本文關(guān)鍵詞： ARID1A 膠質(zhì)瘤 侵襲轉(zhuǎn)移 預(yù)后 轉(zhuǎn)染 分子機制　出處：《河北醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分ARID1A在正常腦組織及腦膠質(zhì)瘤組織中的表達研究目的:檢測ARID1A在人腦膠質(zhì)瘤組織及正常腦組織的表達情況,分析ARID1A在膠質(zhì)瘤中的表達情況及其與臨床病理特征的相關(guān)性。方法:通過RT-PCR和Western blot的方法分別檢測轉(zhuǎn)錄水平和蛋白表達水平人腦膠質(zhì)瘤組織及正常腦組織的ARID1A表達情況,并檢測ARID1A表達與膠質(zhì)瘤患者的年齡、性別、KPS評分、病理級別及復(fù)發(fā)情況的相關(guān)性。結(jié)果:RT-PCR分析結(jié)果顯示,膠質(zhì)瘤組織ARID1A mRNA相對表達量(0.52±0.25)顯著低于正常腦組織(1.23±0.14),差異顯著(P㩳0.001),且膠質(zhì)瘤中ARID1A mRNA的表達水平隨著病理級別的上升而降低(P㩳0.05)。復(fù)發(fā)組膠質(zhì)瘤組織(0.31±0.24)ARID1A表達低于未復(fù)發(fā)組(0.76±0.25),差異有統(tǒng)計學(xué)意義(P㩳0.001)。ARID1A與膠質(zhì)瘤患者的年齡、性別和KPS評分無相關(guān)性。Western blot結(jié)果顯示,ARID1A蛋白在正常腦組織中的表達水平高于腦膠質(zhì)瘤組織,且隨膠質(zhì)瘤惡性程度的升高,ARID1A的蛋白表達水平不斷降低。復(fù)發(fā)組膠質(zhì)瘤組織(0.36±0.21)ARID1A蛋白相對表達量低于未復(fù)發(fā)組(0.71±0.23),差異有統(tǒng)計學(xué)意義(P㩳0.001)。第二部分ARID1A血清學(xué)表達與腦膠質(zhì)瘤臨床病理特征及預(yù)后的相關(guān)性研究目的:研究ARID1A基因在膠質(zhì)瘤患者血清中的表達情況,探討ARID1A在膠質(zhì)瘤患者血清中的表達與臨床病理特征關(guān)系及對預(yù)后的意義。方法:采用高效液相色譜法檢測了83例膠質(zhì)瘤血清樣本和46例健康血清樣品的ARID1A濃度,分析兩者與膠質(zhì)瘤臨床病理特征(包括KPS評分、年齡、病理分級情況、性別、術(shù)前癲癇和腫瘤范圍)及預(yù)后的關(guān)系。并分別對不同的KPS評分、年齡和病理分級情況的ARID1A表達進行了分層Kaplan-Meier分析。結(jié)果:腦膠質(zhì)瘤患者血清中arid1a的濃度(44.98±11.87μg/ml)顯著低于健康人(90.75±12.89μg/ml)。膠質(zhì)瘤患者kps評分、年齡、病理分級情況與arid1a表達水平之間呈現(xiàn)具有顯著相關(guān)性(p㩳0.05),其中較高的kps評分、較大的年齡、較高的病理分級與低水平的arid1a表達密切相關(guān);而性別、術(shù)前癲癇、腫瘤范圍與arid1a表達水平之間沒有具有顯著相關(guān)性(p㧐0.05)。kaplan-meier分析顯示血清中arid1a低表達患者的總生存期短于arid1a高表達患者(對數(shù)秩檢驗,p=0.005)。分層kaplan-meier分析arid1a表達顯示:kps80分組、kps≥80分組、年齡50歲組、年齡≥50歲組、病理i-ii級組和病理iii-iv級組的arid1a高表達患者總生存率均優(yōu)于arid1a低表達患者。多因素分析結(jié)果顯示,arid1a是膠質(zhì)瘤患者預(yù)后的獨立因素(p=0.002,hr=4.992,95%ci=1.831-13.611)。第三部分調(diào)節(jié)arid1a基因表達對人腦膠質(zhì)瘤細胞侵襲與轉(zhuǎn)移的影響及機制研究目的:研究arid1a與人腦膠質(zhì)瘤細胞侵襲與轉(zhuǎn)移的影響及其相關(guān)機制。方法:通過慢性病毒轉(zhuǎn)染技術(shù)建立arid1a(rnai2801)穩(wěn)定低表達的細胞系,同時通過質(zhì)粒轉(zhuǎn)化構(gòu)建arid1a(wv0081)穩(wěn)定過表達的細胞系。分別通過western-blot和qrt-pcr技術(shù)驗證arid1a表達發(fā)生改變后,使用transwell法對細胞遷移和侵襲能力的改變進行研究;使用mtt法對細胞增殖能力的改變進行檢測;流式細胞術(shù)檢測細胞凋亡情況。此外,對arid1a表達發(fā)生變化后相關(guān)細胞中的p53和c-myc的表達變化進行研究;將轉(zhuǎn)染前后的細胞接種于裸鼠體內(nèi)構(gòu)建成瘤模型,研究它們成瘤能力的改變。結(jié)果:建立了能夠穩(wěn)定上調(diào)和下調(diào)arid1a表達的細胞株。arid1a表達發(fā)生改變后,在體外實驗中能夠使得膠質(zhì)瘤細胞的遷移、侵襲、增殖和凋亡能力發(fā)生顯著變化;在體內(nèi)實驗中能夠使得膠質(zhì)瘤細胞的成瘤能力發(fā)生顯著變化。arid1a上調(diào)后,p53基因表達顯著上升,而c-myc基因表達顯著下降;arid1a下調(diào)后,p53基因表達顯著下降,而c-myc基因表達顯著上升。結(jié)論:1 ARID1A基因可能在膠質(zhì)瘤的發(fā)生發(fā)展中發(fā)揮著抑癌基因的作用。2 ARID1A低表達與膠質(zhì)瘤的預(yù)后不良相關(guān)。3上調(diào)/下調(diào)膠質(zhì)瘤細胞ARID1A的表達可以降低/提高膠質(zhì)瘤的侵襲和轉(zhuǎn)移能力。
[Abstract]:Objective to study the expression of ARID1A in the first part of the tumor tissue and normal brain tissue in cerebral gliomas: detection of ARID1A expression in human glioma tissues and normal brain tissues, the expression of ARID1A in gliomas and its correlation with clinical pathological features. Methods: the RT-PCR and Western blot were used to detect the transcription level and the expression level of human glioma tissues and normal brain tissues, the expression of ARID1A, and to detect the expression of ARID1A and glioma patients' age, gender, KPS score, correlation between pathological grade and recurrence of. Results: RT-PCR analysis showed that the relative expression of glioma ARID1A mRNA (0.52 + 0.25) was significantly lower than that of normal the brain tissue (1.23 + 0.14), significant difference (P? 0.001), and the expression level of ARID1A in glioma mRNA with increased pathological grade decreased (P? 0.05). Recurrence of glioma group (0. 