本文選題：膠質(zhì)母細(xì)胞瘤　切入點：泛素特異性蛋白酶44　出處：《第二軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：膠質(zhì)母細(xì)胞瘤是惡性程度最高的中樞神經(jīng)系統(tǒng)原發(fā)性腫瘤之一。由于腫瘤呈浸潤性生長,與周圍腦組織無明顯分解且易于發(fā)生放化療耐受,目前傳統(tǒng)的治療措施療效均不甚理想。新興的個體化基因治療有望成為治愈膠質(zhì)母細(xì)胞瘤的途徑之一,但目前仍缺乏理想可靠的治療靶標(biāo)。因此尋找調(diào)控膠質(zhì)母細(xì)胞瘤惡性生物學(xué)行為的分子靶標(biāo)并明確其在GBM發(fā)病過程中的病理機制,已成為顱腦腫瘤研究領(lǐng)域的熱點之一。細(xì)胞周期的進(jìn)程中,姐妹染色單體準(zhǔn)確適時地分離對細(xì)胞的遺傳學(xué)穩(wěn)定是至關(guān)重要的,紡錘體裝配檢查點(SAC)機制則是確保有絲分裂正常進(jìn)行的重要監(jiān)控機制,其異常是導(dǎo)致非整倍體細(xì)胞出現(xiàn)乃至腫瘤形成的重要原因。在之前的研究報道中我們發(fā)現(xiàn)紡錘體裝配檢查點機制中重要的調(diào)控蛋白——去泛素化酶USP44與腫瘤的發(fā)生密切相關(guān)。去泛素化酶USP44在正常的細(xì)胞周期進(jìn)程中可以穩(wěn)定保護(hù)素的蛋白水平直到全部動粒均與紡錘絲正確匹配,防止細(xì)胞出現(xiàn)不成熟的有絲分裂。抑制小鼠胚胎成纖維細(xì)胞中USP44的表達(dá)水品后能明顯提高非整倍體細(xì)胞的比例及染色體的不穩(wěn)定性,使之更易于發(fā)生惡性轉(zhuǎn)化。USP44是腫瘤促進(jìn)因子Ubc H10的拮抗蛋白,而在部分腫瘤細(xì)胞中USP44卻表達(dá)上調(diào),但因缺乏能有效識別內(nèi)源性USP44的特異性抗體,表達(dá)上調(diào)的USP44是否參與維持腫瘤細(xì)胞的惡性增殖能力及其內(nèi)在的調(diào)控機制目前尚不清楚。本實驗從篩選能特異性識別內(nèi)源性USP44的抗體入手,分析USP44在膠質(zhì)瘤組織中的表達(dá)水平與腫瘤級別及預(yù)后的關(guān)系,USP44與GBM惡性生物學(xué)行為的關(guān)系以及USP44可能參與的部分信號通路。第一部分USP44特異性抗體的篩選與鑒定目的:利用分子生物學(xué)技術(shù)篩選并鑒定能有效識別內(nèi)源性USP44的特異性抗體。方法:預(yù)選三種抗體:Abnova p Ab21808,Santa Cruz sc-337203,Origene TA801913。在U251MG、U87MG及A172等三種細(xì)胞系中利用免疫印跡技術(shù)檢測抗體是否能特異性識別內(nèi)源性USP44;在239T細(xì)胞中過表達(dá)Flag-USP44融合蛋白,分別檢測USP44抗體與Flag抗體的熒光信號;在U251MG細(xì)胞中,利用免疫熒光技術(shù)分別檢測USP44抗體與B23抗體的熒光信號;在U251MG細(xì)胞中利用免疫印跡及免疫熒光技術(shù)檢測USP44敲減后抗體能否識別USP44的表達(dá)下調(diào)。結(jié)果:在免疫印跡實驗中三種抗體均能特異性識別三組細(xì)胞中的內(nèi)源性USP44并形成唯一印跡條帶;抗體p Ab21808的熒光信號能與Flag的熒光信號完全重合;抗體p Ab21808與B23抗體的熒光信號在核仁內(nèi)存在重合;三種抗體均能檢測到USP44敲減后的表達(dá)下調(diào),抗體在USP44敲減后的U251MG細(xì)胞中的信號明顯減弱。結(jié)論:抗體p Ab21808識別內(nèi)源性USP44的特異性最好,能準(zhǔn)確的反映出USP44的亞細(xì)胞定位及si RNA的敲減效率,故我們采用抗體p Ab21808進(jìn)行后續(xù)實驗。第二部分USP44表達(dá)水平與膠質(zhì)瘤病理級別及預(yù)后的關(guān)系目的:在蛋白和m RNA層面上,利用組織標(biāo)本研究USP44表達(dá)水平與膠質(zhì)瘤病理級別及預(yù)后的關(guān)系。方法:利用抗體p Ab21808進(jìn)行免疫組化實驗,分析含61例不同級別膠質(zhì)瘤標(biāo)本的組織芯片,檢測USP44蛋白在組織標(biāo)本中的表達(dá)情況;利用q RT-PCR技術(shù)分析40例不同級別膠質(zhì)瘤冰凍組織中USP44m RNA的表達(dá)水平;結(jié)合患者隨訪資料及q RT-PCR數(shù)據(jù)進(jìn)行生存分析。結(jié)果:USP44的蛋白表達(dá)水平與膠質(zhì)瘤病理級別呈線性相關(guān);在高級別膠質(zhì)瘤組中USP44m RNA的表達(dá)水平明顯高于低級別組;USP44m RNA高表達(dá)組患者的術(shù)后生存時間明顯小于低表達(dá)組。結(jié)論:USP44的表達(dá)水平隨膠質(zhì)瘤病理級別的升高而升高,高表達(dá)的USP44提示患者預(yù)后較差。第三部分USP44對GBM細(xì)胞惡性生物學(xué)行為的影響目的:研究GBM細(xì)胞的增殖、侵襲、周期、凋亡等生物學(xué)行為在USP44敲減后的變化。方法:U251MG和A172細(xì)胞轉(zhuǎn)染USP44-sh RNA慢病毒后進(jìn)行WST-1實驗和平板克隆形成實驗,檢測USP44表達(dá)下調(diào)對細(xì)胞增殖能力的影響;Transwell實驗檢測USP44敲減后U251MG和A172細(xì)胞遷移、侵襲的能力的變化;穩(wěn)定轉(zhuǎn)染的U251MG和A172細(xì)胞經(jīng)碘化丙啶(PI)染色后進(jìn)行流式細(xì)胞周期分析;流式細(xì)胞儀分析熒光染料PE/7-AAD雙染的穩(wěn)定轉(zhuǎn)染細(xì)胞,檢測USP44表達(dá)下調(diào)后GBM細(xì)胞凋亡比例的變化。結(jié)果:USP44表達(dá)下調(diào)能明顯抑制U251MG和A172細(xì)胞的增殖和侵襲能力,同時顯著提高G2/M期細(xì)胞及凋亡細(xì)胞的比例。結(jié)論:敲減USP44能明顯抑制GBM細(xì)胞的惡性生物學(xué)行為,阻滯細(xì)胞周期于G2/M期并誘導(dǎo)細(xì)胞凋亡。第四部分USP44與促癌因子securin之間的相互作用目的:驗證USP44與securin之間是否存在相互作用,USP44是否通過去泛素化作用穩(wěn)定securin。方法:在U251細(xì)胞中利用免疫熒光技術(shù)檢測USP44與securin的熒光信號在細(xì)胞內(nèi)的定位關(guān)系;利用免疫共沉淀技術(shù)驗證USP44能否與securin直接結(jié)合;USP44-sh RNA轉(zhuǎn)染的U251MG細(xì)胞經(jīng)不同濃度的蛋白酶體抑制劑MG132處理后,用western blot技術(shù)檢測securin表達(dá)水平的變化。