本文選題：膠質(zhì)母細(xì)胞瘤　切入點：蛋白質(zhì)組　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景： 膠質(zhì)瘤是由神經(jīng)外胚層分化而來的膠質(zhì)細(xì)胞(星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞和室管膜細(xì)胞等)所形成的腫瘤，是神經(jīng)系統(tǒng)最常見的惡性腫瘤。膠質(zhì)瘤發(fā)病率占惡性腦腫瘤的80%。它的治愈率隨著膠質(zhì)瘤惡性程度的增加而逐步降低。惡性程度最高的膠質(zhì)母細(xì)胞瘤(WHO Ⅳ級)患者的平均生存時間只有12-15個月。這與其他腫瘤治療后5-10年的中位生存期相比差距太大。此外，膠質(zhì)瘤很容易復(fù)發(fā)，且復(fù)發(fā)時經(jīng)常伴有腫瘤惡性等級的升高，，對放、化療藥物敏感性降低或者耐藥性的產(chǎn)生。 目前腫瘤的診斷主要依賴影像學(xué)和組織病理學(xué)。由于膠質(zhì)瘤影像復(fù)雜、多樣及不典型性,影像學(xué)診斷雖然能夠直觀的顯示腫瘤的存在，但不能明確腫瘤的類型及分期。組織病理學(xué)是目前判斷腫瘤類型的常規(guī)手段,但組織活檢具有創(chuàng)傷性,難以實現(xiàn)早期診斷和對療效的實時監(jiān)控。同時，由于組織病理學(xué)的判斷具有很大的主觀性，不同的病理學(xué)醫(yī)生對同一標(biāo)本的判讀經(jīng)常會出現(xiàn)偏差。如果能夠從外周血中探索出膠質(zhì)瘤特異的蛋白分子，無疑將可以在預(yù)后判斷、療效評價以及復(fù)發(fā)監(jiān)測等方面提供更多的信息，以協(xié)助臨床醫(yī)生更好的實現(xiàn)患者的個體化治療。 患者的外周血標(biāo)本雖然獲取容易，但是血漿中的蛋白質(zhì)很難分離和鑒別，質(zhì)譜技術(shù)在這方面具有非常明顯的優(yōu)勢。應(yīng)用二維凝膠電泳（2DE）結(jié)合超高效納升液相色譜電噴霧串聯(lián)質(zhì)譜(Nano-UPLC-nano-ESI-MS/MS)，在血漿中能夠分離并鑒定出近2000種蛋白質(zhì)和多肽。這使得從血漿中篩選出膠質(zhì)瘤特異性的標(biāo)志蛋白成為可能。目的： 本研究應(yīng)用比較蛋白質(zhì)組技術(shù)，通過對原發(fā)性腦膠質(zhì)母細(xì)胞瘤病人和健康人血漿蛋白質(zhì)組的分離、分析和比較，尋找腦膠質(zhì)母細(xì)胞瘤特異性的血漿蛋白標(biāo)志物。為腦膠質(zhì)母細(xì)胞瘤發(fā)病機(jī)理的闡明、靶向治療藥物的篩選和研發(fā)，以及腦膠質(zhì)母細(xì)胞病人的早期診斷、預(yù)后判斷、治療方案選擇、療效評價、復(fù)發(fā)檢測提供有意義的幫助。 方法： 從原發(fā)腦膠質(zhì)母細(xì)胞瘤病人與健康體檢者空腹血漿標(biāo)本中分別隨機(jī)盲選出6例標(biāo)本。先應(yīng)用Bradford法測蛋白質(zhì)含量，并去除非蛋白質(zhì)組分。再采用二維凝膠電泳（2DE）技術(shù)分離差異蛋白質(zhì)點，凝膠銀染色后用BIO-RAD GS-800凝膠成像儀掃描圖像。獲得的圖像經(jīng)PDQuest Advanced-8.0.1分析軟件分析出差異超過2倍的差異點。差異點經(jīng)過膠內(nèi)酶切，應(yīng)用超高效納升液相色譜-電噴霧串聯(lián)質(zhì)譜(NanoUPLC-nano-ESI-MS/MS)技術(shù)分離鑒定。經(jīng)軟件PLGS v2.3處理后應(yīng)用Mascot軟件按照MS/MS離子方式檢索IPI(InternationalProtein Index)Database數(shù)據(jù)庫。經(jīng)過數(shù)據(jù)的比較分析篩選并鑒定出表達(dá)差異的蛋白質(zhì)。 結(jié)果： 通過2DE分離兩組血漿蛋白質(zhì)，經(jīng)過軟件分析，共篩選出表達(dá)量差異大于兩倍的４個差異蛋白質(zhì)點。經(jīng)過NanoUPLC-nano-ESI-MS/MS質(zhì)譜分離并鑒定，Mascot軟件分析數(shù)據(jù)，得到23個表達(dá)上調(diào)的差異蛋白質(zhì)。包括兩個未命名蛋白質(zhì)；人血清白蛋白R218h突變體與甲狀腺素復(fù)合物A鏈；人血清白蛋白與肉豆蔻酸鹽、阿扎丙宗結(jié)合物A鏈；人血清白蛋白與肉豆蔻酸鹽、阿司匹林結(jié)合物A鏈；載脂蛋白D；載脂蛋白前蛋白原；補(bǔ)體C3；溶菌酶前體；第77位氨基酸由半胱氨酸突變?yōu)楸彼岬娜巳芫窤鏈；第96位和109位氨基酸雙突變的人溶菌酶；S100蛋白-A7；S100蛋白-A8；人谷氨酸脫羧酶65單克隆抗體；與疾病有關(guān)的抗人細(xì)胞集落刺激因子自身抗體；組織相容性符合物人類白細(xì)胞抗原A-A1抗多肽/黑色素瘤抗原A1單克隆抗體；與人白介素15結(jié)合的抗白介素15抗體；本周氏蛋白；IGL@protein；人Toll3受體四元復(fù)合物結(jié)合3個Fab片段；神經(jīng)存活因子（抗菌肽）前蛋白原；抗菌肽2抗菌變體；血小板堿性蛋白前蛋白原。 結(jié)論： 本研究首次應(yīng)用了成熟靈敏的二維凝膠電泳技術(shù)，和最先進(jìn)的Nano UPLC-nano-ESI-MS/MS質(zhì)譜技術(shù)。對膠質(zhì)瘤中惡性程度最大的膠質(zhì)母細(xì)胞瘤血漿標(biāo)本進(jìn)行鑒定。本次共篩選出23個膠質(zhì)母細(xì)胞瘤與健康人具有差異的血漿差異蛋白。他們可能共同參與了膠質(zhì)母細(xì)胞瘤的調(diào)控過程。多種差異蛋白的結(jié)合可能構(gòu)成膠質(zhì)母細(xì)胞瘤特異性的診斷模型。對膠質(zhì)母細(xì)胞瘤的早期診斷，預(yù)后判斷、治療方案的選擇、療效評價、復(fù)發(fā)監(jiān)測等方面提供有益的幫助。