31 + 0.24) ARID1A expression was lower than that of the non recurrent group (0.76 + 0.25), the difference was statistically significant (P? 0.001).ARID1A and glioma patients' age, gender and KPS score had no correlation with.Western blot showed that the expression level of ARID1A protein in normal brain tissues was higher than that of brain glioma, with the increased the malignant degree of glioma, the expression level of ARID1A protein decreased. The glioma recurrence group (0.36 + 0.21) ARID1A protein expression was lower than that of the non recurrent group (0.71 + 0.23), the difference was statistically significant (P? 0.001). Part second objective to investigate the expression correlation between serum ARID1A and clinical pathological characteristics and prognosis of brain glioma: the expression of ARID1A gene in serum of patients with gliomas, to explore the relationship between the expression of ARID1A in serum of patients with glioma and clinical pathological features and prognostic significance. Methods: using high performance liquid chromatography. The concentration of ARID1A in 83 cases of glioma and 46 serum samples of healthy serum samples, and analyze their clinical and pathological characteristics of glioma (including KPS score, age, pathologic grade, gender, preoperative epilepsy and range of tumor and prognosis and relationship). According to different KPS scores, age and pathological expression grade ARID1A of the hierarchical Kaplan-Meier analysis. Results: the concentration of arid1a in serum of patients with glioma in the brain (44.98 + 11.87 g/ml) was significantly lower than that of healthy people (90.75 + 12.89 g/ml). Glioma patients KPS score, age, pathological grading situation between the expression level and arid1a showed significant correlation (P? 0.05), the higher the KPS score, age, pathological grade and low levels of high expression of arid1a are closely related; gender, preoperative epilepsy, no significant correlation between the expression level of tumor with arid1a range (P? 0.05).Kapla N-meier analysis showed that the low expression of arid1a in serum of patients with total survival was shorter in patients with high expression of arid1a (log rank test, p=0.005). The expression of arid1a showed that the layered Kaplan-Meier kps80 group, KPS = 80 group, age 50 years, 50 years of age or older group, high expression grade I-II group and grade III-IV group the total survival rate of patients with arid1a were better than those with low arid1a expression. The results of multivariate analysis showed that arid1a was an independent prognostic factor in patients with glioma (p=0.002, hr=4.992,95%ci=1.831-13.611). The third part regulation of arid1a gene expression effect on invasion and metastasis of human glioma cells and the mechanism Objective: To study the effect of arid1a and human glioma cell invasion with the transfer and its related mechanism. Methods: to establish chronic arid1a by transfection technique (rnai2801) stable low expression cell line, at the same time through the construction of arid1a plasmid transformation (wv008 1) stable expression cell line respectively. Expression through Western-blot and qRT-PCR technology to verify the arid1a changes, using the Transwell method on the invasion and migration ability of cell changes were investigated; using MTT method to detect the change of cell proliferation ability; flow cytometry to detect the cell apoptosis. In addition, the research of expression the expression of related cell changes in p53 and c-myc on arid1a; transfected cells before and after inoculation in nude mice tumor model construction, study their tumorigenicity changes. Results: the establishment of stable and downregulation of arid1a expression cell line.Arid1a expression change after, can make the migration of glioma cells the invasion in vitro, proliferation and apoptosis changed significantly; makes the tumorigenic ability of glioma cells significantly changed.Arid1a in vivo After the transfer, the expression of p53 gene increased significantly, while the expression of c-myc gene decreased significantly; the down-regulation of arid1a, p53 gene expression decreased significantly, while the expression of c-myc gene increased significantly. Conclusion: 1 ARID1A gene in the occurrence and development of glioma may play the role of tumor suppressor gene.2 and the low expression of ARID1A in glioma is related to poor prognosis upregulation of.3 / downregulation of ARID1A glioma cells can decrease / increase of glioma invasion and metastasis.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.41

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