本文選題：UCH37 + 膠質(zhì)瘤��； 參考：《第二軍醫(yī)大學》2017年碩士論文

【摘要】：研究背景及目的:腦膠質(zhì)瘤是最常見的成人中樞神經(jīng)系統(tǒng)原發(fā)性難治性惡性腫瘤,發(fā)病率約5—6/10萬,是顱內(nèi)腫瘤患者主要的死亡原因。隨著人口老齡化腦膠質(zhì)瘤發(fā)病率有進一步增加的趨勢,給家庭、社會帶來沉重負擔,嚴重危害人類的健康。世界衛(wèi)生組織(World Health Organization,WHO)將膠質(zhì)瘤分為I-IV級,最高級別的膠質(zhì)母細胞瘤(Glioblastoma,GBM)是惡性程度和發(fā)病率最高的膠質(zhì)瘤,82%的惡性膠質(zhì)瘤病理為膠質(zhì)母細胞瘤。目前膠質(zhì)瘤的標準治療方案為手術(shù)切除聯(lián)合術(shù)后輔助放化療,標準化的診療使患者的總體生存時間(Overall survival,OS)在一定程度上得到延長。但因腦膠質(zhì)瘤惡性增殖、高度復發(fā)、惡性侵襲的特點,替莫唑胺(Temozolomide,,TMZ)等化療藥耐藥,化療耐受性等因素GBM患者總體療效沒有突破性進展,幾乎所有患者不可避免的復發(fā),中位生存期僅12-15個月,5年生存率僅4.7%。改善膠質(zhì)瘤患者的預后,開拓新的治療思路和藥物成為神經(jīng)外科領(lǐng)域的難題和關(guān)注點。腦膠質(zhì)瘤的發(fā)生、發(fā)展、侵襲、復發(fā)涉及很多基因和蛋白的改變。分子生物學、基因組學、蛋白組學的發(fā)展,為探索腫瘤惡性增殖的分子病理機制提供便利的工具。分子靶向治療針對腫瘤發(fā)生的關(guān)鍵分子,促進凋亡,抑制腫瘤形成,成為目前腫瘤治療研究的熱點。新的膠質(zhì)瘤病理分型將組織細胞形態(tài)和基因檢測相結(jié)合,根據(jù)不同基因分型決定不同的靶向用藥。單克隆抗體(貝伐單抗等)和小分子激酶受體抑制劑等是目前靶向藥物的主體,其特異性強,毒副作用小,較傳統(tǒng)細胞毒性化療藥物更有臨床價值。在GBM發(fā)生發(fā)展中存在多基因的突變,靶向單獨分子的療效欠理想,長期使用易產(chǎn)生耐藥性。所以闡明膠質(zhì)瘤惡性增殖的分子機制,尋找調(diào)控膠質(zhì)瘤發(fā)生發(fā)展的關(guān)鍵分子靶點非常重要�；蛐酒�、二代測序等技術(shù)和腫瘤基因數(shù)據(jù)庫為研究腫瘤發(fā)生發(fā)展的分子機制提供有力的支持。泛素-蛋白酶體系統(tǒng)是真核生物大多數(shù)細胞內(nèi)蛋白質(zhì)降解的主要途徑。去泛素化可作用于泛素化蛋白,解離出泛素分子,逆轉(zhuǎn)泛素化過程,使底物不被蛋白酶體降解。去泛素化酶可對泛素前體進行修飾加工,對蛋白酶體復合物及細胞質(zhì)內(nèi)泛素鏈進行水解,從而使泛素分子在細胞內(nèi)穩(wěn)定存在和循環(huán)利用。去泛素化酶參與許多重要的生物過程,如細胞周期調(diào)控、DNA損傷修復、核染色質(zhì)重組、調(diào)節(jié)細胞信號通路等。泛素化和去泛素化的相互作用參與調(diào)節(jié)癌基因、抑癌基因、致癌信號通路關(guān)鍵分子等蛋白的降解和穩(wěn)定,調(diào)控腫瘤的發(fā)生、發(fā)展、遷移、侵襲、復發(fā)等過程。泛素羧基末端水解酶家族(Ubiquitin Carboxy Terminal Hydrolases,UCHs)是一種重要的去泛素化酶,包括UCH-L1、UCH-L3、UCH37、BRCA1相關(guān)蛋白1(BRCA1 Associated Protein 1,BAP1)。研究表明UCH37和卵巢癌、多發(fā)性骨髓瘤、肝癌等惡性腫瘤惡性增殖關(guān)系密切,促進腫瘤的復發(fā)并導致預后不佳,但是尚無報道關(guān)于UCH37和腦膠質(zhì)瘤惡性增殖關(guān)系的實驗研究。因此,本研究的目的是利用多樣本信息驗證UCH37在膠質(zhì)瘤中的差異表達和對膠質(zhì)瘤預后判斷的價值,并利用體外實驗探索UCH37在膠質(zhì)瘤惡性增殖中的作用機制。本論文分為兩部分:第一部分腦膠質(zhì)瘤組織芯片中UCH37基因表達及與患者預后相關(guān)性驗證收集來自上海長征醫(yī)院經(jīng)過病理證實的300例腦膠質(zhì)瘤樣本及16例正常腦組織對照樣本,所有樣本制備成高通量組織芯片。收集入組的患者的臨床資料并對患者術(shù)后放化療及生存時間進行隨訪,結(jié)合免疫組化分析UCH37表達對患者預后的影響。截止隨訪結(jié)束,腦膠質(zhì)瘤患者總體生存期為31.96±26.82個月,無進展生存期為29.19±25.96個月;患者的存活率為39.1%,35.6%患者腫瘤未見影像學進展。免疫組化的結(jié)果顯示UCH37在膠質(zhì)瘤組織表達明顯,對比正常腦組織在腦膠質(zhì)瘤中表達顯著增高。免疫組化UCH37在膠質(zhì)瘤組織中表達評分1.905±1.935,在正常腦組織中評分為0.42±1.04,存在顯著統(tǒng)計學意義(p0.0010)。通過Kaplan-Meier生存曲線研究UCH37表達和患者生存期發(fā)現(xiàn),高、低表達UCH37的膠質(zhì)瘤患者中位OS分別為20個月、40個月,中位無進展生存時間(Progression Free Survival,PFS)分別為19個月、30個月,高表達組的OS、PFS均明顯較低表達組縮短(OS:p0.001;PFS:p0.001)。進一步在原發(fā)膠質(zhì)母細胞瘤患者中研究發(fā)現(xiàn)UCH37顯著高表達于原發(fā)GBM組織(p0.001),高表達UCH的原發(fā)GBM生存期較低表達的短(OS:10個月vs12個月,p0.001;PFS:11個月vs 8個月,p0.001)。Cox多因素風險回歸發(fā)現(xiàn)UCH37是原發(fā)GBM患者OS(HR=2.050,95%CI 1.243-3.380,p=0.005)及PFS(HR=2.135,95%CI 1.291-3.531,p=0.003)預后判斷的獨立因子。接受化療原發(fā)GBM患者中UCH37高表達組生存期較低表達組均縮短(OS:9個月vs15個月,p0.001;PFS:7個月vs11個月,p0.001),接受放療的原發(fā)GBM患者中UCH37高表達組生存期較低表達縮短(OS:10個月vs15個月,p0.001;PFS:7個月vs12個月,p0.001)。結(jié)果提示UCH37表達和原發(fā)GBM患者對放化療的敏感性具有相關(guān)性。第二部分:UCH37基因調(diào)控腦膠質(zhì)瘤惡性增殖的機制研究為研究UCH37在膠質(zhì)瘤增殖過程中發(fā)揮的細胞學功能,我們利用慢病毒轉(zhuǎn)染的方法在腦膠質(zhì)瘤細胞系U87中對UCH37進行干擾和過表達,并設置空載質(zhì)粒作為對照。