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(xì)胞分化特征功能的神經(jīng)上皮細(xì)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(shí)間仍較短及生活質(zhì)量不高，絕大多數(shù)患者最終都會(huì)出現(xiàn)腫瘤復(fù)發(fā)及轉(zhuǎn)移。現(xiàn)有研究表明，基因中致癌過表達(dá)與抑癌失活緊密相關(guān)，而現(xiàn)如今對(duì)如何致癌的分子機(jī)制不明。 CENP-W于2007年首次被報(bào)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(xì)胞具有高致瘤性故以一個(gè)假定的致癌基因并命名為CUG2（Cancer-upregulated gene2）；同時(shí)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(dá)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(xì)胞株中的表達(dá)與否、以及表達(dá)程度與病理級(jí)別（WHO分級(jí)）的關(guān)系，同時(shí)分析CENP-W的表達(dá)與膠質(zhì)瘤患者生存時(shí)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(jìn)一步在治療腦膠質(zhì)瘤方面提供一個(gè)新的方向。 方法：本實(shí)驗(yàn)設(shè)計(jì)分為三步 1.第一步通過Real-Time PCR、Western blotting、免疫組化檢測(cè)對(duì)照組中正常腦組織（非瘤性腦組織）、實(shí)驗(yàn)組中4個(gè)不同病理級(jí)別腦膠質(zhì)瘤的CENP-WmRNA與蛋白表達(dá)差異，收集數(shù)據(jù)并應(yīng)用統(tǒng)計(jì)學(xué)方法進(jìn)行分析。 2.第二步應(yīng)用Western blotting檢測(cè)方法檢測(cè)A172、LN308、U118、U87、U251、LN229、SNB19、TJ899、TJ905九類膠質(zhì)瘤細(xì)胞株中CENP-W與GAPDH蛋白表達(dá)對(duì)比情況，收集數(shù)據(jù)并應(yīng)用統(tǒng)計(jì)學(xué)方法進(jìn)行分析。 3.第三步所收集腫瘤標(biāo)本的患者均行術(shù)后隨訪資料，將腫瘤患者各項(xiàng)相關(guān)因素收集后進(jìn)行相關(guān)統(tǒng)計(jì)學(xué)分析，，并分析腦膠質(zhì)瘤組織中CENP-W的表達(dá)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(gè)單峰，結(jié)果較好。mRNA表達(dá)水平：正常組織（1.0±0.19），I級(jí)（3.3±0.31），II級(jí)（5.4±0.43），III級(jí)（3.5±0.42），IV級(jí)（8.2±0.49）。CENP-W在不同病理級(jí)別的腦膠質(zhì)瘤組織中與正常腦組織組對(duì)比，差異具有統(tǒng)計(jì)學(xué)意義。同時(shí)高級(jí)別的膠質(zhì)瘤中CENP-W mRNA的表達(dá)水平明顯高于I、II、III級(jí)膠質(zhì)瘤。 2、CENP-W在腦膠質(zhì)瘤組織中蛋白相對(duì)表達(dá)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(duì)表達(dá)量高，病理IV級(jí)的蛋白相對(duì)表達(dá)量是正常對(duì)照組2倍左右。 3、總蛋白主要從上述細(xì)胞株中提取，并與內(nèi)參GAPDH對(duì)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(xì)胞株較HEK293組（0.24±0.08）相對(duì)蛋白表達(dá)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(duì)表達(dá)量小；由上結(jié)果在大部分膠質(zhì)瘤細(xì)胞株內(nèi)CENP-W呈中-高表達(dá)。利用灰度軟件進(jìn)行掃描對(duì)比，P0.05差異具有統(tǒng)計(jì)學(xué)意義。 4、在人腦膠質(zhì)瘤中CENP-W進(jìn)一步用免疫組化方法進(jìn)行驗(yàn)證。結(jié)果顯示CENP-W在膠質(zhì)瘤中表達(dá)陽性，細(xì)胞核呈陽性。而正常腦組織中表達(dá)陰性。利用卡方檢驗(yàn)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(jì)學(xué)意義。統(tǒng)計(jì)結(jié)果示在腦膠質(zhì)瘤中CENP-W陽性率74.0%，其中陽性表達(dá)中高惡性組占81.4%，低惡性組占18.6%。 5、統(tǒng)計(jì)分析膠質(zhì)瘤患者預(yù)后各項(xiàng)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(duì)膠質(zhì)瘤患者的生存時(shí)間存在相關(guān)性顯著，余因素對(duì)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(dá)是陰性的膠質(zhì)瘤患者生存時(shí)間較長（HR=0.325，P=0.005），而CENP-W陽性患者的生存時(shí)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(dá)量有隨著膠質(zhì)瘤病理級(jí)別升高有漸增高趨勢(shì)。 3、腦膠質(zhì)瘤組織的免疫組化中CENP-W表達(dá)陽性對(duì)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(hào)：1497683