nNavl．5在人脑星形细胞瘤中的表达及意义

目的：研究nNav1．5mRNA及蛋白在人脑星形细胞瘤组织和正常脑组织中的表达差异。方法：采用逆转录聚合酶链反应（RT—PCR）及western blot法分别检测nNav1．5mRNA及蛋白在30例人脑星形细胞瘤组织和8例正常脑组织中的表达水平。结果：人脑星形细胞瘤组织中nNav1．5基因及蛋白的表达值显著高于正常脑组织（P〈0．01）,且随病理级别的增高而升高。结论：nNav1．5mRNA及蛋白在人脑星形细胞瘤组织中的表达水平显著升高,这可能与星形细胞瘤细胞增殖活跃及侵袭性生长等生物特性有关。     

Livin在人脑星形细胞瘤中的表达及意义

目的：探讨凋亡抑制蛋白（inhibitor of apoptosis protein，IAP）家族成员livinα和livinβ在人脑星形细胞瘤中的表达及与星形细胞瘤临床病理特征之间的关系。方法：采用逆转录聚合酶链反应（RT-PCR）检测31例人脑星形细胞瘤、18例炎性肿块、10例正常脑组织中livinα和livinβ的mRNA表达水平，并用western blot检测2种livin蛋白的表达情况。结果：29例人脑星形细胞瘤有livinα和livinβ的mRNA表达，16例炎性肿块、9例正常脑组织中有livinα或livinβ的mRNA表达，三者表达率无统计学意义，但livinα和livinβ在星形细胞瘤中的表达值显著高于炎性肿块和正常脑组织（P〈0．01）。不同级别星形细胞瘤间livin的表达无统计学意义。结论：人脑星形细胞瘤中livin在mRNA及蛋白水平的表达均明显增高，两者结果一致．可能与星形细胞瘤的形成和细胞增殖活跃有关。     

EMP1基因在人脑胶质瘤中的表达及其临床意义

目的:探讨上皮膜蛋白1基因(epithelial membrane protein 1,EMP1)在人脑胶质瘤中的表达及其与肿瘤恶性程度的相互关系。方法:肿瘤组织标本均取自上海长征医院神经外科2005至2006年部分手术病例,正常脑组织来源于捐献,分别采用逆转录酶聚合酶链反应(RT-PCR)和免疫组化染色方法检测33例星形细胞瘤、3例少枝胶质细胞瘤、2例室管膜瘤以及7例正常脑组织标本中EMP1 mRNA及蛋白的表达。结果:EMP1无论在基因还是在蛋白表达水平上,在胶质瘤和正常脑组织中均有不同程度表达,但在星形细胞瘤中表达的含量明显高于正常脑组织,且在星形细胞瘤中含量随着其病理分级的增高而增高,在低级别(WHOⅠ～Ⅱ级)与高级别(Ⅲ～Ⅳ级)之间存在显著差异(蛋白P<0.05;基因P<0.01)。结论:基因EMP1在人脑胶质瘤中显著高表达,并且与星形细胞瘤的恶性程度密切相关。     

不同病理分级星形细胞瘤中TRPML-2的表达

目的 探讨瞬时受体电势阳离子通道mucolipin亚家族-2(transient receptor potential cation channel,mu-colipin subfamily,member 2,TRPML-2)通道蛋白在不同病理分级星形细胞瘤中的表达水平.方法 收集经病理诊断证实的56例不同病理分级的星形细胞瘤组织和10例正常脑组织标本,通过实时荧光定量PCR(real-time quanti-tative PCR,qRT-PCR)和免疫组织化学法检测TRPML-2 mRNA和蛋白水平.结果 高级别星形细胞瘤与正常脑组织和低级别星形细胞瘤比较在TRPML-2 mRNA和蛋白表达上差异均具有统计学意义(均P<0.05).Spearman等级相关分析显示,TRPML-2的表达水平与星形细胞瘤的病理分级呈正相关关系(r=0.688,P<0.05).结论TRPML-2表达水平随星形细胞瘤的恶性程度增高而增高.     