利用二代測序方法驗證UCH37干擾和過表達的序列,通過定量聚合酶鏈式反應(Polymerase Chain Reaction,PCR)實驗和Western Blot實驗驗證UCH37干擾和過表達后的m RNA和蛋白的表達效果,流式細胞儀檢測細胞周期比例變化,通過CCK-8法繪制細胞增殖曲線,克隆形成率實驗檢測克隆能力。我們發(fā)現(xiàn)與空載質(zhì)粒的對照組相比,UCH37過表達后膠質(zhì)瘤細胞增殖、克隆能力顯著提高;UCH37敲減后膠質(zhì)瘤細胞增殖及克隆能力明顯下降,且細胞周期被阻滯在S期。結(jié)論:綜上所述,我們通過分析組織芯片結(jié)合臨床組織樣本篩選,驗證基因UCH37表達于腦膠質(zhì)瘤組織中并和患者臨床預后具有相關(guān)性,可判斷膠質(zhì)瘤患者臨床預后,并是原發(fā)GBM患者預后的獨立預測因子并影響原發(fā)GBM患者對放化療的敏感性。通過體外實驗驗證UCH37可促進膠質(zhì)瘤細胞的惡性增殖。因此,UCH37可作為預測腦膠質(zhì)瘤患者臨床預后的分子靶點而進行深入的研究。
[Abstract]:Background and objective: glioma is the most common primary intractable malignant tumor in the adult central nervous system. The incidence of the disease is about 5 - 6/10 million. It is the main cause of death in the patients with intracranial tumors. With the increasing trend of the incidence of brain glioma in the population, it has brought a heavy burden to the family and society, which seriously endangers human beings. Health. The WHO (World Health Organization, WHO) divides the glioma into the I-IV grade, the highest grade of glioblastoma (Glioblastoma, GBM) is the highest malignancy and the highest incidence of glioma, and 82% of the malignant glioma is glioblastoma. The standard treatment for glioma is currently assisted by surgical resection combined with surgery. The overall survival time (Overall survival, OS) of patients was extended to a certain extent by radiotherapy and chemotherapy. However, the overall efficacy of GBM patients was almost no breakthrough due to the malignant proliferation, high recurrence, malignant invasion of glioma, the drug resistance of temozolomide (Temozolomide, TMZ) and other factors in chemical therapy. All patients have an inevitable recurrence. The median survival time is only 12-15 months. The 5 year survival rate is only 4.7%. to improve the prognosis of the glioma patients. The development of new treatment ideas and drugs has become a difficult problem and concern in the Department of neurosurgery. The occurrence, development, invasion, and recurrence of glioma involve many changes in genes and proteins. Molecular biology, genome The development of proteomics provides a convenient tool for the exploration of the molecular pathological mechanism of malignant tumor proliferation. Molecular targeting therapy is a hot spot in the research of tumor therapy, which is the key molecule of cancer, promoting apoptosis and inhibiting the formation of tumor. Different genotyping determines different targeting drugs. Monoclonal antibodies (bevacizumab) and small molecular kinase receptor inhibitors are the main body of target drugs at present. They are more specific and less toxic and side effects. They are more valuable than traditional cytotoxic chemotherapeutic drugs. There are multiple gene mutations in the development of GBM and the therapy target to individual molecules. Therefore, it is very important to elucidate the molecular mechanism of malignant proliferation of glioma and to find the key molecular targets to regulate the development of glioma. The gene