結(jié)果:USP44與securin的熒光信號在分裂相的細(xì)胞中明顯重合;內(nèi)源性USP44能與securin直接結(jié)合;MG132能逆轉(zhuǎn)由敲減USP44引起的securin表達(dá)下調(diào)且不影響USP44本身的表達(dá)水平。結(jié)論:Securin是USP44的催化底物,USP44通過去泛素化作用穩(wěn)定securin并抑制其被蛋白酶體降解。第五部分USP44表達(dá)下調(diào)對U87MG細(xì)胞體內(nèi)成瘤能力的影響目的:驗證USP44表達(dá)下調(diào)對GBM細(xì)胞體內(nèi)成瘤能力的影響。方法:連續(xù)培養(yǎng)USP44-sh RNA穩(wěn)定轉(zhuǎn)染的U87MG細(xì)胞;選擇5周齡雄性裸鼠5只,分別在裸鼠兩側(cè)后肢近端的內(nèi)側(cè)皮下注射上述細(xì)胞及正常U87MG細(xì)胞(107cells)。每2天用游標(biāo)卡尺測量腫瘤的長短徑一次。至腫瘤長徑超過10mm時將裸鼠脫頸處死,取出腫瘤;用裸鼠腫瘤標(biāo)本制作石蠟切片進(jìn)行免疫組化實驗,對細(xì)胞形態(tài)和Ki-67的表達(dá)進(jìn)行檢測。結(jié)果:USP44敲減組和對照組相比,腫瘤的終體積和生長速率均明顯減小;免疫組化結(jié)果表明敲減組Ki-67的表達(dá)水平明顯低于對照組。結(jié)論:USP44表達(dá)下調(diào)能明顯抑制U87MG細(xì)胞的體內(nèi)成瘤能力。
[Abstract]:Glioblastoma is one of the central nervous system of most malignant primary tumor. The tumor showed invasive growth, no significant decomposition and prone to chemotherapy tolerance and surrounding brain tissue, the curative effect of the traditional treatment measures are not ideal. Individual gene therapy is expected to become one of the new ways to cure glioma cells tumor, but there is still a lack of reliable ideal therapeutic target. So looking for molecular target for malignant biological behavior regulation of glioblastoma and clear in the pathogenesis of GBM pathogenesis, has become a hot topic in the research field of brain tumor. The process of cell cycle, sister chromatid separation timely accurate genetics the stable cell is crucial, spindle assembly checkpoint (SAC) is an important mechanism to ensure the normal monitoring mechanism of mitosis, the abnormality is caused by non An important reason for aneuploidy cells and tumor formation. Research reports before we found the important regulatory proteins of the spindle assembly checkpoint mechanism -- deubiquitinase USP44 closely associated with tumor. Deubiquitinase USP44 protein level could be stable until all kinetochores are ofosteoprotegerin and spindle right matching in the normal cell cycle progression, cells appeared to prevent premature mitosis. Inhibition of mouse embryonic expression of USP44 cells in the water can be significantly improved when the instability of non proportional and chromosome aneuploid cells, making it more prone to malignant transformation of.USP44 is a tumor promoting factor Ubc antagonist H10, and USP44 in tumor cells is up-regulated, but due to lack of specific antibodies can effectively identify endogenous USP44 expression, USP44 is involved in the maintenance of tumor Cell malignant proliferation and regulation mechanism is unclear. This experiment from screening antibody specific identification of endogenous USP44 with the analysis of the relationship between the level and the prognosis of USP44 expression in glioma tissues and tumor, part of the signal pathways of USP44 and GBM for the malignant biological behavior and the relationship between the USP44 may be involved in the the first part. Screening and identification of USP44 specific antibodies: using molecular biology techniques can effectively identify the screening and