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ARID1A在人脑胶质瘤中的表达及其机制研究

发布时间:2018-02-27 07:00

  本文关键词: ARID1A 胶质瘤 侵袭转移 预后 转染 分子机制 出处:《河北医科大学》2016年博士论文 论文类型:学位论文


【摘要】:第一部分ARID1A在正常脑组织及脑胶质瘤组织中的表达研究目的:检测ARID1A在人脑胶质瘤组织及正常脑组织的表达情况,分析ARID1A在胶质瘤中的表达情况及其与临床病理特征的相关性。方法:通过RT-PCR和Western blot的方法分别检测转录水平和蛋白表达水平人脑胶质瘤组织及正常脑组织的ARID1A表达情况,并检测ARID1A表达与胶质瘤患者的年龄、性别、KPS评分、病理级别及复发情况的相关性。结果:RT-PCR分析结果显示,胶质瘤组织ARID1A mRNA相对表达量(0.52±0.25)显著低于正常脑组织(1.23±0.14),差异显著(P㩳0.001),且胶质瘤中ARID1A mRNA的表达水平随着病理级别的上升而降低(P㩳0.05)。复发组胶质瘤组织(0.31±0.24)ARID1A表达低于未复发组(0.76±0.25),差异有统计学意义(P㩳0.001)。ARID1A与胶质瘤患者的年龄、性别和KPS评分无相关性。Western blot结果显示,ARID1A蛋白在正常脑组织中的表达水平高于脑胶质瘤组织,且随胶质瘤恶性程度的升高,ARID1A的蛋白表达水平不断降低。复发组胶质瘤组织(0.36±0.21)ARID1A蛋白相对表达量低于未复发组(0.71±0.23),差异有统计学意义(P㩳0.001)。第二部分ARID1A血清学表达与脑胶质瘤临床病理特征及预后的相关性研究目的:研究ARID1A基因在胶质瘤患者血清中的表达情况,探讨ARID1A在胶质瘤患者血清中的表达与临床病理特征关系及对预后的意义。方法:采用高效液相色谱法检测了83例胶质瘤血清样本和46例健康血清样品的ARID1A浓度,分析两者与胶质瘤临床病理特征(包括KPS评分、年龄、病理分级情况、性别、术前癫痫和肿瘤范围)及预后的关系。并分别对不同的KPS评分、年龄和病理分级情况的ARID1A表达进行了分层Kaplan-Meier分析。结果:脑胶质瘤患者血清中arid1a的浓度(44.98±11.87μg/ml)显著低于健康人(90.75±12.89μg/ml)。胶质瘤患者kps评分、年龄、病理分级情况与arid1a表达水平之间呈现具有显著相关性(p㩳0.05),其中较高的kps评分、较大的年龄、较高的病理分级与低水平的arid1a表达密切相关;而性别、术前癫痫、肿瘤范围与arid1a表达水平之间没有具有显著相关性(p㧐0.05)。kaplan-meier分析显示血清中arid1a低表达患者的总生存期短于arid1a高表达患者(对数秩检验,p=0.005)。分层kaplan-meier分析arid1a表达显示:kps80分组、kps≥80分组、年龄50岁组、年龄≥50岁组、病理i-ii级组和病理iii-iv级组的arid1a高表达患者总生存率均优于arid1a低表达患者。多因素分析结果显示,arid1a是胶质瘤患者预后的独立因素(p=0.002,hr=4.992,95%ci=1.831-13.611)。第三部分调节arid1a基因表达对人脑胶质瘤细胞侵袭与转移的影响及机制研究目的:研究arid1a与人脑胶质瘤细胞侵袭与转移的影响及其相关机制。方法:通过慢性病毒转染技术建立arid1a(rnai2801)稳定低表达的细胞系,同时通过质粒转化构建arid1a(wv0081)稳定过表达的细胞系。分别通过western-blot和qrt-pcr技术验证arid1a表达发生改变后,使用transwell法对细胞迁移和侵袭能力的改变进行研究;使用mtt法对细胞增殖能力的改变进行检测;流式细胞术检测细胞凋亡情况。此外,对arid1a表达发生变化后相关细胞中的p53和c-myc的表达变化进行研究;将转染前后的细胞接种于裸鼠体内构建成瘤模型,研究它们成瘤能力的改变。结果:建立了能够稳定上调和下调arid1a表达的细胞株。arid1a表达发生改变后,在体外实验中能够使得胶质瘤细胞的迁移、侵袭、增殖和凋亡能力发生显著变化;在体内实验中能够使得胶质瘤细胞的成瘤能力发生显著变化。arid1a上调后,p53基因表达显著上升,而c-myc基因表达显著下降;arid1a下调后,p53基因表达显著下降,而c-myc基因表达显著上升。结论:1 ARID1A基因可能在胶质瘤的发生发展中发挥着抑癌基因的作用。2 ARID1A低表达与胶质瘤的预后不良相关。3上调/下调胶质瘤细胞ARID1A的表达可以降低/提高胶质瘤的侵袭和转移能力。
[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.

【学位授予单位】:河北医科大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:R739.41

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