脑胶质母细胞瘤血浆差异蛋白质分析

发布时间：2018-03-16 04:00

  本文选题：胶质母细胞瘤　切入点：蛋白质组　出处：《中国人民解放军军事医学科学院》2014年硕士论文　论文类型：学位论文

【摘要】：背景： 胶质瘤是由神经外胚层分化而来的胶质细胞(星形胶质细胞、少突胶质细胞和室管膜细胞等)所形成的肿瘤，是神经系统最常见的恶性肿瘤。胶质瘤发病率占恶性脑肿瘤的80%。它的治愈率随着胶质瘤恶性程度的增加而逐步降低。恶性程度最高的胶质母细胞瘤(WHO Ⅳ级)患者的平均生存时间只有12-15个月。这与其他肿瘤治疗后5-10年的中位生存期相比差距太大。此外，胶质瘤很容易复发，且复发时经常伴有肿瘤恶性等级的升高，，对放、化疗药物敏感性降低或者耐药性的产生。 目前肿瘤的诊断主要依赖影像学和组织病理学。由于胶质瘤影像复杂、多样及不典型性,影像学诊断虽然能够直观的显示肿瘤的存在，但不能明确肿瘤的类型及分期。组织病理学是目前判断肿瘤类型的常规手段,但组织活检具有创伤性,难以实现早期诊断和对疗效的实时监控。同时，由于组织病理学的判断具有很大的主观性，不同的病理学医生对同一标本的判读经常会出现偏差。如果能够从外周血中探索出胶质瘤特异的蛋白分子，无疑将可以在预后判断、疗效评价以及复发监测等方面提供更多的信息，以协助临床医生更好的实现患者的个体化治疗。 患者的外周血标本虽然获取容易，但是血浆中的蛋白质很难分离和鉴别，质谱技术在这方面具有非常明显的优势。应用二维凝胶电泳（2DE）结合超高效纳升液相色谱电喷雾串联质谱(Nano-UPLC-nano-ESI-MS/MS)，在血浆中能够分离并鉴定出近2000种蛋白质和多肽。这使得从血浆中筛选出胶质瘤特异性的标志蛋白成为可能。目的： 本研究应用比较蛋白质组技术，通过对原发性脑胶质母细胞瘤病人和健康人血浆蛋白质组的分离、分析和比较，寻找脑胶质母细胞瘤特异性的血浆蛋白标志物。为脑胶质母细胞瘤发病机理的阐明、靶向治疗药物的筛选和研发，以及脑胶质母细胞病人的早期诊断、预后判断、治疗方案选择、疗效评价、复发检测提供有意义的帮助。 方法： 从原发脑胶质母细胞瘤病人与健康体检者空腹血浆标本中分别随机盲选出6例标本。先应用Bradford法测蛋白质含量，并去除非蛋白质组分。再采用二维凝胶电泳（2DE）技术分离差异蛋白质点，凝胶银染色后用BIO-RAD GS-800凝胶成像仪扫描图像。获得的图像经PDQuest Advanced-8.0.1分析软件分析出差异超过2倍的差异点。差异点经过胶内酶切，应用超高效纳升液相色谱-电喷雾串联质谱(NanoUPLC-nano-ESI-MS/MS)技术分离鉴定。经软件PLGS v2.3处理后应用Mascot软件按照MS/MS离子方式检索IPI(InternationalProtein Index)Database数据库。经过数据的比较分析筛选并鉴定出表达差异的蛋白质。 结果： 通过2DE分离两组血浆蛋白质，经过软件分析，共筛选出表达量差异大于两倍的４个差异蛋白质点。经过NanoUPLC-nano-ESI-MS/MS质谱分离并鉴定，Mascot软件分析数据，得到23个表达上调的差异蛋白质。包括两个未命名蛋白质；人血清白蛋白R218h突变体与甲状腺素复合物A链；人血清白蛋白与肉豆蔻酸盐、阿扎丙宗结合物A链；人血清白蛋白与肉豆蔻酸盐、阿司匹林结合物A链；载脂蛋白D；载脂蛋白前蛋白原；补体C3；溶菌酶前体；第77位氨基酸由半胱氨酸突变为丙氨酸的人溶菌酶A链；第96位和109位氨基酸双突变的人溶菌酶；S100蛋白-A7；S100蛋白-A8；人谷氨酸脱羧酶65单克隆抗体；与疾病有关的抗人细胞集落刺激因子自身抗体；组织相容性符合物人类白细胞抗原A-A1抗多肽/黑色素瘤抗原A1单克隆抗体；与人白介素15结合的抗白介素15抗体；本周氏蛋白；IGL@protein；人Toll3受体四元复合物结合3个Fab片段；神经存活因子（抗菌肽）前蛋白原；抗菌肽2抗菌变体；血小板碱性蛋白前蛋白原。 结论： 本研究首次应用了成熟灵敏的二维凝胶电泳技术，和最先进的Nano UPLC-nano-ESI-MS/MS质谱技术。对胶质瘤中恶性程度最大的胶质母细胞瘤血浆标本进行鉴定。本次共筛选出23个胶质母细胞瘤与健康人具有差异的血浆差异蛋白。他们可能共同参与了胶质母细胞瘤的调控过程。多种差异蛋白的结合可能构成胶质母细胞瘤特异性的诊断模型。对胶质母细胞瘤的早期诊断，预后判断、治疗方案的选择、疗效评价、复发监测等方面提供有益的帮助。
[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.
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