本文關(guān)鍵詞： 膠質(zhì)瘤 著絲粒蛋白W CENP-W CUG2　出處：《南昌大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的： 腦膠質(zhì)瘤是最為常見惡性腦腫瘤，它是一組能向膠質(zhì)細胞分化特征功能的神經(jīng)上皮細胞腫瘤總稱，發(fā)病率占顱內(nèi)腫瘤全部的50%-60%。近年來，盡管各種不同治療方法綜合應(yīng)用，臨床療效有一定提高，但生存時間仍較短及生活質(zhì)量不高，絕大多數(shù)患者最終都會出現(xiàn)腫瘤復(fù)發(fā)及轉(zhuǎn)移�，F(xiàn)有研究表明，基因中致癌過表達與抑癌失活緊密相關(guān)，而現(xiàn)如今對如何致癌的分子機制不明。 CENP-W于2007年首次被報道，其屬于CCAN（constitutive centromereassociated network）家族，起初因發(fā)現(xiàn)在多種癌性組織及小鼠成纖維細胞具有高致瘤性故以一個假定的致癌基因并命名為CUG2（Cancer-upregulated gene2）；同時CENP-W在中樞神經(jīng)系統(tǒng)發(fā)育中扮演重要角色并參與神經(jīng)元的功能維持，敲除CENP-W可導(dǎo)致斑馬魚胚胎神經(jīng)退行性變。國內(nèi)外尚未深入探討CENP-W在腦膠質(zhì)瘤表達及與其生存預(yù)后之間的關(guān)系。 本研究擬初步探討CENP-W在膠質(zhì)瘤組織及細胞株中的表達與否、以及表達程度與病理級別（WHO分級）的關(guān)系，同時分析CENP-W的表達與膠質(zhì)瘤患者生存時間預(yù)后關(guān)系，為下一步研究CENP-W在腦膠質(zhì)瘤發(fā)生、發(fā)展過程可能所參與調(diào)節(jié)過程，為進一步在治療腦膠質(zhì)瘤方面提供一個新的方向。 方法：本實驗設(shè)計分為三步 1.第一步通過Real-Time PCR、Western blotting、免疫組化檢測對照組中正常腦組織（非瘤性腦組織）、實驗組中4個不同病理級別腦膠質(zhì)瘤的CENP-WmRNA與蛋白表達差異，收集數(shù)據(jù)并應(yīng)用統(tǒng)計學(xué)方法進行分析。 2.第二步應(yīng)用Western blotting檢測方法檢測A172、LN308、U118、U87、U251、LN229、SNB19、TJ899、TJ905九類膠質(zhì)瘤細胞株中CENP-W與GAPDH蛋白表達對比情況，收集數(shù)據(jù)并應(yīng)用統(tǒng)計學(xué)方法進行分析。 3.第三步所收集腫瘤標本的患者均行術(shù)后隨訪資料，將腫瘤患者各項相關(guān)因素收集后進行相關(guān)統(tǒng)計學(xué)分析，，并分析腦膠質(zhì)瘤組織中CENP-W的表達與患者的預(yù)后生存之間的關(guān)系。 結(jié)果： 1、RT-PCR熔解曲線能分析到CENP-W和β-actin分別只有一個單峰，結(jié)果較好。mRNA表達水平：正常組織（1.0±0.19），I級（3.3±0.31），II級（5.4±0.43），III級（3.5±0.42），IV級（8.2±0.49）。CENP-W在不同病理級別的腦膠質(zhì)瘤組織中與正常腦組織組對比，差異具有統(tǒng)計學(xué)意義。同時高級別的膠質(zhì)瘤中CENP-W mRNA的表達水平明顯高于I、II、III級膠質(zhì)瘤。 2、CENP-W在腦膠質(zhì)瘤組織中蛋白相對表達量結(jié)果：正常（0.46±0.062），I型（0.66±0.099），II型（0.85±0.069），III型（0.70±0.05），IV型（1.02±0.058），人腦膠質(zhì)瘤組織中CENP-W蛋白相對表達量高，病理IV級的蛋白相對表達量是正常對照組2倍左右。 3、總蛋白主要從上述細胞株中提取，并與內(nèi)參GAPDH對比，其中A172（0.89±0.11）、U87（0.71±0.08）、U251（0.71±0.07）、TJ899（0.83±0.09）、TJ905（1.08±0.11）5種膠質(zhì)瘤細胞株較HEK293組（0.24±0.08）相對蛋白表達量明顯增高，LN308（0.46±0.07）、LN229（0.39±0.09）則差異性較柔和，而U118（0.21±0.07）、SNB19（0.22±0.12）較HEK293相對表達量��；由上結(jié)果在大部分膠質(zhì)瘤細胞株內(nèi)CENP-W呈中-高表達。利用灰度軟件進行掃描對比，P0.05差異具有統(tǒng)計學(xué)意義。 4、在人腦膠質(zhì)瘤中CENP-W進一步用免疫組化方法進行驗證。結(jié)果顯示CENP-W在膠質(zhì)瘤中表達陽性，細胞核呈陽性。而正常腦組織中表達陰性。利用卡方檢驗分析CENP-W與兩組膠質(zhì)瘤關(guān)系，P=0.0140.05，差異具有統(tǒng)計學(xué)意義。統(tǒng)計結(jié)果示在腦膠質(zhì)瘤中CENP-W陽性率74.0%，其中陽性表達中高惡性組占81.4%，低惡性組占18.6%。 5、統(tǒng)計分析膠質(zhì)瘤患者預(yù)后各項因素，從多因素分析上看，其中Grade、CENP-W的P值分別為0.00和0.005，故Grade、CENP-W對膠質(zhì)瘤患者的生存時間存在相關(guān)性顯著，余因素對生存預(yù)后影響不大。高惡性組較低惡性組的預(yù)后明顯差。CENP-W表達是陰性的膠質(zhì)瘤患者生存時間較長（HR=0.325，P=0.005），而CENP-W陽性患者的生存時間短。 結(jié)論： 1、CENP-W在腦膠質(zhì)瘤中是上調(diào)致癌基因。 2、CENP-WmRNA、蛋白表達量有隨著膠質(zhì)瘤病理級別升高有漸增高趨勢。 3、腦膠質(zhì)瘤組織的免疫組化中CENP-W表達陽性對提示腦膠質(zhì)瘤的惡性程度有一定的意義。
[Abstract]:Objective:
