【作者】 王春琳；

【导师】 卢亦成；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 背景脑胶质瘤在成人中枢神经系统肿瘤中约占颅内肿瘤的40-50%,脑胶质瘤呈弥漫性、侵袭性生长,预后较差、平均生存期较短。目前以手术切除、放射治疗、化学治疗等综合性治疗。但治疗效果并不理想。特别是胶质母细胞瘤(WHOⅣ级)平均生存期约1年左右。化疗是胶质瘤治疗重要辅助手段,但相当部分胶质瘤易对化学治疗产生耐药性,无法达到理想疗效,胶质瘤细胞对化疗药物产生耐药性成为了对胶质瘤进行化疗的重大阻碍。临床上常可见相同病理级别、手术切除程度,并均行放射治疗和化学治疗的的患者预后却不尽相同。这种生存时间差异很有可能与个体对放化疗敏感程度不同相关。但目前针对人脑胶质瘤的化疗耐药相关靶标及分子机制尚未被完全阐明,现阶段认为恶性胶质瘤化疗的耐药机制主要与以下几个方面有关:(1)O~6-甲基鸟嘌呤-DNA-甲基转移酶(O~6-Methylguanine Methyltransferase,MGMT);(2)错配修复;(3)多腺苷二磷酸多聚酶(Poly(ADP-ribose)polymerase);(4)凋亡相关基因;(5)转运蛋白。我们前期工作通过对胶质瘤病例进行基因芯片检测和临床随访,根据生存时间对芯片数据进行聚类分析,并依据基因与疾病相关数据库,通过生物信息学技术筛选与胶质瘤化疗耐药相关的差异基因,这些差异表达基因可能参与了胶质瘤细胞对化疗敏感性的机制,为后续临床化疗方案的个体化制定和分子靶向化疗提供实验基础。鼠双微基因4(Mouse double minute 4,MDM4)是前期由基因芯片行生物信息分析后筛选出的在生存期长短中有显著差异,并与化疗药物相关的其中一条基因。MDM4是p53的一个关键调节因子,通过调控p53的转录功能或线粒体途径来发挥影响细胞凋亡的作用。泛素特异性蛋白酶2a(Ubiquitin-specifc protease 2a,USP2a)是一种去泛素化酶(Deubiquitinating enzymes,DUBs) ,MDM4是USP2a的一种底物。MDM4和USP2a在生存期长的临床标本中比在生存期短的临床标本中表达高,通过患者预后指标的统计学分析表明,在胶质母细胞瘤中,MDM4与患者的生存时间呈正相关;在脑胶质瘤患者的临床标本和细胞株中,MDM4和USP2a能够发生免疫共沉淀形成复合物,MDM4和p53Ser46~P共定位于线粒体中;在胶质瘤细胞株U87MG中,在人工诱导凋亡条件下,MDM4促进凋亡依赖于p53线粒体通路,而非依赖于p21。USP2a具有促进MDM4稳定性的作用,在USP2a稳定MDM4后,通过p53依赖性线粒体通路促进了UV照射后的U87MG细胞凋亡。第一部分MDM4在人脑胶质母细胞瘤组织中的表达及意义目的:探讨在人脑胶质母细胞瘤中MDM4的mRNA水平表达及蛋白水平表达及意义。方法:采用实时荧光定量PCR的方法检测MDM4的mRNA表达水平,通过Western blot检测MDM4的蛋白表达水平。并采用免疫组化的方法检测MDM4在胶质母细胞瘤中的表达,并分析MDM4的蛋白表达与患者生存时间的关系及意义。结果:1、在人脑胶质母细胞瘤中,MDM4基因的实时荧光定量PCR结果显示在预后较差的患者胶质瘤组织和预后较好的胶质瘤标本中MDM4 mRNA相对表达水平无显著性差异;2、蛋白免疫印迹提示预后良好组中MDM4的蛋白表达较预后较差组高;在生存期长的患者样本的线粒体中MDM4表达比生存期短的患者高,线粒体及胞质中p53Ser46磷酸化升高,线粒体中细胞色素C(Cytochrome C,Cytc)表达减少;3、在人脑胶质母细胞瘤中USP2a与MDM4能够在胞质中发生免疫共沉淀,MDM4与p53Ser46~P能够在线粒体中发生免疫共沉淀;4、免疫组化实验发现,MDM4蛋白主要表达于阳性染色细胞(呈棕黄色显色细胞)的细胞浆中,MDM4高表达的患者生存期较低表达的患者生存期长,MDM4阳性表达与USP2a和p53Ser46~P表达存在相关性,与p53未发现显著性相关;结论:1、在人脑胶质母细胞瘤中,USP2a与MDM4可能是在胞质中发生作用;MDM4与p53Ser46~P可能是在线粒体中发生作用;2、MDM4蛋白水平的上调很有可能是通过转录后调控而提高,推测在胶质瘤中MDM4上调是通过USP2a上调导致的结果,USP2a—MDM4—p53Ser46~P这条通路在人胶质母细胞瘤中可能是存在的;3、发生细胞凋亡时,线粒体外膜的通透性增加,Cytc由线粒体内部向胞质中释放进一步促进凋亡的发生;4、MDM4为胶质母细胞瘤患者预后的独立预测因子。第二部分MDM4在人脑胶质瘤细胞株U87MG中的表达及意义目的:研究MDM4在人脑胶质瘤细胞株U87MG中的表达及其与USP2a和p53等相关蛋白的作用,为进一步研究MDM4在胶质瘤中作用的分子机制提供基础。方法:采用免疫共沉淀和免疫荧光激光共聚焦的方法检测MDM4在人脑胶质母细胞瘤细胞株中的表达及其与USP2a和p53等相关蛋白的关系。结果:1、在U87MG细胞系中通过内源性免疫共沉淀(Co-IP)发现,在MDM4沉淀下来的全细胞组分(whole cell lysate,WCL)中能够检测到USP2a蛋白;在MDM4沉淀下来的线粒体蛋白组分中,未检测到USP2a蛋白,但能够检测到p53Ser46~P蛋白;用紫外线照射诱导U87MG细胞凋亡条件下,提取线粒体蛋白行内源性免疫共沉淀发现,U87MG细胞经UV照射后MDM4与Bcl-2和p53Ser46~P结合较正常细胞生长环境下明显增强;2、行U87MG细胞内激光共聚焦发现,在U87MG细胞中过表达GFP-p53、Flag-MDM4及Myc-USP2a后,MDM4在胞质中能够与p53及USP2a发生共定位;过表达Flag-MDM4后,发现MDM4与内源性p53Ser46~P也可发生共定位。结论:1、在U87MG细胞系中,MDM4与USP2a在胞质中线粒体外结合,MDM4能与p53Ser46~P在线粒体中发生结合;MDM4能与p53Ser46~P、USP2a发生共定位;2、在U87MG细胞中,USP2a与MDM4具备发生相互作用的条件,MDM4与p53Ser46~P也具备发生相互作用的条件。第三部分MDM4在人胶质瘤细胞株U87MG中对细胞凋亡的影响及作用机制研究目的:在人脑胶质瘤中研究MDM4在细胞凋亡中作用及分子通路,探讨其发挥肿瘤调控作用的机制。方法:在体外细胞系中采用慢病毒干扰及过表达各相关靶标,观察MDM4与凋亡通路中各蛋白间的关系。