【作者】 王伟；

【导师】 杨学军；

【作者基本信息】 天津医科大学 ， 外科学 神外（专业学位）， 2017， 硕士

【摘要】 胶质母细胞瘤(glioblastoma,GBM)是中枢神经系统中最常见的原发性肿瘤[1],具有细胞高度增殖、血管形成异常、向周围侵袭迁移等生物学特点[2,3]。随着新的影像学辅助技术的不断发展和微创手术理念的不断完善[4,5],肿瘤的影像学切除率得到极大程度的提高,患者的中位生存期获得延长,生存质量得以改善,然而由于胶质母细胞侵袭浸润性生长的生物学特性,导致肿瘤极易发生侵袭迁移,引起肿瘤的复发播散,严重制约了治疗效果的提高,也导致了患者不良的预后[6]。肿瘤微环境是一个复杂的动态局部环境,包括肿瘤细胞、肿瘤基质细胞(免疫细胞、成纤维细胞等)、细胞外基质以及微环境内细胞通过旁分泌或自分泌产生的多种细胞外因子,在肿瘤的发生发展、增殖以及侵袭迁移中发挥着重要作用[7,8]。由于肿瘤组织内细胞增殖迅速、血管形成异常、能量代谢异常的特点,导致了肿瘤细胞营养缺乏以及肿瘤微环境中肿瘤细胞外酸内碱的状态[9,10],肿瘤细胞可能为了破解这种生存环境的压力而向远处转移,侵袭到营养相对丰富更适宜的区域,造成肿瘤的播散。钠氢交换体1(sodium hydrogen exchanger 1,NHE1)是钠氢交换体家族中的一员,是广泛分布于各类细胞细胞膜的一离子通道种,生理状态下,NHE1的主要功能在于维持细胞内外酸碱平衡以及细胞体积的调节[11]。研究表明,NHE1是肿瘤细胞微环境酸化的主要调节因子[12],在多种肿瘤如肝癌[13,14]、宫颈癌[15,16]、乳腺癌[17,18]等中表达上调或激活,增加了肿瘤细胞侵袭迁移的能力。而在胶质瘤研究中其发挥怎样的作用尚不明确。本课题研究胶质瘤组织NHE1的表达及其与预后的关系,并通过体外实验在高表达NHE1的人恶性胶质瘤细胞系中探究其在胶质瘤细胞侵袭迁移运动的作用及其可能机制。课题研究分为以下两个部分:1、NHE1在不同级别胶质瘤组织中的表达情况及其与临床预后的关系。首先应用癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中GBM及较低级别胶质瘤(lower grade glioma,LGG)患者的样本数据及其对应的临床数据,分析NHE1在GBM及LGG患者中的差异性表达,并依据NHE1表达情况的差异,分析GBM患者的临床预后;然后应用免疫组织化学染色的方法对我院手术采集的40例不同病理级别的胶质瘤组织样本及10例正常对照脑组织样本进行检测,分析样本中NHE1表达情况的差异;同时对不同病理级别的胶质瘤组织样本及对照脑组织中NHE1 mRNA水平和蛋白的表达量应用Real-time PCR和western blot的方法进行定量分析。然后采用western blot的方法筛选出表达NHE1蛋白较高的恶性胶质瘤细胞系进行后续体外实验。结果显示,TCGA数据库中172例GBM患者样本中NHE1的表达水平明显高于350例LGG患者样本中NEH1的表达水平,NHE1的表达水平与GBM患者的预后存在显著相关性,表达水平越高,患者预后越差,生存期越短。本院采集的手术组织样本中,NHE1在对照脑组织样本中不表达或弱阳性表达,在高级别胶质瘤组织样本中表达增高,并且阳性率随着肿瘤病理级别的增高而增高。Real-time PCR和western blot的检测结果显示与TCGA数据库分析结果及免疫组化染色结果相符合。在U87和SNB19两个胶质瘤细胞系中western blot检测显示NHE1蛋白表达量较高,用于后续体外实验研究。2、研究NHE1在胶质瘤细胞迁移和侵袭中的作用。应用含有0.2%胎牛血清的DMEM培养基培养细胞,在U87和SNB19两种细胞系中,应用干扰NHE1表达的siRNA对细胞进行转染,分别分为siRNA-NHE1组、siRNA-NC组(转入无义序列)和control组,在不同的处理组中,NHE1 mRNA及蛋白表达水平分别采用Real-time PCR和western blot检测,细胞免疫荧光技术检测NHE1的表达情况与细胞伪足形成情况,Transwell实验和划痕实验分别检测胶质瘤细胞的侵袭和迁移能力,荧光基质胶降解实验观察NHE1对于胶质瘤细胞侵袭性伪足形成和荧光基质胶降解的影响,western blot技术检测胶质瘤细胞MMP-9、MMP-2、cortactin、p-cortactin的蛋白表达量。结果显示:应用siRNA敲低NHE1的表达,抑制了胶质瘤细胞的侵袭迁移的能力;NHE1蛋白在细胞的侵袭性伪足前端富集;敲低NHE1的表达后其侵袭性伪足形成和基质胶降解能力显著下降(P<0.05);相较于control组和siRNA-NC组,si RNA-NHE1组胶质瘤细胞MMP-9、MMP-2、p-cortactin表达明显降低(P<0.05),cortactin无明显变化(P>0.05)。结论:1、胶质瘤组织样本中NHE1表达增高,高级别胶质瘤组织样本中表达明显高于正常对照脑组织样本与低级别胶质瘤组织样本,并且在GBM患者中NHE1的表达水平与患者预后相关,表达越高,预后越差。2、抑制NHE1蛋白的表达,可抑制胶质瘤细胞MMP-2和MMP-9的表达以及侵袭性伪足的形成,进而影响胶质瘤细胞侵袭和迁移的能力。3、NHE1富集于侵袭性伪足前端,可能通过改变侵袭伪足局部pH值影响cortactin的磷酸化,从而影响侵袭性伪足的形成。4、针对NHE1的靶向药物有望抑制胶质瘤细胞的侵袭迁移。更多还原

