AJAP1在脑胶质瘤中异常表达及抑制细胞增殖与侵袭的分子机制研究

发布时间：2018-07-14 11:00

【摘要】：脑胶质瘤是最常见的颅内原发性肿瘤，其中2/3以上为恶性胶质瘤，占成人全身肿瘤约2%，胶质母细胞瘤中位生存期仅1年，5年的生存期几乎为零[1-2]。手术切除，术后化疗和放疗综合疗效仍不是很理想，目前尚无根治方法。脑胶质瘤侵袭性生长的特性是胶质瘤难以根治和复发的根本原因。因此胶质瘤治疗策略的优化不仅应着眼在原发部位肿瘤的治疗，还应着力控制肿瘤细胞向正常脑组织的侵袭和转移。寻找具有抑制胶质瘤细胞侵袭、转移特性的相关基因，深入揭示其作用机制，对胶质瘤治疗具有重要意义。越来越多的证据表明在人类染色体1p36区域编码着许多类似肿瘤抑制基因[3-4]。最近研究报告发现粘附连接相关蛋白-1(Adherens Junctional AssociatedProtein-1，AJAP1,也称为Shrew-1)作为另一个类似抑癌基因定位于人类染色体1p36区域。Radlwimmer研究组对胶质母细胞瘤患者进行基因组杂交和微阵列分析比较，AJAP1首先被描述为一种与胶质母细胞发生的生物标记物[5]。随后,Zhang课题组证实在SVHVC, LOVO, SW480,MCF-7等多种细胞系中，AJAP1启动子区存在着高度甲基化[6]。但是，在胶质瘤细胞中改变AJAP1的表达水平影响细胞侵袭和增殖的机制仍然不清楚。本课题重点研究AJAP1在胶质瘤中的异常表达情况，探讨其与胶质瘤级别之间的关系，应用AJAP1重组腺病毒转染技术改变胶质瘤细胞中AJAP1的表达水平，初步探讨研究AJAP1对胶质瘤生物学表型的影响及其潜在机制。方法第一部分应用免疫组织化学染色以及免疫荧光共聚焦成像方法检测48例胶质瘤标本、5例正常脑组织标本中AJAP1的表达水平，采用Western blot方法对8个胶质瘤细胞系、1个正常细胞系HEK293中AJAP1蛋白表达水平进行检测，初步研究分析AJAP1与胶质瘤级别之间的关系。第二部分侧重于研究人脑胶质瘤细胞转染AJAP1重组腺病毒后对胶质瘤细胞恶性生物学表型及对细胞骨架影响的体外研究，并初步探讨其潜在机制。U87、U251细胞转染AJAP1重组腺病毒质粒后，Transewell实验检测胶质瘤细胞侵袭能力的改变；平板克隆形成试验检测细胞的增殖能力；划痕实验评价细胞迁移能力。Western blot方法检测胶质瘤细胞增殖、侵袭等相关指标的变化，同时应用免疫荧光方法检测过表达AJAP1后对细胞骨架的变化。第三部分则利用颅脑立体定向技术将同时转染AJAP1重组腺病毒和荧光素酶慢病毒的U87胶质瘤细胞接种在5周大小的BALB/c-nu（SPF）雌性裸鼠尾状核建立颅内胶质瘤模型，设空白对照组，利用活体生物发光成像系统（IVISLumina Imaging System）动态观察肿瘤生长情况及裸鼠的生存期的改变，应用组织病理学方法检测两组动物肿瘤标本中增殖、侵袭等蛋白表达水平的变化。结果通过免疫组化对石蜡切片检测显示：胶质瘤样本中AJAP1的表达水平显著低于正常脑组织，并且随着胶质瘤级别的增高，AJAP1的表达随着降低。组织免疫荧光试验也同样证实在高级别的胶质瘤标本中，AJAP1的表达明显低于低级别胶质瘤标本，与免疫组化结果相符，AJAP1定位于胞浆。因此AJAP1的表达水平与胶质瘤病理分级呈负相关。在胶质瘤细胞系中，Western blot检测显示在人胚肾上皮细胞HEK293中呈高表达，而在大部分胶质瘤细胞系中显著低表达，以A172、TJ905、TJ899低表达最为显著。随后，利用AJAP1重组腺病毒转染U87、U251胶质瘤细胞，通过提高细胞中AJAP1的蛋白表达水平，AJAP1组与空白对照组及转染空载体vector组相比较，平板克隆形成试验显示AJAP1组肿瘤细胞的克隆数形成减低，transwell试验显示AJAP1组细胞的侵袭能力受到明显抑制，划痕试验显示细胞的迁移能力下降，Western Blot检测Ki-67、MMP-9蛋白的表达水平均有明显的下调。在细胞免疫荧光试验中，过表达AJAP1后，，U87、U251细胞骨架结构发生改变，AJAP1组与vector组相比较，细胞生长模式发生改变，由原来的球状聚集生长形态变为片状生长，丝状伪足明显减少。在体内动物实验中，活体动物生物荧光成像结果证实U87-AJAP1组成瘤明显小于U87-vector组，且vector组于32d内全部死亡，AJAP1组32d内仅死亡4只。免疫组化结果表明AJAP1的表达明显上调，而Ki-67、MMP-9的表达明显下调，与细胞实验结果相符。结论 1.以正常脑组织做对照，AJAP1基因在胶质瘤标本和恶性胶质瘤细胞系中存在低表达，与肿瘤级别呈负相关，提示AJAP1可能作为抑癌基因参与胶质瘤的发生与发展。 2.应用AJAP1重组腺病毒载体转染人脑胶质瘤U87和U251细胞后，可有效抑制肿瘤细胞的增殖、侵袭和迁移能力，细胞免疫荧光提示在人脑胶质瘤细胞中过表达AJAP1后，可能通过对细胞骨架的重建、丝状伪足的减少，从而降低胶质瘤细胞侵袭、迁移能力。 3.成功建立U87细胞颅内胶质瘤模型，在体内进一步证实了AJAP1的抑瘤作用，与体外实验结果相符。 4.通过对AJAP1对胶质瘤增殖、侵袭、迁移等抑制作用的分子机制的初步研究，表明AJAP1可作为胶质瘤基因治疗的潜在靶点。AJAP1与细胞骨架关系密切，值得进一步深入探讨。