[Abstract]:Background:
Glioma is from neuroectodermal glial cells to differentiation (astrocytes, oligodendrocytes and ependymal cells) formed by tumor is the most common malignant tumors in the central nervous system. 80%. glioma incidence rate of malignant brain tumors and its cure rate increased with the malignant degree of glioma and gradually reduce at the highest level of malignant glioblastoma (WHO IV) the average survival time of only 12-15 months. The median survival time compared with the other 5-10 years after cancer treatment the gap is too big. In addition, glioma is easy recurrence, and recurrence of malignant tumor is often accompanied by increased levels of. The sensitivity to chemotherapeutic drugs or reduce drug resistance.
The tumor diagnosis mainly depends on image and pathology of glioma. Because the image is complex, diverse and atypical, imaging diagnosis can show the presence of a tumor directly, but can not clear the tumor type and stage. Histopathology is the judgment of conventional means tumor type, but biopsy is traumatic and it is difficult to achieve early diagnosis and real-time monitoring of the effect. At the same time, because it is very subjective histopathological judgment, different pathologists interpretation on the same specimens often appear deviation. If to explore glioma specific protein from peripheral blood, will undoubtedly can in the prognosis. On the evaluation of the curative effect and recurrence monitoring to provide more information to realize the individualized treatment of patients to assist clinicians better.
Patients with peripheral Xuebiao although this is easy to obtain, but it is difficult to plasma protein separation and identification, mass spectrometry technology has obvious advantages in this aspect. The application of two-dimensional gel electrophoresis (2DE) combined with ultra high performance nano liquid chromatography electrospray tandem mass spectrometry (Nano-UPLC-nano-ESI-MS/MS), can be separated and identified nearly 2000 proteins and peptide in plasma. The plasma from the selected marker of glioma specific protein is possible:
The application of comparative proteomics, the primary glioblastoma patients and healthy human plasma proteome separation, analysis and comparison of plasma protein markers for glioblastoma specific. In order to elucidate the pathogenesis of glioblastoma, targeted screening and therapeutic drug development the early diagnosis, and the patient's brain glioblastoma prognosis, treatment selection, evaluation of curative effect, recurrence detection provide significant help.
Method錛

本文編號：1618206