【Abstract】 Glioblastoma is the most common primary tumor in central nervous system,with the characteristics of highly proliferation,abnormal angiogenesis and invasion and migration to the surrounding areas.Despite the continuous improvement of new imaging technologies and development of surgery,the extent of resection for gliomas has been greatly increased and the patients’ median survival was prolonged.However,due to the characteristics of invasion and migration,the patients might face inevitable relapse and poor prognosis.Tumor microenvironment is a complex dynamic local environment composing of tumor cells,stromal cells(immune cells,fibroblasts,etc.),extracellular matrix and a variety of extracellular factors which plays an important role in tumor cell development,proliferation,and invasion and migration.Rapid proliferation of tumor cells,abnormal blood vessels and abnormal energy metabolism would result in tumor cells nutrition deficiency and acid extracellular environment as well.In order to evade this environmental stress,migration to relatively nutrient rich region might be an optimal choice to survive for tumor cells.Sodium hydrogen exchanger 1(NHE1)is a member of sodium hydrogen exchangers family,which is widely expressed in cell membrane of various type of cells.In physiological condition,NHE1 plays a critical role in intracellular pH(pHi)and cell volume homeostasis.NHE1 is also an important regulator in extracellular acidification and upregulated or activated in a variety of tumors and leads to invasion and migration,such as hepatocellular carcinoma,cervical carcinoma,breast cancer and so on.However,the role of NHE1 remains unclear in glioma.This study aims to investigate the expression of NHE1 in gliomas and the underlying mechanisms of NHE1 in invasion and migration of glioma cells,including two parts:1.To detect the expression of NHE1 in gliomas and analyze the relationship with clinical prognosis.First of all,we downloaded the gene expression data and clinical data of patients with GBM and lower grade glioma patients(LGG),including grade WHOⅠ-Ⅲ in TCGA database and analyzed the expression of NHE1,statistical assessment was performed for the relationship between NHE1 expression and prognosis in GBM patients.In addition,40 glioma samples with different pathological grades and 10 control brain tissue samples were obtained from surgey for immunohistochemical staining;At the same time,Real-time PCR and western blot were employed to detect the expression of NHE1 at mRNA level and protein level.Then,western blot was used to detect the expression of NHE1 in four human malignant glioma cell lines,and the cell lines with higher expression of NHE1 were selected for the later in vitro experiments.The results showed that NHE1 expression in 172 GBMs was significantly higher than that in 350 LGGs,prognosis of patients with GBM was negatively correlated with the expression of NHE1.The immunohistochemical staining demonstrated that the NHE1 expression was elevated in GBM,and the expression level increased positively with tumor pathological grade.The results of Real-time PCR and western blot were also consistent with immunohistochemical results.In U87 and SNB19 glioma cell lines,the expression of NHE1 protein was increased significantly,which was selected for in vitro experiments.2.To investigate the role of NHE1 in glioma cell migration and invasion.In low serum culture conditions,siRNA aimed at silencing NHE1 was transfected into U87 and SNB19 cell lines to knock down the NHE1 expression.Those cells were assigned into three group:siRNA-NHE1 group,siRNA-NC group and control group.The expression of NHE1 was detected at mRNA and protein level respectively using Real-time PCR and western blot,transwell invasion assay and wound healing assay were performed to determine the migration and invasion of glioma cells,the expression of MMP-2,MMP-9,cortactin and p-cortactin were detected by western blot,the invadopodia and NHE1 location of glioma cells were observed by immunofluorescence,the formation of invadopodia and the invasion ability were detected by fluorescent Matrigel degradation experiments.The results showed that NHE1 was enriched in cell barbed end of invadopodia,compared with control group and siRNA-NC group.The expression of NHE1 was inhibited at both mRNA and protein level after transfected,the ability of migration and invasion of glioma cells was markedly impeded.In the siRNA-NHE1 group the expression of MMP-2,MMP-9 and p-cortactin were decreased significantly,while cortactin did not change significantly,the invadopodia formation and the degraded area of matrix for each of the tumor cells also decreased significantly.Conclusion:1.Expression of NHE1 in glioma tissues compared with control brain tissue,and the expression is significantly increased in GBM tissue than that in lower grade glioma tissue,the expression is negatively correlated with the prognosis of patients with GBM.2.Knocking down NHE1 expression can inhibit the expression of MMP-2 and MMP-9 in glioma cells and decrease formation of invadopodia and ability of invasion.3.NHE1 is enriched in cell barbed end of invadopodia,which might affect the formation of invadopodia by altering the local pH of invadopodia.4.Targeting at NHE1 is expected to inhibit the invasion and migration of glioma cells.更多还原