转化生长因子α及其受体在人脑星形细胞瘤中的表达及意义

目的　研究转化生长因子α(transforminggrowthfactoralpha ,TGF α)及其表皮生长因子受体 (epidermalgrowthfactorreceptor ,EGFR)在人脑星形细胞瘤中的表达及意义。方法　采用免疫组织化学方法检测 5 0例人脑星形细胞瘤标本和 10例正常人脑组织中TGF α和EGFR蛋白表达。结果　 5 0例人脑星形细胞瘤TGF α和EGFR蛋白表达总阳性率分别为 64 .0 % ( 3 2 /5 0 )和 68.0 % ( 3 4/5 0 ) ,正常脑组织未见阳性表达 (P <0 .0 1) ;二者密切相关 (P <0 .0 1) ,并与星形细胞瘤病理分级有显著相关性 ,其中Ⅰ～Ⅱ级者阳性率显著低于Ⅲ、Ⅳ级者 (P <0 .0 5 ) ;结论　TGF α和EGFR形成的自分泌环在人脑星形细胞瘤发生发展过程中起重要作用 ,同时检测它们可作为判断人脑星形细胞瘤临床分期、预后的重要指标     

人脑胶质瘤高表达nNav1.5促进肿瘤细胞侵袭

目的：观察电压-门控钠离子通道（VGSCs）亚型nNav1.5在人脑胶质瘤组织中的表达,探讨nNav1.5表达对胶质瘤U251细胞迁移及侵袭的影响。方法：收集本科室手术切除的脑胶质瘤标本68例,应用免疫组织化学S-P法检测胶质瘤组织中nNav1.5的表达。设计合成nNav1.5特异性小干扰RNA（small interfering RNA,siRNA）,用脂质体转染至U251细胞,应用Real-time RT-PCR和Western blot法分别检测nNav1.5的mRNA和蛋白表达水平的变化,应用划痕实验和Matrigel侵袭实验检测U251细胞迁移和侵袭能力。结果：与正常组织相比,nNav1.5在胶质瘤组织中表达率明显升高（72.6% vs 23.0%,P < 0.01）,并且其在低级别胶质瘤中（WHOⅠ～Ⅱ级）明显高于其在高级别胶质瘤（WHO Ⅲ～Ⅳ级）中的表达率（52.9% vs 85.8%,P < 0.01）;siRNA显著抑制U251细胞中nNav1.5的mRNA和蛋白表达并明显降低U251细胞的迁移侵袭能力。结论：电压-门控钠离子通道nNav1.5在胶质瘤中高表达并促进U251细胞的迁移和侵袭能力,nNav1.5是胶质瘤恶性侵袭的调控因子,有望成为胶质瘤的新标记物和治疗靶点。     

人脑胶质瘤组织高表达nNav1.5促进肿瘤细胞的迁移和侵袭

目的：观察电压-门控钠离子通道（voltage-gated sodium channel, VGSC）亚型nNav1.5在人脑胶质瘤组织中的表达,并探讨其对脑胶质瘤U251细胞迁移及侵袭的影响。方法：收集中国医科大学附属第一医院神经外科于2011年10月至2012年10月手术切除并经病理证实的脑胶质瘤组织标本68例,应用免疫组织化学S-P法检测脑胶质瘤组织中nNav1.5的表达。设计并化学合成nNav1.5基因特异性小干扰RNA（nNav1.5-siRNA）,用脂质体介导转染胶质瘤U251细胞,应用Real-time PCR和Western blotting法分别检测U251细胞中nNav1.5 mRNA和蛋白的表达水平,并采用细胞划痕实验和Transwell侵袭实验检测U251细胞迁移和侵袭能力的变化。结果：nNav1.5在人脑胶质瘤组织中表达的阳性率显著高于正常组织（72.6% vs 23.0%,P<0.01）,并且其在高级别胶质瘤（WHO Ⅲ～Ⅳ级）组织中的阳性率明显高于低级别胶质瘤（WHOⅠ～Ⅱ级）组织（85.8% vs 52.9%,P<0.01）。nNav1.5-siRNA转染可显著抑制U251细胞中nNav1.5 mRNA和蛋白的表达（P<0.01）;转染后U251细胞的迁移距离明显小于未转染细胞\[(0.019±0.015) vs（0.223±0.031）mm,P<0.01\],且其侵袭指数明显低于未转染细胞\[(2.99±0.15)% vs（6.77±0.26）%,P<0.01\]。〖JP2〗结论：nNav1.5在人脑胶质瘤组织中高表达,干扰nNav1.5表达可显著抑制胶质瘤细胞的迁移和侵袭能力,nNav1.5是胶质瘤恶性侵袭的调控因子并有望成为胶质瘤的新标志物和治疗靶点。     