[Abstract]:Glioma is the most common primary intracranial tumor, of which more than 2/3 is malignant glioma, which accounts for about 2% of adult tumor, the median survival time of glioblastoma is only 1 years, the survival time of 5 years is almost zero [1-2]., and the comprehensive curative effect of postoperative chemotherapy and radiotherapy is still not very ideal. There is no radical cure. The long characteristic is the root cause of glioma which is difficult to cure and relapse. Therefore, the optimization of the treatment strategy of glioma should not only focus on the treatment of primary tumor, but also focus on controlling the invasion and metastasis of tumor cells to normal brain tissue. To find the related genes that can inhibit the invasion and metastasis of glioma cells, and to reveal its role in depth. The mechanism is of great significance for the treatment of glioma.
More and more evidence suggests that a number of recent studies in the human chromosome 1p36 region encode a number of tumor suppressor genes, [3-4]., the adhesion associated protein -1 (Adherens Junctional AssociatedProtein-1, AJAP1, also known as Shrew-1) as another similar tumor suppressor gene located in the human chromosome 1p36 region.Radlwimmer study The group of patients with glioblastoma were compared with genomic hybridization and microarray analysis. AJAP1 was first described as a biomarker [5]. associated with glioblastoma. The Zhang topic group proved that the AJAP1 promoter region was highly methylated [6]. but in the various cell lines, such as SVHVC, LOVO, SW480, MCF-7, but in glioma cells. The mechanism of changing the expression level of AJAP1 on invasion and proliferation of cells is still unclear.
This topic focuses on the abnormal expression of AJAP1 in glioma, discusses its relationship with the grade of glioma, and changes the expression level of AJAP1 in glioma cells by AJAP1 recombinant adenovirus transfection technology, and preliminarily studies the effect of AJAP1 on the biological phenotype of glioma and its potential mechanism.
Method
In the first part, immunohistochemical staining and immunofluorescence confocal imaging were used to detect the expression level of AJAP1 in 48 specimens of glioma and 5 normal brain tissue specimens. The level of AJAP1 protein expression in 8 glioma cell lines and 1 normal cell lines HEK293 was detected by Western blot method, and AJAP1 was preliminarily studied and analyzed. The relationship between the grade of glioma.
