白。 3、电泳后经硝酸银染色,应用ImageMaster 2-DE Platinum 5.0软件对凝胶进行分析,表达水平相差2倍以上的蛋白质点为差异蛋白点,切取胶上的差异蛋白质点,并行脱色、酶解、萃取,为后续质谱分析奠定基础。 4、应用基质辅助激光解吸/电离飞行时间串联质谱(MALDI-TOF-MS)技术对选取的差异蛋白斑点,进行蛋白质鉴定分析。在Mascot蛋白数据库中检索,进行蛋白的鉴定,通过人工检索文献数据库,深入分析蛋白质的功能。【研究结果】 1、采用通用裂解液裂解细胞,等电聚焦前在样品中加入Destreak试剂,可以获得稳定性好、图像清晰、分辨率高的双向电泳图谱。 2、通过双向凝胶电泳技术,使具有稳定放射抗拒性细胞株CNE-2R及其亲本细胞株CNE-2的总蛋白得到较好的分离。运用ImageMaster 2-DE Platinum 5.0软件筛选出放射抗拒细胞系CNE-2R和亲本细胞系CNE-2之间差异在2倍以上的差异蛋白质点32个。 3、对筛选出的32个表达差异的蛋白质点进一步行质谱鉴定,其中11个蛋白质点被成功鉴定,并进一步行生物信息学分析,发现大多数的蛋白为信号转导相关蛋白、分子伴侣、细胞骨架成分,在生物学功能方面主要与凋亡调节、细胞周期调控、RNA转录、信号转导、细胞骨架组成和辐射应激反应等有关。这些蛋白可作为放射治疗的潜在靶点,成为提高肿瘤放射敏感性的有效途径,具有深入研究的价值,为鼻咽癌的放射治疗研究提供新的研究思路和方法。【研究结论】 2-DE结合MALDI-TOF-MS技术能够有效地识别和鉴定鼻咽癌放疗抗拒细胞与其亲本细胞系之间差异表达的蛋白质。本研究发现11个差异表达蛋白质,其功能涉及多种生理代谢和调节过程,提示鼻咽癌的放射抗拒性可能是多种蛋白质共同作用的结果,其中可能参与鼻咽癌放射抗拒性的发生。
[Abstract]:[research background and purpose]
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in China, especially in the southern region. Radiotherapy is considered to be the first choice for treatment. However, local residual and local recurrence occur in some patients after radiotherapy, and the radiation resistance of tumor cells is one of the important reasons. A lot of studies have been done on the mechanism of tumor radiation resistance, which have been found to be related to cell cycle regulation, cell apoptosis and DNA damage repair and other related genes and proteins. But it is not found that the genes or proteins that can predict the tumor's radiological resistance and prognosis in the expression of nasopharyngeal carcinoma have not been found. It is presumed that the main reason may be the expression of mRNA and its abundance. The inconsistency of the corresponding protein expression level is caused. Thus, it is found that the occurrence of the radiation resistance mechanism of nasopharyngeal carcinoma is not caused by a single gene or protein change, but a result of the interaction of molecular networks composed of multiple genes or proteins. Therefore, proteomics technology is used to further explore nasopharyngeal carcinoma from protein expression level. The mechanism of radiation resistance provides the possibility.
At present, the proteomics research of nasopharyngeal carcinoma is mainly focused on the early diagnosis of nasopharyngeal carcinoma and the screening of tumor markers. The tumor cells cultured in vitro are the research object, which can avoid the tissue heterogeneity caused by the clinical tissue samples, the influencing factors are complex, the positive results are easily lost and so on. Tumor cell lines begin to gradually transition to the clinic and become one of the main methods to study the radiation resistance mechanism.
The purpose of this study is to find the candidate proteins associated with the radiation resistance of nasopharyngeal carcinoma by proteomics technology. By establishing and optimizing the preparation methods of the cell protein samples, the proteomic analysis of the cell lines and their parent cell lines with stable radiation resistance is then compared, and the resistance to radiation is found. The differentially expressed protein is intended to elucidate the molecular mechanism of the radiation resistance of nasopharyngeal carcinoma at the level of protein expression, which lays the foundation for the biological, functional verification and animal experiments of the subsequent candidate proteins, and provides a new idea for the future clinical prediction of the individual radiosensitivity of nasopharyngeal cancer patients and the implementation of the individualized radiotherapy.
[research methods]
1, three methods were used to extract and analyze the total protein of human nasopharyngeal carcinoma cell CNE-2, and to establish a method for the preparation of protein samples with good stability and reproducibility.
2, in vitro culture with stable radiological resistance cell line CNE-2R and its parent cell line CNE2, the total protein of CNE-2R and CNE2 cells in logarithmic growth period was extracted respectively. Two dimensional gel electrophoresis (2-DE), the first phase solid phase pH gradient electrophoresis, and second to SDS- polyacrylamide gel electrophoresis, were used to separate the total protein of cell CNE-2R and CNE-2.
3, after electrophoresis with silver nitrate, the gel was analyzed with ImageMaster 2-DE Platinum 5 software. The protein points above 2 times of the difference of the expression level were different protein points, and the differential protein points on the gel were cut, and the decolorization, enzymatic hydrolysis and extraction were used to lay the foundation for the subsequent mass spectrometry analysis.
4, the matrix assisted laser desorption / ionization time of flight tandem mass spectrometry (MALDI-TOF-MS) was used to identify the protein spots and identify the protein. In the Mascot protein database, the protein was identified and the function of the protein was analyzed by the manual retrieval of the literature database.
[results]
1, a two way electrophoresis map with good stability, clear image and high resolution can be obtained by using a common lysate to crack cells and adding Destreak reagent to the sample before isoelectric focusing.
2, through two-dimensional gel electrophoresis, the total protein of the stable radiological resistance cell line CNE-2R and its parent cell line CNE-2 was better separated. Using the ImageMaster 2-DE Platinum 5 software, the difference of the difference between the radiological resistance cell line CNE-2R and the parent cell line CNE-2 was found to be more than 2 times the difference of the protein points.
3, the protein points of the 32 differentially expressed proteins were further identified by mass spectrometry, of which 11 proteins were identified successfully, and further bioinformatics analysis showed that most of the proteins were signal transduction related proteins, molecular chaperones, cytoskeleton components, and regulation of apoptosis and cell cycle regulation in biological function. RNA transcription, signal transduction, cytoskeleton composition and radiation stress response are related. These proteins can be used as potential targets for radiation therapy and become an effective way to improve the radiosensitivity of tumor. It is of great value to study and provide new research ideas and methods for the study of radiotherapy for nasopharyngeal carcinoma.
[Conclusion]
2-DE combined with MALDI-TOF-MS technology can effectively identify and identify proteins that are differentially expressed between the radiation resistant cells of nasopharyngeal carcinoma and their parent cell lines. This study found 11 differentially expressed proteins whose function involves a variety of physiological metabolism and regulatory processes, suggesting that the radioactivity resistance of nasopharyngeal carcinoma may be a common effect of a variety of proteins. The results may be involved in the occurrence of radiopharyngeal resistance in nasopharyngeal carcinoma.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

【参考文献】