Glioma is the most common malignant brain tumor, it is a set of features to glial cell differentiation function of tumor neural epithelial cells in general, the incidence of intracranial tumors accounted for all of the 50%-60%. in recent years, in spite of the different treatment methods of comprehensive application, clinical curative effect is enhanced, but the survival time is shorter and the quality of life is not the high, most patients will eventually relapse and metastasis. The existing research indicates that gene expression and carcinogenesis suppressor inactivation is closely related to, and now on the molecular mechanism of how cancer is unknown.
CENP-W was first reported in 2007, which belongs to the CCAN (constitutive centromereassociated network) family, at first because it was found that fibroblasts are highly tumorigenic so a putative oncogenic gene named CUG2 in a variety of cancer tissues and mice (Cancer-upregulated Gene2); and CENP-W in the central nervous system play an important role in the development and involved in neuronal function maintenance, knockdown of CENP-W in zebrafish embryos can lead to neurodegeneration. At home and abroad has not yet in-depth study of CENP-W expression in glioma and its survival prognosis.
This study was designed to investigate the expression of CENP-W in glioma tissues and cell lines or not, and the degree of expression and pathological grade (WHO grade) relationship, and expression and prognosis of survival time of patients with glioma CENP-W analysis, the next step for the research of CENP-W in glioma occurrence, development process may be involved in the adjustment process, in order to further provide a new direction in the treatment of brain glioma.
Methods: the experimental design is divided into three steps
1., the first step was to detect the difference of CENP-WmRNA and protein expression between 4 normal brain tissues (non tumor brain tissues) in the control group and the 4 pathological grades of gliomas in the control group by Real-Time PCR, Western blotting and immunohistochemistry.
2., the second step is to detect A172, LN308, U118, U87, U251, LN229, SNB19, TJ899 and TJ905 in nine kinds of glioma cell lines by using Western blotting detection method, and collect data and analyze them by statistical methods.
3. the patients in the third step collected tumor specimens were followed up after operation, and the related factors of tumor patients were collected for statistical analysis. The relationship between the expression of CENP-W in glioma tissues and the prognosis and survival of patients was analyzed.
Result錛

本文編號：1497683