结果:1、在紫外线诱导U87MG细胞凋亡条件下,过表达USP2a后,细胞凋亡明显增加;敲减USP2a后,细胞凋亡减少;在过表达USP2a时敲减p53,细胞凋亡明显减少;2、在正常生长状态下的U87MG细胞中,过表达USP2a时,MDM4蛋白水平上调,但MDM2和p53无明显变化;敲减USP2a后,MDM4蛋白水平明显减低,MDM2也有所降低,但p53水平无明显降低;3、在正常生长状态下的U87MG细胞中,过表达USP2a时引起线粒体中p53和p53Ser46~P蛋白水平升高;过表达p21对线粒体中p53不产生影响,敲减p21不能阻断USP2a对线粒体中p53的作用;敲减MDM4能阻断USP2a对线粒体中p53的作用;4、在紫外线诱导U87MG细胞凋亡条件下,敲减USP2a后Bcl-2蛋白表达升高、Caspase3蛋白表达降低;而在敲减USP2a时过表达MDM4,能够阻断Bcl-2和Caspase3发生变化;5、在紫外线诱导U87MG细胞凋亡条件下,过表达MDM4后,细胞凋亡明显增加;当敲减MDM4后,细胞凋亡明显减少;6、在紫外线诱导U87MG细胞凋亡条件下,过表达MDM4同时敲减p53与单纯过表达MDM4相比,细胞凋亡比例明显减少;7、在紫外线诱导U87MG细胞凋亡条件下,敲减MDM4后,细胞线粒体中p53和p53Ser46~P表达水平明显降低,同时伴线粒体中细胞色素C明显增多、胞质中细胞色素C明显减少;8、在紫外线诱导U87MG细胞凋亡条件下,敲减p21细胞凋亡比例无明显变化;9、在紫外线诱导U87MG细胞凋亡条件下,过表达MDM4时,Bcl-2表达减少,Caspase3表达升高;而在过表达MDM4同时敲减p53时能够阻断Bcl-2和Caspase3发生变化;在过表达MDM4同时敲减p21与单纯过表达MDM4相比, Bcl-2、Caspase3表达无明显变化;过表达MDM2对Bcl-2和Caspase3无明显影响;结论:1、在紫外线诱导U87MG细胞凋亡条件下,USP2a促凋亡作用是通过p53介导的;2、在U87MG细胞正常生长状态下,USP2a对MDM4有上调作用,但对p53无明显作用,说明USP2a有稳定MDM4蛋白水平的作用,而对全细胞组分中的p53总蛋白表达并未产生明显影响;3、在U87MG细胞正常生长状态下,USP2a对线粒体中MDM4有上调作用,但对线粒体中p53和p53Ser46~P有上调作用,p21对线粒体中p53无影响。这说明USP2a对线粒体中p53的影响未通过p21发生作用,而可能通过MDM4介导对p53产生作用的;4、在紫外线诱导U87MG细胞凋亡条件下,敲减USP2a引起凋亡通路中Bcl-2表达升高、Caspase3降低,可能是通过MDM4介导发生作用的;5、在紫外线诱导U87MG细胞凋亡条件下,MDM4的表达可能与细胞凋亡呈正相关;6、在紫外线诱导U87MG细胞凋亡条件下,敲减p53能够阻断MDM4的促凋亡作用,MDM4的促凋亡作用需要通过p53来完成;7、在紫外线诱导U87MG细胞凋亡条件下,敲减MDM4后引起线粒体凋亡通路中线粒体p53、p53Ser46~P和细胞色素C的变化。这提示在致命性应激条件下,MDM4是在线粒体中与p53Ser46~P发生作用,并影响细胞色素C的改变,敲减MDM4使线粒体中p53Ser46磷酸化水平下降,细胞色素C由线粒体向外释放减少,抑制了细胞凋亡;8、在紫外线诱导U87MG细胞凋亡条件下,敲减p21对细胞凋亡无明显影响;9、在紫外线诱导U87MG细胞凋亡条件下,MDM4对凋亡相关蛋白(Bcl-2、Caspase3)产生作用是通过p53起作用而不是通过p21起作用;过表达MDM2对Bcl-2和Caspase3无明显影响;综上所述,我们认为在紫外线诱导U87MG细胞凋亡条件下,MDM4促进凋亡依赖于p53线粒体通路,而非依赖于p21。USP2a具有促进MDM4稳定性的作用,在USP2a稳定MDM4后,通过p53依赖性线粒体通路促进了UV照射后的U87细胞凋亡。更多还原

【Abstract】 BackgroundGlioma in the adult central nervous system tumors account for about 40-50% of intracranial tumors, gliomas showed diffuse, invasive growth and poor prognosis, with an average survival time is short. Current treatment programs include surgery, radiation therapy, chemotherapy, and other comprehensive treatment. But the treatment is not satisfactory. In particular, the average survival time of glioblastoma (WHOⅣ) patients were approximately 1 year.Chemotherapy is an important adjunct to the treatment of glioma, but a considerable part of glioma which easily develop resistance to chemotherapy can not achieve the desired effect, chemotherapeutic drug resistance of glioma has become the major obstacles . We Often see the prognosis for patients with the same clinical pathological grade, extent of surgical resection, and underwent radiation therapy and chemotherapy is not the same. This difference in survival time with the individual’s probably depended on sensitivity to chemotherapy. But molecular mechanisms of glioma chemotherapy resistance and related target have not yet been fully