HIF-1α、VEGF、EphB4/ephrinB2在人脑星形细胞瘤中的表达及临床意义

目的 研究HIF-1α、VEGF、EphB4及其配体ephrinB2在人脑星形细胞瘤中的表达,探讨其与星形细胞瘤临床病理特征的关系.方法 应用组织芯片技术及免疫组化SP法检测60例人脑星形细胞瘤和13例正常脑组织中HIF-1α、 VEGF、EphB4/ephrinB2表达及微血管密度(MVD).结果 HIF-1α、VEGF、EphB4/ephrinB2蛋白在星形细胞瘤表达水平及MVDs与肿瘤临床病理分级显著相关(P＜0.05),与患者年龄、性别及肿瘤的大小无关.HIF-1α、VEGF、EphB4/ephrinB2两两间均呈正相关.结论 HIF-1α、 VEGF、EphB4/ephrinB2过表达与肿瘤临床病理分级关系密切,可能成为判断星形细胞瘤恶性进展和预后的重要指标.     

uPA和VEGF在脑星形细胞瘤侵袭中的作用及其相关性的研究

 目的　探讨尿激酶型纤溶酶原激活剂 (uPA)和血管内皮细胞生长因子 (VEGF)在人脑星形细胞瘤中的表达特征及意义。方法　用免疫组化的方法检测uPA和VEGF在 6 4例星形细胞瘤、4例垂体腺瘤、5例正常脑组织中的表达情况。结果　 6 4例星形细胞瘤中均有uPA和VEGF的表达 ,且随恶性程度的升高 ,其表达也随之升高。在垂体腺瘤中低度表达 ,在正常脑组织中无表达。uPA和VEGF在星形细胞瘤中的表达呈正相关 (r =0 .92 )。结论　uPA和VEGF的表达与星形细胞瘤的侵袭性和血管的生成等有关。uPA和VEGF在星形细胞瘤中的表达具有协同性     

间变性星形细胞瘤中DR4及DR5的表达

目的研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)的死亡受体(death receptor,DR)DR4和DR5在间变性星形细胞瘤中的表达,并探讨其临床意义.方法联合采用免疫组化和原位杂交方法检测24间变性星形细胞瘤和16例正常脑组织中DR的表达.结果免疫组化染色显示,24例间变性星形细胞瘤均大量表达死亡受体DR4和DR5,而16例正常脑组织中7例(43.8%)表达DR4,5例(31.3%)表达DR5.间变性星形细胞瘤组织中DR蛋白的表达显著高于正常脑组织中DR蛋白的表达,两者差异有显著性(P<0.01).原位杂交显示,DR在全部24例间变性星形细胞瘤组织和大部分正常脑组织中均呈强阳性表达,二者差异无显著性(P>0.05).结论间变性星形细胞瘤细胞中普遍存在DR的高表达,这可能为间变性星形细胞瘤的凋亡诱导治疗提供新的靶点.DR蛋白在正常脑组织和间变性星形细胞瘤中的表达差异,可能是TRAIL选择性诱导凋亡的机制之一.     

