CMG2对胶质瘤细胞侵袭能力的促进作用及其机制

发布时间：2018-03-19 08:39

本文选题：脑胶质瘤　切入点：CMG2　出处：《第三军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的胶质瘤是中枢神经系统常见的恶性肿瘤。尽管近年来其治疗方法有了长足的进步,但其疗效仍不尽人意。高度侵袭性是导致病人预后差的重要原因之一。因此,深入研究胶质瘤侵袭的分子机制,对于发展有效的胶质瘤治疗新策略具有重要意义。毛细血管形态发生基因(Capillary morphogenesis gene 2,cmg2)因其在血管形成过程显著上调表达而被发现,因其编码的蛋白能介导炭疽杆菌毒素进入细胞内,故又称其为炭疽毒素受体2(Anthrax Toxin Receptor 2,ANTXR2),并被证明是炭疽杆菌毒素亲和力最强的受体。业已阐明CMG2突变会导致幼年透明性纤维瘤病(Juvenile Hyaline Fibromatosis)。但CMG2确切的生理功能尚不十分清楚。近年来,已有关于CMG2与肿瘤关系的报道,CMG2高表达能促进前列腺癌的转移。正常脑组织中不表达CMG2,至今也无CMG2与胶质瘤关系的研究报道,但本课题组前期研究发现CMG2在胶质瘤中显著高表达,并与肿瘤级别呈正相关而与病人预后呈负相关。鉴于侵袭性是胶质瘤最显著的恶性生物学行为之一,本课题研究的目的在于探索CMG2在胶质瘤侵袭中的作用及可能的的分子机制,从而有望加深对胶质瘤侵袭机制的认识,也可能为胶质瘤的预后判断提供新的指标并为临床治疗提供新的靶点。主要方法1.利用慢病毒方法,构建稳定过表达CMG2的胶质瘤细胞U87 OE和091214 OE以及稳定敲低CMG2表达的胶质瘤细胞U87-sh2。2.Matrigel-transwell侵袭实验观察过表达/敲低CMG2细胞侵袭能力的差异,划痕实验观察其迁移能力的改变。3.建立小鼠原位移植瘤模型,观察过表达CMG2与否的胶质瘤细胞在体内的侵袭能力的差异以及对小鼠生存期的影响。4.细胞免疫荧光方法用于观察不同CMG2表达状态胶质瘤细胞丝状伪足和层状伪足的差异。5.Western Blot检测比较CMG2过表达与否的胶质瘤细胞中YAP及p-YAP的表达水平。6.免疫组织化学方法分析72例不同级别的胶质瘤病人肿瘤组织中YAP的表达特征,采用Kaplan-Meier法和LogRank检验分析YAP在胶质瘤组织中的表达与病人预后的关系。结果1.CMG2促进胶质瘤细胞侵袭和迁移能力(1)体外Matrigel-transwell侵袭实验和划痕实验显示:与对照组相比较,过表达CMG2增强胶质瘤细胞侵袭和迁移能力(p0.05),而敲低CMG2则降低胶质瘤细胞的迁移能力(p0.05)。(2)小鼠体内原位移植瘤实验显示:与对照组相比较,种植了过表达CMG2的胶质瘤细胞的小鼠肿瘤组织前沿具明显侵袭现象,小鼠生存期缩短(p0.05);种植CMG2下调表达细胞的小鼠生存期延长(p0.05)。2.CMG2促进胶质瘤细胞层状伪足和丝状伪足的形成细胞免疫荧光染色分析发现:与对照组相比较,过表达CMG2的胶质瘤细胞能形成更多的层状伪足和丝状伪足。3.CMG2上调胶质瘤细胞中YAP的表达Western Blot检测分析发现:伴随过表达CMG2的胶质瘤细胞伪足形成增多,Hippo通路的关键效应蛋白YAP的表达上调。4.胶质瘤组织中YAP的表达与肿瘤级别呈正相关而与患者预后呈负相关(1)免疫组织化学分析显示:YAP在高级别胶质瘤中的表达高于低级别(p0.05)。(2)生存分析显示:YAP高表达的胶质瘤病人预后不良(p0.05)。结论1.CMG2对胶质瘤细胞侵袭能力具有促进作用。2.CMG2可能通过上调YAP表达诱导伪足形成进而促进胶质瘤细胞侵袭。3.人胶质瘤组织中YAP的表达与肿瘤级别呈正相关,与胶质瘤病人预后呈负相关。
[Abstract]:Background and objective: glioma is the most common malignant tumors of the central nervous system. In recent years although the treatment method has made great progress, but its effect is still unsatisfactory. Highly invasive is an important cause of poor prognosis of patients. Therefore, further study of molecular mechanism of glioma invasion, is of great significance for the new strategy for the treatment of glioma the development of tumor effectively. Capillary morphogenesis gene (Capillary morphogenesis gene 2, cmg2) due to the upregulation of vascular formation was found, because of its encoding protein can mediate the anthrax toxin into cells, it is also known as anthrax toxin receptor 2 (Anthrax Toxin 2 Receptor, ANTXR2), and anthrax toxin was proved to be the highest affinity receptor. It is already illustrated CMG2 mutations can lead to juvenile hyaline fibromatosis (Juvenile Hyaline Fibromatosis). But the exact physiological CMG2 Function is not very clear. In recent years, has been reported about the relationship between CMG2 and tumor, the high expression of CMG2 can promote the metastasis of prostate cancer. The expression of CMG2 in normal brain tissue, the research report has no relationship between CMG2 and glioma, but the previous study found that CMG2 in glioma tissues, and was positively correlated with tumor grade and negatively correlated with the prognosis of the patients. In view of the invasive biological behavior of malignant glioma is one of the most significant, the purpose of this study is to explore