elucidated. At this stage, the mechanisms of resistance to chemotherapy of malignant glioma are as follows: ( 1 ) O~6-Methylguanine Methyltransferase(MGMT);(2) Mismatch Repair;(3) Poly(ADP-ribose)polymerase;(4)Apoptosis-Regulating Genes;(5)Transporter Proteins.Our previous work carried out some genes which were associated with chemotherapy resistance by gene chip detection of glioma cases and clinical follow-up, MDM4 (Mouse double minute 4) is one of chemotherapy-related genes which were selected from the gene chip analysis of biological information.MDM4 is a key regulator of p53, which was affected on apoptosis through regulation of p53 transcriptional function or the mitochondrial pathway. USP2a (Ubiquitin-specifc protease 2a) is one of Deubiquitinating enzymes(DUBs), MDM4 is a substrate of USP2a. the protein expressions of MDM4 and USP2a in the clinical specimens from longer survival patients were higher than that in clinical specimens from shorter survival patients. In clinical glioma specimens and cell lines, MDM4 and USP2a can happen immunoprecipitation and form complex. MDM4 and p53Ser46~P were co-localized in the mitochondria. In the artificial induction of apoptosis conditions, MDM4 promote mitochondrial pathway of apoptosis depends on p53, but not p21. USP2a can promote stability of MDM4.Part I: Study on the expression and significance of MDM4 in human glioblastomaObjective: To investigate the expression and significance of MDM4 mRNA and protein in brain glioblastoma.Methods: The mRNA expression of MDM4 in 8 cases of human glioblastoma tissues were detected with quantitative real-time PCR. The protein expression in these samples were detected using Western blot. The protein location and relationship of MDM4 and prognosis of patients were detected by IHC.Results: The expression of MDM4 mRNA was no difference in patient of poor prognosis and in patient of good prognosis. The expression of MDM4 protein is higher in patient of good prognosis than in patient of poor prognosis. Survival in patients with MDM4 high expression were longer than in patients with MDM4 low expression. The positive expression of MDM4 was associated with USP2a,but not p53.Conclusion: In human glioblastoma of brain, USP2a and MDM4 may take effect with each other in the cytoplasm; MDM4 and p53Ser46~P may take effect with each other in the mitochondria. The elevated levels of MDM4 protein expression may be due to post-transcriptional regulation by USP2a. MDM4 was an independent predictor of prognosis of glioblastoma patients.PartⅡ: Study on the expression of MDM4 and relationship with USP2a and p53 in human glioblastoma cell line U87MGObjective: To detect the expression of MDM4 and relationship with USP2a and p53.Methods: By immunoprecipitation and confocal immunofluorescence