chip, two generation sequencing and the tumor gene database provide strong support for the molecular mechanism of tumor development. The body system is the main pathway of protein degradation in most cells in eukaryotes. Deubiquitination can act on ubiquitin protein, dissociate ubiquitin molecules, reverse the ubiquitination process, and degrade the substrate by proteasome. Deubiquitination enzyme can modify the ubiquitin precursor and carry out the proteasome complex and the ubiquitin chain in the cytoplasm. Hydrolysis, thus making ubiquitin molecules stable and circulatory in cells. De ubiquitinase participates in many important biological processes, such as cell cycle regulation, DNA damage repair, nuclear chromatin recombination, regulation of cell signaling pathways, and so on. The interaction of ubiquitination and de generalization can regulate oncogene, tumor suppressor gene, and carcinogenic signal pathway. Ubiquitin Carboxy Terminal Hydrolases (UCHs) is an important deubiquitination enzyme, including UCH-L1, UCH-L3, UCH37, BRCA1 related protein 1 (BRCA1 Associated Protein 1,). H37 is closely related to malignant proliferation of malignant tumors such as ovarian cancer, multiple myeloma and liver cancer, which promotes the recurrence of tumor and leads to poor prognosis. However, there is no report about the relationship between UCH37 and glioma proliferation. Therefore, the purpose of this study is to verify the differential expression of UCH37 in glioma by using multi sample information. The prognostic value of glioma and the mechanism of UCH37 in the malignant proliferation of glioma were explored in vitro. This paper is divided into two parts: the first part is the expression of UCH37 gene in the tissue microarray of brain glioma and the correlation of the prognosis of the patients with the prognosis of the patients, which are collected from 300 cases of glioma confirmed by the Shanghai Changzheng Hospital and 1 6 normal brain tissue control samples, all samples were prepared into high throughput tissue chips. The clinical data of the patients in the group were collected and followed up for postoperative radiotherapy and chemotherapy and survival time. The effect of UCH37 expression on the prognosis of the patients was analyzed with immunohistochemical analysis. The total survival period of the patients with glioma was 31.96 + 26.82 months by the end of the follow-up. The survival rate of progression free was 29.19 + 25.96 months, the survival rate of the patients was 39.1%, and there was no imaging progress in the 35.6% patients. The results of immunohistochemistry showed that the expression of UCH37 in glioma tissues was obvious, and the expression of normal brain tissue in glioma was significantly higher than that of normal brain tissue. The expression of immuno group UCH37 in glioma tissues was 1.905 + 1.935. The score of the normal brain tissue was 0.42 + 1.04, and there was a significant statistical significance (p0.0010). Through the Kaplan-Meier survival curve, the