發(fā)布時(shí)間：2018-05-11 22:12

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

【摘要】：【研究背景及目的】 鼻咽癌(nasopharyngeal carcinoma,NPC)是我國(guó)常見的惡性腫瘤之一,以南方地區(qū)尤為高發(fā),放療被認(rèn)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(fù)發(fā),腫瘤細(xì)胞的放射抗拒性是重要原因之一。目前國(guó)內(nèi)外在基因水平上對(duì)腫瘤放射抗拒機(jī)制進(jìn)行了大量的研究,發(fā)現(xiàn)與細(xì)胞周期調(diào)控、細(xì)胞凋亡及DNA損傷修復(fù)等相關(guān)基因及蛋白有關(guān),但尚未發(fā)現(xiàn)在鼻咽癌表達(dá)中能良好預(yù)測(cè)腫瘤放射抗拒性和預(yù)后的基因或蛋白,據(jù)推測(cè)主要原因可能為mRNA的表達(dá)豐度與其相應(yīng)的蛋白質(zhì)表達(dá)水平的不一致性造成的。由此得知,鼻咽癌放射抗拒機(jī)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(gòu)成的分子網(wǎng)絡(luò)相互作用的結(jié)果。因此,應(yīng)用蛋白質(zhì)組學(xué)技術(shù)為從蛋白質(zhì)表達(dá)水平進(jìn)一步探討鼻咽癌放射抗拒機(jī)制提供可能性。 目前,鼻咽癌的蛋白質(zhì)組學(xué)研究主要集中于鼻咽癌的早期診斷和篩選腫瘤標(biāo)志物等方面。以體外培養(yǎng)的腫瘤細(xì)胞為研究對(duì)象,可以避免臨床組織樣本引起的組織異質(zhì)性、影響因素復(fù)雜、陽性結(jié)果易丟失等諸多缺點(diǎn)。因此,從體外培養(yǎng)的腫瘤細(xì)胞系開始逐步過渡到臨床成為當(dāng)今研究放射抗拒機(jī)制的主要方法之一。 本研究旨在應(yīng)用蛋白質(zhì)組學(xué)技術(shù)尋找與鼻咽癌放射抗拒發(fā)生相關(guān)的候選蛋白,通過建立和優(yōu)化細(xì)胞蛋白質(zhì)樣品的制備方法,再對(duì)具有穩(wěn)定放射抗拒性的鼻咽癌細(xì)胞系及其親本細(xì)胞系進(jìn)行比較蛋白質(zhì)組學(xué)分析,從而找到與放射抗拒相關(guān)的差異表達(dá)蛋白。擬在蛋白質(zhì)表達(dá)水平初步闡明鼻咽癌放射抗拒的分子機(jī)制,為后續(xù)候選蛋白的生物學(xué)、功能驗(yàn)證及動(dòng)物實(shí)驗(yàn)奠定基礎(chǔ),為今后臨床預(yù)測(cè)鼻咽癌病人個(gè)體放射敏感性并實(shí)施個(gè)體化放療提供新的思路。 【研究方法】 1、分別采用三種方法對(duì)人鼻咽癌細(xì)胞CNE-2進(jìn)行總蛋白質(zhì)的提取和雙向電泳分析,建立穩(wěn)定性與重復(fù)性俱佳的細(xì)胞蛋白質(zhì)樣品制備方法。 2、體外培養(yǎng)具有穩(wěn)定放射抗拒性細(xì)胞株CNE-2R及其親本細(xì)胞株CNE2,分別提取處于對(duì)數(shù)生長(zhǎng)期的CNE-2R、CNE2細(xì)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(xì)胞CNE-2R、CNE-2的總蛋白。 3、電泳后經(jīng)硝酸銀染色,應(yīng)用ImageMaster 2-DE Platinum 5.0軟件對(duì)凝膠進(jìn)行分析,表達(dá)水平相差2倍以上的蛋白質(zhì)點(diǎn)為差異蛋白點(diǎn),切取膠上的差異蛋白質(zhì)點(diǎn),并行脫色、酶解、萃取,為后續(xù)質(zhì)譜分析奠定基礎(chǔ)。 4、應(yīng)用基質(zhì)輔助激光解吸/電離飛行時(shí)間串聯(lián)質(zhì)譜(MALDI-TOF-MS)技術(shù)對(duì)選取的差異蛋白斑點(diǎn),進(jìn)行蛋白質(zhì)鑒定分析。在Mascot蛋白數(shù)據(jù)庫中檢索,進(jìn)行蛋白的鑒定,通過人工檢索文獻(xiàn)數(shù)據(jù)庫,深入分析蛋白質(zhì)的功能。 【研究結(jié)果】 1、采用通用裂解液裂解細(xì)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(shù),使具有穩(wěn)定放射抗拒性細(xì)胞株CNE-2R及其親本細(xì)胞株CNE-2的總蛋白得到較好的分離。運(yùn)用ImageMaster 2-DE Platinum 5.0軟件篩選出放射抗拒細(xì)胞系CNE-2R和親本細(xì)胞系CNE-2之間差異在2倍以上的差異蛋白質(zhì)點(diǎn)32個(gè)。 3、對(duì)篩選出的32個(gè)表達(dá)差異的蛋白質(zhì)點(diǎn)進(jìn)一步行質(zhì)譜鑒定,其中11個(gè)蛋白質(zhì)點(diǎn)被成功鑒定,并進(jìn)一步行生物信息學(xué)分析,發(fā)現(xiàn)大多數(shù)的蛋白為信號(hào)轉(zhuǎn)導(dǎo)相關(guān)蛋白、分子伴侶、細(xì)胞骨架成分,在生物學(xué)功能方面主要與凋亡調(diào)節(jié)、細(xì)胞周期調(diào)控、RNA轉(zhuǎn)錄、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞骨架組成和輻射應(yīng)激反應(yīng)等有關(guān)。這些蛋白可作為放射治療的潛在靶點(diǎn),成為提高腫瘤放射敏感性的有效途徑,具有深入研究的價(jià)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(jié)論】 2-DE結(jié)合MALDI-TOF-MS技術(shù)能夠有效地識(shí)別和鑒定鼻咽癌放療抗拒細(xì)胞與其親本細(xì)胞系之間差異表達(dá)的蛋白質(zhì)。本研究發(fā)現(xiàn)11個(gè)差異表達(dá)蛋白質(zhì),其功能涉及多種生理代謝和調(diào)節(jié)過程,提示鼻咽癌的放射抗拒性可能是多種蛋白質(zhì)共同作用的結(jié)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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.63

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本文編號(hào)：1875835