The second part focuses on the study of the human glioma cells transfected with AJAP1 recombinant adenovirus to the malignant biological phenotype of glioma cells and the effect of the cytoskeleton on the cytoskeleton in vitro. The potential mechanism.U87, U251 cells transfected with AJAP1 recombinant adenovirus plasmid, the Transewell test was used to detect the invasion ability of glioma cells. The proliferation ability of the cells was detected by the plate cloning test, and the scratch test was used to evaluate the cell migration ability.Western blot method to detect the changes of the proliferation and invasion of glioma cells, and the changes of cytoskeleton after the expression of AJAP1 were detected by immunofluorescence.
In the third part, the U87 glioma cells transfected with AJAP1 recombinant adenovirus and luciferase lentivirus were injected into the caudate nucleus of BALB/c-nu (SPF) female nude mice at the size of 5 weeks to establish the intracranial glioma model, and a blank control group was set up with the dynamic bioluminescence imaging system (IVISLumina Imaging System) dynamics. The growth of tumor and the change of the survival period of nude mice were observed. Histopathological methods were used to detect the changes in the expression of proliferation and invasion of the two groups of animal tumor specimens.
The detection of paraffin section by immunohistochemistry showed that the expression level of AJAP1 in the glioma samples was significantly lower than that of the normal brain tissue, and the expression of AJAP1 decreased with the increase of glioma level. The tissue immunofluorescence test also confirmed that the expression of AJAP1 in the high grade glioma specimens was significantly lower than that of the low grade glioma. In accordance with the immunohistochemical results, AJAP1 was located in the cytoplasm. Therefore, the expression level of AJAP1 was negatively correlated with the pathological grade of glioma. In the glioma cell line, the Western blot detection showed high expression in the human embryonic renal epithelial cell HEK293, but in most glioma cell lines, the low expression was the most low expression of A172, TJ905, TJ899. Then, U87, U251 glioma cells were transfected with AJAP1 recombinant adenovirus. By improving the protein expression level of AJAP1 in the cells, the AJAP1 group was compared with the blank control group and the empty carrier vector group. The clone formation test showed that the number of clones in the AJAP1 group was reduced, and the Transwell test showed the invasion of the AJAP1 group. The attack ability was obviously inhibited, the scratch test showed that the cell migration ability decreased, Western Blot detected Ki-67, and the expression level of MMP-9 protein decreased obviously. In the cell immunofluorescence test, after overexpression of AJAP1, the structure of U87 and U251 cytoskeleton changed, and the cell growth pattern of AJAP1 group was compared with the vector group, and the cell growth pattern changed. The original globular growth morphology changed to lamellar growth and filamentous foot decreased significantly.
In vivo animal experiments, the results of biological fluorescence imaging of living animals confirmed that the U87-AJAP1 formation tumor was significantly less than the U87-vector group, and the vector group died within 32D, and only 4 in AJAP1 group 32D died. The immunohistochemical results showed that the expression of AJAP1 was obviously up-regulated, and the table of Ki-67 and MMP-9 decreased obviously, which was in accordance with the results of cell experiment.
conclusion
1. the low expression of AJAP1 gene in glioma specimens and malignant glioma cell lines is negatively correlated with normal brain tissue, suggesting that AJAP1 may be used as a tumor suppressor gene to participate in the occurrence and development of glioma.
2. transfection of AJAP1 recombinant adenovirus vector to human glioma U87 and U251 cells can effectively inhibit the proliferation, invasion and migration of tumor cells. Cell immunofluorescence suggests that after overexpression of AJAP1 in human glioma cells, it may be possible to reduce the invasion and migration of glioma cells by reconstructing the cytoskeleton and reducing the filamentous pseudo foot. Ability to move.
3. the U87 cell glioma model was successfully established, which further confirmed the inhibitory effect of AJAP1 in vivo.
4. the preliminary study of the molecular mechanism of AJAP1 on the proliferation, invasion and migration of glioma shows that AJAP1 can be a potential target of gene therapy for glioma,.AJAP1 is closely related to cytoskeleton, and it is worth further exploring.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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