FOXJ2在脑胶质瘤中的表达及其对胶质瘤细胞迁移作用的研究

发布时间：2018-08-28 19:13

【摘要】：目的:脑胶质瘤是中枢神经系统最常见的恶性肿瘤。包括神经外科手术、放射治疗及化疗等多模式治疗相结合,但脑胶质瘤患者的预后仍然不令人满意。局部复发是治疗失败的主要原因。胶质瘤细胞具有侵袭和迁移能力是导致局部复发的重要原因。FOXJ2是转录因子叉头框(Forkhead box,FOX)蛋白家族成员之一,FOXJ2涉及细胞周期的调控以及肿瘤的发生发展。已有研究表明FOXJ2表达的上调可抑制乳腺癌的迁移和侵袭能力。本研究旨在初步了解FOXJ2在脑胶质瘤中的表达情况及其与胶质瘤患者临床病例因素的关系,检测胶质瘤细胞中FOXJ2、E-钙黏蛋白(E-cadherin)的表达,分析其相互的关系以及对胶质瘤细胞迁移的影响,探讨FOXJ2的异常表达在胶质瘤发生、发展过程中的意义。方法:1.从组织学水平,本研究首先通过Western Blot方法,检测在两组人正常脑组织和七组不同级别胶质瘤组织中FOXJ2的表达情况;然后,运用免疫组化方法检测80例胶质瘤组织标本中FOXJ2和E-cadherin的表达情况,并采用统计学方法分析FOXJ2、E-cadherin的表达情况与胶质瘤患者的年龄、性别、肿瘤位置、大小及肿瘤组织学分级等临床病理因素之间的相关性。通过Kaplan-Meier法分析FOXJ2的表达与胶质瘤患者的生存率之间的相关性;2.从细胞分子水平,本研究选取U87MG,U373,H4,A172和U251MG等五种胶质瘤细胞株,用Western Blot技术检测FOXJ2、E-cadherin和波形蛋白(Vimentin)的表达。选取U87胶质瘤细胞株,瞬时转染FOXJ2过表达和干扰质粒,通过Western Blot和细胞免疫荧光检测胶质瘤细胞E-cadherin和Vimentin的表达;用划痕实验和Transwell迁移实验检测细胞迁移能力。明确FOXJ2对胶质瘤的迁移的影响。结果:1.Western Blot及免疫组化结果均显示,FOXJ2在胶质瘤组织中的表达随胶质瘤病理级别的增高而降低,提示FOXJ2的表达强度与胶质瘤组织学分级(P=0.012)之间的关系有统计学意义,而与患者的年龄、性别、肿瘤体积大小、位置、是否发生坏死等临床病理特征间无差异。Kaplan-Meier生存曲线分析显示FOXJ2在胶质瘤中的低表达患者的总生存率明显低于FOXJ2高表达的患者(P0.01)。2.Western Blot技术检测U87,U373,H4,A172,U251胶质瘤细胞株FOXJ2的表达,结果显示FOXJ2在迁移能力弱的H4细胞中表达较高,而在迁移能力强的U251细胞中表达偏低。选取U87和U251细胞株,检测上皮间质转化(epithelial-mesenchymal transition,EMT)标记,观察到FOXJ2与E-cadherin表达呈正相关(r=0.303;P0.01),Vimentin的表达情况与FOXJ2的表达情况相反。3.通过对转染过表达FOXJ2质粒的U87胶质瘤细胞,进行Western Blot和细胞免疫荧光检测,观察到FOXJ2过表达可促进胶质瘤细胞E-cadherin的表达,抑制Vimentin的表达。划痕实验和Transwell迁移实验,观察到FOXJ2过表达可抑制胶质瘤细胞的迁移。4.通过对转染sh RNA FOXJ2质粒的U87胶质瘤细胞,进行Western Blot和细胞免疫荧光检测,观察到干扰FOXJ2表达抑制胶质瘤细胞E-cadherin的表达,促进Vimentin的表达。划痕实验和Transwell迁移实验,观察到干扰FOXJ2表达可促进胶质瘤细胞的迁移。结论:1.FOXJ2在胶质瘤中的表达随病理分级的提高而降低。FOXJ2的低表达提示胶质瘤患者的预后不良。2.FOXJ2的表达可影响E-cadherin、Vimentin的表达,影响EMT的进程,从而抑制胶质瘤细胞迁移。
[Abstract]:Objective:Glioma is the most common malignant tumor of the central nervous system.It includes neurosurgery,radiotherapy and chemotherapy,but the prognosis of the patients with glioma is still unsatisfactory.Local recurrence is the main cause of treatment failure.The ability of glioma cells to invade and migrate leads to local recurrence. FOXJ2 is a member of the Forkhead box (FOX) protein family. FOXJ2 is involved in cell cycle regulation and tumor development. Upregulation of FOXJ2 expression has been shown to inhibit the migration and invasion of breast cancer. The expression of FOXJ2 and E-cadherin in glioma cells was detected, the relationship between FOXJ2 and E-cadherin and the migration of glioma cells was analyzed, and the significance of the abnormal expression of FOXJ2 in the occurrence and development of glioma was explored. Methods: 1. From the histological level, this study was first carried out by W. Estn Blot method was used to detect the expression of FOXJ2 in normal brain tissues of two groups and seven groups of glioma tissues of different grades. Immunohistochemical method was used to detect the expression of FOXJ2 and E-cadherin in 80 glioma tissues, and statistical method was used to analyze the expression of FOXJ2 and E-cadherin and the age of glioma patients. Kaplan-Meier method was used to analyze the correlation between FOXJ 2 expression and survival rate of glioma patients. 