the effect of CMG2 on the invasion of glioma cells and the possible molecular mechanism, which is expected to deepen the understanding on the mechanism of the invasion of glioma, may provide new indicators and provide a new target for clinical treatment for glioma prognosis judgment. The 1. main methods of using lentiviral method, construct the stable over expression of CMG2 glioma cells U87 OE and 091214 OE and stability On U87-sh2.2.Matrigel-transwell glioma cells with low CMG2 expression of invasion experiment observed differential expression / invasive ability of CMG2 cells at low, scratch test to observe the migration ability change.3. mice orthotopic transplantation tumor model, observe the expression of CMG2 and glioma cells in vivo invasion ability difference and the survival of the mice.4. immunofluorescence methods were used to analyze the expression features of 72 cases with different levels of glioma patients in tumor tissue YAP expression level of.6. immunohistochemical method between.5.Western Blot detection to observe the different expression of CMG2 in glioma cells and filopodia between CMG2 YAP lamellar pseudopodia and the expression of p-YAP and glioma cells, the relationship between by using Kaplan-Meier method and LogRank test to analyze the expression of YAP and prognosis in glioma tissues. The results showed that 1.CMG2 promotes glioma Tumor cell invasion and migration (1) showed that in vitro Matrigel-transwell assay and scratch test: compared with the control group, overexpression of CMG2 enhances the invasion and migration of glioma cells (P0.05), while knockdown of CMG2 decreased the migration ability of glioma cells (P0.05). (2) showed in mice results: compared with the control group, planting overexpression glioma cells CMG2 mice tumor tissue invasion frontier has obvious phenomenon, shorten the survival time of mice (P0.05); cultivation of down regulated expression of CMG2 cells of mice prolonged survival (P0.05).2.CMG2 promotes glioma cells and filopodia formation of lamellar pseudopodia cell immunofluorescence staining the analysis found that: compared with the control group, expression of Western Blot detected CMG2 glioma cells can form more lamellar pseudopodia and filopodia upregulation of.3.CMG2 glioma cells in YAP analysis Now with over expression of glioma cell pseudopod formation of CMG2 was increased, and the expression of tumor associated protein YAP level key effect of Hippo pathway up-regulated the expression of.4. in human glioma YAP and prognosis in patients with negative correlation (1) immunohistochemical analysis showed that the expression of YAP in high grade gliomas was higher than that of low level (P0.05). (2) survival analysis showed that the prognosis of patients with glioma YAP high expression of bad (P0.05). Conclusion 1.CMG2 can promote the.2.CMG2 may regulate YAP expression and tumor grade was positively induced podosome formation and promote invasion of.3. human glioma tissues YAP glioma cells related to glioma cell invasion ability, and negatively related to the prognosis of glioma patients.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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