was used to detect the expression of MDM4 and relationship with USP2a and p53.Results: In the U87MG cell line, MDM4 could combined with USP2a in the cytoplasm, MDM4 combined with p53Ser46~P in the mitochondria; MDM4 was co-localized with p53Ser46~P, USP2a. Conclusion: USP2a and MDM4 could interact with each other, and MDM4 and p53Ser46~P could also interact with each other in the U87MG cell line.Part III Study on effects of MDM4 on apoptosis of U87MG and mechanism of itObjective: To investigate effects of MDM4 on apoptosis of U87MG and to explore the mechanism of regulating tumors.Methods: In vitro cell lines, to observe the relationship between MDM4 and the various proteins on the apoptosis pathway using lentiviral interference and overexpression of relevant target.Results: 1. Under the conditions of UV induced U87MG cells apoptosis, after overexpression of USP2a, apoptosis increased significantly; after USP2a knocked down, apoptosis decreased; p53 knocked down can block the effect of it. 2. Under condition of normal growth of U87MG cells, after overexpression of USP2a, the expression of MDM4 protein increased significantly; after USP2a knocked down, the expression of MDM4 protein decreased. 3. Under condition of normal growth of U87MG cells, after overexpression of USP2a , the expression of p53 and p53Ser46~P proteins in the mitochondria increased; MDM4 knocked down can block the effect of it,but not p21. 4. Under the condition of UV induced U87MG cells apoptosis, after USP2a knocked down, the expression of Bcl-2 protein increased and the expression of Caspase3 protein decreased; Overexpression of MDM4 can block the effect of it. 5. Under the condition of UV induced U87MG cells apoptosis, after overexpression of MDM4, apoptosis increased significantly; after MDM4 knocked down, apoptosis decreased; p53 knocked down can block the effect of it. 6. Under the condition of UV induced U87MG cells apoptosis, after USP2a knocked down, the expression of p53, p53Ser46~P and Cytochrome C proteins in the mitochondria increased and the expression of Cytochrome C protein which was outside of mitochondria decreased.7. Under the condition of UV induced U87MG cells apoptosis, after p21 knocked down, apoptosis did not change significantly. 8. Under the condition of UV induced U87MG cells apoptosis, after overexpression of MDM4 , the expression of Bcl-2 protein decreased and the expression of Caspase3 protein increased; p53 knocked down can block the effect of it,but not p21. After Overexpression of MDM2, the expression of Bcl-2 protein and Caspase3 protein did not change significantly.Conclusion: Under the condition of UV induced U87MG cells apoptosis, MDM4 could promote apoptosis by its effects on p53 mitochondrial pathway; USP2a could promote stability of MDM4.更多还原