2. At the cellular and molecular level, five glioma cell lines, including U87MG, U373, H4, A172 and U251MG, were selected for Western Blot assay. The expression of FOXJ2, E-cadherin and Vimentin was detected. U87 glioma cell line was transfected with FOXJ2 overexpression and interfering plasmid. The expression of E-cadherin and Vimentin was detected by Western Blot and immunofluorescence assay. The migration ability of glioma cells was detected by scratch test and Transwell migration test. Western Blot and immunohistochemical results showed that FOXJ2 expression in glioma tissues decreased with the increase of pathological grade, suggesting that FOXJ2 expression intensity and histological grade of glioma (P = 0.012) were statistically significant, but with the age, sex, tumor size and location of patients. Kaplan-Meier survival curve analysis showed that the overall survival rate of patients with low FOXJ2 expression in gliomas was significantly lower than that of patients with high FOXJ2 expression (P 0.01). 2 Western Blot assay showed that the expression of FOXJ2 in U87, U373, H4, A172, U251 gliomas was significantly lower than that of patients with high FOXJ2 expression (P 0.01). U87 and U251 cell lines were selected to detect epithelial-mesenchymal transition (EMT) markers. FOXJ2 was positively correlated with E-cadherin expression (r = 0.303; P 0.01). Vimentin expression was contrary to FOXJ2 expression. Western Blot and immunofluorescence assay were performed on U87 glioma cells transfected with FOXJ2 plasmid. Overexpression of FOXJ2 could promote the expression of E-cadherin and inhibit the expression of Vimentin. Scratch test and Transwell migration test showed that over-expression of FOXJ2 could inhibit the migration of glioma cells. Western Blot and immunofluorescence were used to detect the expression of E-cadherin and Vimentin in U87 glioma cells infected with SH RNA FOXJ2 plasmid. Scratch test and Transwell migration test showed that interfering with FOXJ2 expression could promote the migration of glioma cells. The expression of FOXJ 2 decreased with the increase of pathological grade. The low expression of FOXJ 2 suggested that the prognosis of glioma patients was poor. 2. The expression of FOXJ 2 could affect the expression of E-cadherin and Vimentin, affect the process of EMT and inhibit the migration of glioma cells.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41

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