本文選題：鼻咽癌 + 放射抗拒　； 參考：《廣西醫(yī)科大學》2011年碩士論文

【摘要】：【研究背景及目的】 鼻咽癌(nasopharyngeal carcinoma,NPC)是我國常見的惡性腫瘤之一,以南方地區(qū)尤為高發(fā),放療被認為是首選的治療手段。然而,臨床上部分病人放療后出現(xiàn)局部殘留與局部復發(fā),腫瘤細胞的放射抗拒性是重要原因之一。目前國內外在基因水平上對腫瘤放射抗拒機制進行了大量的研究,發(fā)現(xiàn)與細胞周期調控、細胞凋亡及DNA損傷修復等相關基因及蛋白有關,但尚未發(fā)現(xiàn)在鼻咽癌表達中能良好預測腫瘤放射抗拒性和預后的基因或蛋白,據(jù)推測主要原因可能為mRNA的表達豐度與其相應的蛋白質表達水平的不一致性造成的。由此得知,鼻咽癌放射抗拒機制的發(fā)生并非由單一基因或蛋白改變引起,而是多基因或蛋白構成的分子網(wǎng)絡相互作用的結果。因此,應用蛋白質組學技術為從蛋白質表達水平進一步探討鼻咽癌放射抗拒機制提供可能性。 目前,鼻咽癌的蛋白質組學研究主要集中于鼻咽癌的早期診斷和篩選腫瘤標志物等方面。以體外培養(yǎng)的腫瘤細胞為研究對象,可以避免臨床組織樣本引起的組織異質性、影響因素復雜、陽性結果易丟失等諸多缺點。因此,從體外培養(yǎng)的腫瘤細胞系開始逐步過渡到臨床成為當今研究放射抗拒機制的主要方法之一。 本研究旨在應用蛋白質組學技術尋找與鼻咽癌放射抗拒發(fā)生相關的候選蛋白,通過建立和優(yōu)化細胞蛋白質樣品的制備方法,再對具有穩(wěn)定放射抗拒性的鼻咽癌細胞系及其親本細胞系進行比較蛋白質組學分析,從而找到與放射抗拒相關的差異表達蛋白。擬在蛋白質表達水平初步闡明鼻咽癌放射抗拒的分子機制,為后續(xù)候選蛋白的生物學、功能驗證及動物實驗奠定基礎,為今后臨床預測鼻咽癌病人個體放射敏感性并實施個體化放療提供新的思路。 【研究方法】 1、分別采用三種方法對人鼻咽癌細胞CNE-2進行總蛋白質的提取和雙向電泳分析,建立穩(wěn)定性與重復性俱佳的細胞蛋白質樣品制備方法。 2、體外培養(yǎng)具有穩(wěn)定放射抗拒性細胞株CNE-2R及其親本細胞株CNE2,分別提取處于對數(shù)生長期的CNE-2R、CNE2細胞總蛋白。采用雙向凝膠電泳(2-DE)即第一向固相pH梯度電泳、第二向SDS-聚丙烯酰胺凝膠電泳。分離細胞CNE-2R、CNE-2的總蛋白。 3、電泳后經(jīng)硝酸銀染色,應用ImageMaster 2-DE Platinum 5.0軟件對凝膠進行分析,表達水平相差2倍以上的蛋白質點為差異蛋白點,切取膠上的差異蛋白質點,并行脫色、酶解、萃取,為后續(xù)質譜分析奠定基礎。 4、應用基質輔助激光解吸/電離飛行時間串聯(lián)質譜(MALDI-TOF-MS)技術對選取的差異蛋白斑點,進行蛋白質鑒定分析。在Mascot蛋白數(shù)據(jù)庫中檢索,進行蛋白的鑒定,通過人工檢索文獻數(shù)據(jù)庫,深入分析蛋白質的功能。 【研究結果】 1、采用通用裂解液裂解細胞,等電聚焦前在樣品中加入Destreak試劑,可以獲得穩(wěn)定性好、圖像清晰、分辨率高的雙向電泳圖譜。 2、通過雙向凝膠電泳技術,使具有穩(wěn)定放射抗拒性細胞株CNE-2R及其親本細胞株CNE-2的總蛋白得到較好的分離。運用ImageMaster 2-DE Platinum 5.0軟件篩選出放射抗拒細胞系CNE-2R和親本細胞系CNE-2之間差異在2倍以上的差異蛋白質點32個。 3、對篩選出的32個表達差異的蛋白質點進一步行質譜鑒定,其中11個蛋白質點被成功鑒定,并進一步行生物信息學分析,發(fā)現(xiàn)大多數(shù)的蛋白為信號轉導相關蛋白、分子伴侶、細胞骨架成分,在生物學功能方面主要與凋亡調節(jié)、細胞周期調控、RNA轉錄、信號轉導、細胞骨架組成和輻射應激反應等有關。這些蛋白可作為放射治療的潛在靶點,成為提高腫瘤放射敏感性的有效途徑,具有深入研究的價值,為鼻咽癌的放射治療研究提供新的研究思路和方法。 【研究結論】 2-DE結合MALDI-TOF-MS技術能夠有效地識別和鑒定鼻咽癌放療抗拒細胞與其親本細胞系之間差異表達的蛋白質。本研究發(fā)現(xiàn)11個差異表達蛋白質,其功能涉及多種生理代謝和調節(jié)過程,提示鼻咽癌的放射抗拒性可能是多種蛋白質共同作用的結果,其中可能參與鼻咽癌放射抗拒性的發(fā)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.

【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R739.63

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