Invasion and Metastasis Suppression by Anti-Neonatal Nav1.5 Antibodies in Breast Cancer

Background: Detectable neonatal Nav1.5 (nNav1.5) expression in tumour breast tissue positive for lymph node metastasis and triple-negative subtype serves as a valid tumour-associated antigen to target and prevent breast cancer invasion and metastasis. Therapeutic antibodies against tumour antigens have become the predominant class of new drugs in cancer therapy because of their fewer adverse effects and high specificity. Objective: This study was designed to investigate the therapeutic and anti-metastatic potential of the two newly obtained anti-nNav1.5 antibodies, polyclonal anti-nNav1.5 (pAb-nNav1.5) and monoclonal anti-nNav1.5 (mAb-nNav1.5), on breast cancer invasion and metastasis. Methods: MDA-MB-231 and 4T1 cells were used as in vitro models to study the effect of pAb-nNav1.5 (59.2 µg/ml) and mAb-nNav1.5 (10 µg/ml) (24 hours treatment) on cell invasion. 4T1-induced mammary tumours in BALB/c female mice were used as an in vivo model to study the effect of a single dose of intravenous pAb-nNav1.5 (1 mg/ml) and mAb-nNav1.5 (1 mg/ml) on the occurrence of metastasis. Real-time PCR and immunofluorescence staining were conducted to assess the effect of antibody treatment on nNav1.5 mRNA and protein expression, respectively. The animals’ body weight, organs, lesions, and tumour mass were also measured and compared. Results: pAb-nNav1.5 and mAb-nNav1.5 treatments effectively suppressed the invasion of MDA-MB-231 and 4T1 cells in the 3D spheroid invasion assay. Both antibodies significantly reduced nNav1.5 gene and protein expression in these cell lines. Treatment with pAb-nNav1.5 and mAb-nNav1.5 successfully reduced mammary tumour tissue size and mass and prevented lesions in vital organs of the mammary tumour animal model whilst maintaining the animal’s healthy weight. mRNA expression of nNav1.5 in mammary tumour tissues was only reduced by mAb-nNav1.5. Conclusion: Overall, this work verifies the uniqueness of targeting nNav1.5 in breast cancer invasion and metastasis prevention, but more importantly, humanised versions of mAb-nNav1.5 may be valuable passive immunotherapeutic agents to target nNav1.5 in breast cancer.     

Hormonal therapy for postmenopausal breast cancer: the science of sequencing

Upregulation of functional voltage-gated Na⁺ channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the ‘neonatal’ splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by ∼90%) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (∼30%), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33%, and significantly increased the cells’ sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43%, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42%, in a dose-dependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.     

Expression and aberrant promoter methylation of Wnt inhibitory factor-1 in human astrocytomas

Background

Wnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the expression and promoter methylation status of WIF-1 gene in human astrocytomas.

Methods

The tissue samples consisted of 53 astrocytomas and 6 normal brain tissues. The expression levels of WIF-1 were determined by immunohistochemistry and semiquantitative RT-PCR. The results were analyzed in correlation with clinicopathological data. Methylation status of WIF-1 gene promoter was investigated using methylation specific PCR. The relationship between methylation and expression of the genes was analyzed.

Results

The average expression levels of WIF-1 protein and mRNA in astrocytomas were decreased significantly compared with normal control tissues. The protein and mRNA expression of WIF-1 gene in astrocytomas was decreased with the increase of pathological grade. Furthermore, WIF-1 promoter methylation was observed by MS-PCR in astrocytomas which showed significant reduction of WIF-1 expression. The WIF-1 promoter hypermethylation was associated with reduced expression of WIF-1 expression.

Conclusion

Our results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation. This may be an important mechanism in astrocytoma carcinogenesis.     

肝细胞生长因子及其受体mRNA在星形细胞瘤中的表达

目的　探讨肝细胞生长因子(HGF)及其受体(c Met)mRNA在正常脑组织和脑星形细胞瘤中的表达规律,探讨其与肿瘤增殖、肿瘤血管生成、临床病理及生存时间之间的关系。方法　用原位杂交法检测57例脑星形细胞瘤和5例正常脑组织标本HGF、c Met mRNA的表达;用免疫组织化学方法检测PCNA在肿瘤细胞中的表达,CD34 免疫组化反应检测并计算微血管密度(MVD),并结合临床病理资料进行分析。结果　在正常脑组织中未见HGF 和c Met 表达,而在脑星形细胞瘤中HGF、c Met、PCNA及CD34表达随病理级别的增高而明显增强(P<0.05),随生存期的延长而明显减弱(P<0.05),并且与患者的性别、年龄、肿瘤部位、肿瘤直径无关(P>0.05);HGF表达与c Met、PCNA的表达及MVD之间显著相关。结论　HGF/c Met在脑星形细胞瘤的形成与发展过程中起重要作用,它可促进肿瘤增殖及瘤组织内微血管的发生,并对预后判断有指导意义。     