UCH37 expression and the patient's survival time were found. The middle OS of the patients with glioma with high and low expression of UCH37 was 20 months, 40 months, and the median progression free survival time (Progression Free Survival, PFS) was 19 months, 30 months, respectively. The OS and PFS in the high expression group were significantly shorter in the lower expression group (OS:p0.001; PFS:p0.001). Further studies in the patients with primary glioblastoma showed that UCH37 was highly expressed in the primary GBM tissue (p0.001), and the primary GBM survival period of the high expression UCH was shorter (OS:10 month vs12 month, p0.001; 8 months of UCH months) Factor risk regression found that UCH37 was an independent factor in predicting the prognosis of OS (HR=2.050,95%CI 1.243-3.380, p=0.005) and PFS (HR=2.135,95%CI 1.291-3.531, p=0.003) in the primary GBM patients. The low expression of UCH37 high expression group was shortened in the high expression group of GBM patients with radiotherapy (OS:10 months vs15 months, p0.001; PFS:7 month vs12 months, p0.001). The results suggest that the expression of UCH37 expression and the primary GBM patients are related to the sensitivity of radiotherapy and chemotherapy. The second part: the mechanism of UCH37 gene regulating the malignant proliferation of glioma is to study UCH37 In the proliferation process of glioma, we used the method of lentivirus transfection to interfere and overexpress UCH37 in the glioma cell line U87, and set the empty plasmid as the control. The two generation sequencing method was used to verify the UCH37 interference and overexpressed sequence, and the quantitative polymerase chain reaction (Polymerase Chain) was used. Reaction, PCR) experiment and Western Blot test verified the expression of M RNA and protein after UCH37 interference and overexpression. Flow cytometry was used to detect cell cycle ratio. Cell proliferation curve was plotted by CCK-8 method and clone formation rate test was used to detect cloning ability. The proliferation and cloning ability of the tumor cells increased significantly, and the proliferation and clone ability of glioma cells decreased significantly after UCH37 knockout, and the cell cycle was blocked in the S phase. Conclusion: in summary, we screened the tissue chip and clinical tissue samples to verify that the gene UCH37 was expressed in the brain glioma tissue and had a phase with the patient's clinical prognosis. The clinical prognosis of the patients with glioma can be judged and the independent predictor of the prognosis of the primary GBM patients and the sensitivity of the primary GBM patients to radiotherapy and chemotherapy. It is proved that UCH37 can promote the malignant proliferation of glioma cells through in vitro experiments. Therefore, UCH37 can be used as a molecular target to predict the clinical prognosis of glioma patients. Research.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

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本文編號：1828032