identification of endogenous USP44 specific antibody. Methods: three primary antibodies: Abnova P Ab21808, Santa Cruz sc-337203, Origene TA801913. in U251MG U87MG by Western blot and A172 three cell line detection whether the antibodies can specifically recognize endogenous USP44; overexpression of Flag-USP44 fusion protein in 239T cells, USP44 antibody and Flag antibody were detected by fluorescent signal No; in U251MG cells, were used to detect the fluorescence signal of USP44 antibody and B23 antibody by immunofluorescence technique; expression detection of USP44 antibody can recognize USP44 knockdown in U251MG cells by Western blot and immunofluorescence technique. Results: in immunoblotting experiments of three kinds of antibodies can specifically recognize endogenous USP44 three group of cells and the formation of the only blot bands; the fluorescence signal of Ab21808 and P antibody can completely overlapped fluorescence signal Flag; fluorescence signal Ab21808 and antibody P B23 antibodies in the presence of coincidence in the nucleoli of three; antibody was detected USP44 mRNA knockdown, antibody in USP44 knock signal after reduction the U251MG cells decreased significantly. Conclusion: P Ab21808 specific antibody recognition of endogenous USP44 best, can accurately reflect the subcellular localization of USP44 and Si RNA knockdown efficiency, so we adopt the anti P Ab2 1808 for subsequent experiments. The relation between the second part of the USP44 expression and the pathological grades of gliomas and prognosis in M protein and RNA level, the relationship between the expression level of USP44 of tissue samples with pathological grade and prognosis. Methods: immunohistochemical experiments using P antibody Ab21808 analysis of tissue microarray containing 61 patients with different grade gliomas. The expression of USP44 protein was detected in tissue samples; analysis of the expression level of 40 cases of different grade gliomas in frozen tissue using Q USP44m RNA RT-PCR; survival analysis combined with follow-up data and Q RT-PCR data. Results: the expression of USP44 was correlated with glioma pathological grade; in high grade glioma group in the expression level of USP44m RNA was significantly higher than low level group; the group of patients with high USP44m RNA expression after the survival time was less than the low expression group Conclusion: the expression level of USP44 increased with the pathological grade of glioma, the high expression of USP44 were associated with poor prognosis. The third part: the effect of USP44 on the malignant biological behavior of GBM cells Objective: To study the GBM cell proliferation, invasion, apoptosis in the cycle, the biological behavior changes after knockdown of USP44. Methods: U251MG USP44-sh RNA and A172 cells transfected with lentivirus after WST-1 assay and colony formation assay, detect the effect of USP44 knockdown on cell proliferation; Transwell USP44 and A172 U251MG assay on cell migration after reduction, change ability of invasion; stably transfected U251MG and A172 cells by propidium iodide (PI) staining after FCM analysis; analysis of transfected cells stable fluorescent dye PE/7-AAD double staining flow cytometry to detect the expression of USP44, change the ratio of GBM cell apoptosis after the cut. Results: the expression of USP44 Downregulation could significantly inhibit the proliferation and invasion of U251MG cells and A172 cells, and significantly improve the cell apoptosis and the ratio of G2/M phase cells. Conclusion: knockdown of USP44 can inhibit the malignant biological behavior of GBM cells, G2/M cell cycle arrest and apoptosis. The fourth part USP44 and promote the interaction between cancer factor securin the Objective: to verify the interaction between USP44 and securin, by USP44 to ubiquitination of stable securin. methods: located in U251 cells by fluorescence signal immunofluorescence detection of USP44 and securin in cells; verify whether USP44 directly combined with securin by CO immunoprecipitation; USP44-sh RNA transfection U251MG cells with proteasome inhibitor MG132 with different concentration, the expression of securin was detected by Western blot. Results: USP44 and securin Obviously the coincidence of the fluorescence signal in mitotic cells; endogenous USP44 can be directly combined with securin; MG132 can reverse caused by knockdown of USP44 securin expression without affecting the expression level of USP44 itself. Conclusion: Securin is catalyzed by USP44, USP44 by ubiquitination of stable securin and inhibited by the proteasome the fifth part of the USP44 expression. The degradation effect of down-regulation of tumorigenicity of U87MG cells Objective: to verify the effect of down-regulation of USP44 expression on the tumorigenicity of GBM cells in vivo. Methods: Cultured USP44-sh RNA transfected U87MG cells; 5 week old male nude mice 5, respectively, on both sides of the proximal hindlimb in nude mice subcutaneous inside the injection of cells and normal U87MG cells (107cells). Every 2 days measured with vernier caliper once. The length of tumor diameter to tumor size more than 10mm mice were sacrificed, remove the tumor; naked Paraffin sections of rat tumor specimens by immunohistochemical experiment, detect the expression on cell morphology and Ki-67. Results: USP44 knockdown group compared with the control group, the final volume and tumor growth rate were significantly decreased; immunohistochemistry showed that the expression level of knockdown of Ki-67 group was significantly lower than the control group. Conclusion: USP44 expression of U87MG cells was significantly inhibited in vivo tumorigenicity.

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

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李燾;劉朝奇;王雄偉;;泛素化在人表皮生長因子受體降解中的研究進(jìn)展[J];重慶醫(yī)學(xué);2012年04期

2 馬祖祥;趙維玲;李永柏;高曉潔;楊軍;孫平;李成榮;;特發(fā)性腎病綜合征患兒外周血單個核細(xì)胞泛素的表達(dá)及意義[J];中國中西醫(yī)結(jié)合腎病雜志;2006年10期

3 王素霞;劉媛;吳慧娟;張志剛;;去泛素化酶的研究及其進(jìn)展[J];臨床與實驗病理學(xué)雜志;2008年06期

4 吳怡;劉新;;蛋白質(zhì)泛素化、去泛素化與腫瘤發(fā)生的關(guān)系[J];沈陽醫(yī)學(xué)院學(xué)報;2008年01期

5 茅幸;張志剛;吳慧娟;;泛素特異性加工酶2的研究進(jìn)展[J];國際病理科學(xué)與臨床雜志;2012年05期

6 何洪智;趙曉航;張立勇;吳e，

本文編號：1611073