MSP58基因表达水平与人脑胶质瘤恶性度的关系

目的:观察MSP58基因表达水平与人脑胶质瘤恶性度的关系.方法:在41例按WHO分类和分级标准为Ⅰ-Ⅳ级的人脑胶质瘤标本、4株人脑胶质瘤细胞系(U251,U87,BT325和SHG44)以及6例正常脑组织标本中,运用半定量RT - PCR及蛋白质印迹法检测MSP58 mRNA和蛋白的表达水平.结果:MSP58 mR-NA和蛋白在人脑胶质瘤组织中存在表达,其表达水平随人脑胶质瘤恶性度的增加而升高.在正常脑组织和恶性脑胶质瘤之间,以及脑胶质瘤Ⅰ-Ⅳ级病理级别间比较均有显著差异.MSP58 mRNA和蛋白在U251、U87、BT325和SHG44细胞中均有高表达.结论:MSP58 mRNA和蛋白在人脑胶质瘤中高表达,其表达水平与脑胶质瘤的恶性度有关,提示MSP58在脑胶质瘤的形成和恶性演进中具有重要作用.     

Expression of insulin-like growth factors I and II and their receptor mRNAs in primary human astrocytomas and meningiomas; in vivo studies using in situ hybridization and immunocytochemistry.

These studies demonstrate the expression of IGF-1, IGF-11, and their respective receptor mRNAs in primary human astrocytomas and meningiomas. In situ hybridization and immunocytochemistry have localized a strong expression of both IGF-1 and IGF-11 mRNAs and of their protein products in the tumor cells of astrocytomas and meningiomas. The expression of IGF-1 and IGF-11 mRNAs in the tumor cells was accompanied by the co-expression of their respective type-1 and type-11 IGF receptor mRNAs. Control, non-malignant human brain expressed IGF-1 mRNA and IGF-1 and IGF-11 receptor mRNAs. There was no significant expression of IGF-11 MRNA in the control brain specimens. Control pachymeninges (dura mater) expressed low levels of IGF-1 mRNA and IGF-1 receptor mRNA. There was no significant expression of IGF-11 and IGF-11 receptor mRNAs in pachymeninges. The co-expression of IGFs and their receptors in brain tumors may contribute in their development and maintenance. The strong inappropriate expression of IGF-11 mRNA and its protein product in the tumor cells of astrocytomas and meningiomas, but not in normal brain specimens, may serve as molecular markers for the early detection of these tumors.     

Expression of N-cadherin and alpha-catenin in astrocytomas and glioblastomas.

We examined levels of mRNA and protein for N-cadherin, the predominant cadherin in neural tissues, and mRNA levels for the cadherin-associated protein, alpha-catenin, in a series of gliomas and in glioblastoma cell lines. mRNA levels for N-cadherin and alpha-catenin were significantly higher in glioblastomas than in low-grade astrocytomas or normal brain, while the levels of intact N-cadherin protein were similar in glioblastomas, low-grade astrocytomas and brain. In addition, there was no consistent relationship between invasiveness and expression of N-cadherin and alpha-catenin in highly invasive vs minimally invasive tumours within the same histopathological grade. To assess further the relationship between cadherin expression and neural tumour invasion, we measured N-cadherin expression, calcium-dependent cell adhesion and motility of several glioblastoma cell lines. While all N-cadherin-expressing lines were adhesive, no correlation was seen between the level of N-cadherin expression and cell motility. Together, these findings imply that, in contrast to the role played by E-cadherin in carcinomas, N-cadherin does not restrict the invasion of glioblastomas.