本文關(guān)鍵詞： 核蛋白 熒光標(biāo)記雙向差異凝膠電泳 基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜 Jurkat T淋巴細(xì)胞　出處：《吉林大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】： JurkatT細(xì)胞株為白血病T淋巴細(xì)胞的一種,利用該類細(xì)胞作為研究對(duì)象可進(jìn)行外界損傷性因素導(dǎo)致細(xì)胞壞死和凋亡的研究。電離輻射能夠?qū)е录?xì)胞不可逆性損傷,是惡性腫瘤的疾病的重要治療方法。蛋白組學(xué)是研究細(xì)胞內(nèi)蛋白構(gòu)成以及在外界條件作用下發(fā)生生理或病理變化的實(shí)驗(yàn)室研究技術(shù)。將電離輻射與蛋白組學(xué)實(shí)驗(yàn)技術(shù)相結(jié)合,研究核內(nèi)蛋白差異蛋白質(zhì)變化規(guī)律,對(duì)尋求對(duì)白血病治療有益的關(guān)鍵性核內(nèi)調(diào)控蛋白因子十分重要。 本研究驗(yàn)證了目前應(yīng)用普遍的蛋白提取方法,建立了熒光免疫雙向差異電泳結(jié)合質(zhì)譜學(xué)分離和鑒定核內(nèi)蛋白的實(shí)驗(yàn)技術(shù)平臺(tái),并對(duì)分離的差異蛋白質(zhì)進(jìn)行鑒定,共得到14個(gè)差異蛋白質(zhì)點(diǎn),為進(jìn)一步全面了解核內(nèi)蛋白質(zhì)表達(dá)差異奠定了基礎(chǔ)。 T淋巴細(xì)胞在輻射強(qiáng)度為6Gy條件下進(jìn)行輻射誘發(fā)細(xì)胞損傷,在不同的時(shí)間點(diǎn)收集細(xì)胞,提取核內(nèi)蛋白,應(yīng)用2D-DIGE電泳進(jìn)行分離,并利用質(zhì)譜進(jìn)行分析,根據(jù)質(zhì)譜分析結(jié)果和MSCOT數(shù)據(jù)庫檢索,對(duì)細(xì)胞核蛋白做整體、全面的分析,鑒定了表達(dá)差異蛋白,共得到5組10個(gè)與T淋巴細(xì)胞細(xì)胞核內(nèi)物質(zhì)表達(dá)以及核外轉(zhuǎn)運(yùn)、參與整個(gè)細(xì)胞生長(zhǎng)、增殖和轉(zhuǎn)歸的關(guān)鍵性蛋白。其分類如下:①淋巴細(xì)胞核內(nèi)腫瘤相關(guān)抗原包括腫瘤生長(zhǎng)因子-β受體相關(guān)蛋白呈現(xiàn)上調(diào)表達(dá)、細(xì)胞生長(zhǎng)因子結(jié)合蛋白和核糖核酸酶—血管生長(zhǎng)因子抑制劑呈現(xiàn)下調(diào)表達(dá),而腫瘤排斥抗原表現(xiàn)出統(tǒng)計(jì)學(xué)意義的下調(diào)表達(dá),在整個(gè)細(xì)胞試驗(yàn)流程中表達(dá)比較恒定。②淋巴細(xì)胞代謝相關(guān)核內(nèi)相關(guān)蛋白包括泛素羧基末端水解酶,二硫化物異構(gòu)酶蛋白和谷胱甘肽S-轉(zhuǎn)移酶均呈現(xiàn)下調(diào)表達(dá)。③淋巴細(xì)胞核內(nèi)遺傳物質(zhì)相關(guān)蛋白包括不均-核糖核蛋白K和G1期/S期轉(zhuǎn)換控制蛋白呈現(xiàn)下調(diào)表達(dá),核RNA解旋酶呈上調(diào)表達(dá)。④淋巴細(xì)胞細(xì)胞核穩(wěn)定性相關(guān)蛋白包括蛋白酶體激活蛋白PA28,在0~8小時(shí)時(shí)程范圍內(nèi)呈下調(diào)表達(dá),在8~16小時(shí)范圍內(nèi)呈現(xiàn)明顯上調(diào)表達(dá),隨后的16~24小時(shí)范圍內(nèi)蛋白表達(dá)呈現(xiàn)下降趨勢(shì)。 本研究為進(jìn)一步研究電離輻射損傷淋巴細(xì)胞打下了理論基礎(chǔ)。
[Abstract]:JurkatT cell line is one of the leukemic T lymphocytes, which can be used as a research object to study the apoptosis and necrosis caused by external injury. Ionizing radiation can lead to irreversibly damaged cells. Proteomics is a laboratory technique for the study of intracellular protein composition and physiological or pathological changes under external conditions. It combines ionizing radiation with proteomics. It is very important to study the variation of differential protein in nuclear proteins in order to seek the key factors which are beneficial to the treatment of leukemia. In this study, we established an experimental platform for the separation and identification of nuclear proteins by fluorescence immunotwo-dimensional differential electrophoresis combined with mass spectrometry, and identified the isolated differential proteins. A total of 14 differentially expressed protein spots were obtained, which laid a foundation for further understanding the differences in protein expression in the nucleus. T lymphocytes were induced by radiation at a radiation intensity of 6 Gy. The cells were collected at different time points, the nuclear proteins were extracted, separated by 2D-DIGE electrophoresis, and analyzed by mass spectrometry. According to the results of mass spectrometry analysis and MSCOT database retrieval, the nuclear proteins were analyzed, and the differentially expressed proteins were identified. A total of 10 groups and 10 groups were obtained, which expressed substance in the nucleus of T lymphocytes and transported them outside the nucleus. The key proteins involved in cell growth, proliferation and outcome are classified as follows: 1. Tumor associated antigens, including tumor growth factor- 尾 receptor associated proteins, are up-regulated, The expression of cell growth factor binding protein and ribonuclease-vascular growth factor inhibitor was down-regulated, while tumor rejection antigen showed a statistically significant down-regulation. In the whole cell test process, the expression of relatively constant .2 lymphocyte metabolism-related nuclear related proteins, including ubiquitin carboxyl terminal hydrolase, Both disulfide isomerase protein and glutathione S-transferase showed down-regulated expression, including heterogeneous ribonucleoprotein K and G1 phase r-S phase transition control protein. The expression of nuclear RNA helicase was up-regulated, including proteasome activator PA28, which was down-regulated in 0h and 816h, respectively. The protein expression showed a downward trend in the 24-hour range. This study provides a theoretical basis for the further study of lymphocytes damaged by ionizing radiation.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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