神经母细胞瘤中TrkA基因三种异构体表达及其临床意义*

目的：探讨神经母细胞瘤（NB）中TrkA基因三种异构体的表达及其临床意义。方法：回顾性分析随访资料较完整的首诊为NB的患儿39例。采用SYRB Green I 荧光定量PCR 方法检测39例NB手术切除标本中TrkA异构体mRNA 的表达。t 检验比较不同临床分期中TrkA异构体的表达差异。按首次就诊时临床资料（年龄、性别、原发部位、转移部位、临床分期）,TrkAⅠ/Ⅱ与TrkAⅢ的表达比值等因素进行分组。Kaplan-Meier 法计算5 年生存率,单因素分析用Kaplan-Meier 生存曲线比较组间生存率,显著性检验采用Log-Rank法。COX 回归模型进行多因素分析。结果：TrkAⅠ/Ⅱ的表达在低分期组明显大于高分期组（P<0.01）,而TrkAⅢ的表达在低分期组明显低于高分期组（P<0.01）,TrkAⅠ/Ⅱ与TrkAⅢ的表达比值在低分期组明显高于高分期组（P<0.01）。 单因素分析发现年龄>1 岁、腹部原发、骨骼转移、Evans第Ⅲ/Ⅳ期、TrkAⅠ/Ⅱ与TrkAⅢ表达比值较低组别的5 年生存率,明显低于其对照组。性别对生存率无影响。COX 模型多因素分析显示骨骼转移、TrkAⅠ/Ⅱ与TrkAⅢ表达比值低对5 年生存率产生不良影响。结论：TrkAⅠ/Ⅱ高表达示NB患者后良好,TrkAⅢ高表达示NB患者预后较差。早期对肿瘤行TrkA基因检测及骨骼扫描,对NB患者预后预测具有重要意义。     

神经母细胞瘤血管形成兔角膜模型观察与抑制实验

[目的]建立兔角膜神经母细胞瘤种植模型 ,观察血管形成过程并用TNP 470和环磷酰胺 (CTX)干扰肿瘤血管形成 ,探讨通过抗血管形成治疗神经母细胞瘤的新途径。[方法]15只新西兰白兔角膜接种一人神经母细胞瘤 ,观察肿瘤生长情况及血管密度计数。用TNP 470和CTX干预 ,观察血管形成和肿瘤生长抑制情况并检测血管内皮生长因子的表达情况。[结果]术后2天就有血管向肿瘤方向出芽生长 ,1周时达高峰形成血管丛。肿瘤生长明显分成两个期 ,即血管前期和血管期 ,在血管前期中 ,肿瘤生长缓慢 ,血管期 ,肿瘤开始明显生长。TNP 470、CTX药物干预血管形成 ,血管生长曲线明显被压低。用药后一周出现抑制肿瘤疗效 ,两药之间无明显差别(P>0.05)。VEGF在神经母细胞瘤和用TNP 470后均有高表达(P>0.05) ,而CTX可抑制其表达(P<0.05) ,图像分析值分别为31.27±5.81、25.17±7.61和16.76±4.38。[结论]神经母细胞瘤有较高的血管依赖性。TNP 470和CTX对抑制肿瘤血管形成、限制肿瘤的生长 ,有明显的作用。     

Experimental Research on the TrkA Gene Inhibition of Angiogenesis in Human Neuroblastoma

Objective This study was designed to investigate the feasibility of gene therapy for human neuroblastoma (NB) with the TrkA gene inhibition of tumor angiogenesis, growth and metastasis. Methods Three groups of cells including SY5Y, SY5Y-TrkA and SY5Y-Vec NB cells, were cultured by routine methods. Comparison of oncogenicity was performed among the three groups of cells. Tumor volume and angiogenesis in nude mice were also compared with VEGFmRNA expression (by RT-PCR analysis), immunohistochemistry and microvessel counting. Results The TrkA-transfected SY5Y NB cells showed significantly reduced oncogenicity and tumor angiogenesis. Tumor volumes were statistically different among the control, Empty-Vec and the experimental group, namely 1.736±0.485cm³, 1.803±0.751cm³ and 0.395±0.015cm³, respectively (P< 0.01). The difference of vascular endothelial growth factor (VEGF) expression between the experimental group and the control group was significant (P<0.01 ). Microvessel density (MVD) of the control, Empty-Vec and the experimental group were 27.21±14.58, 27.76±14.15 and 4.08±4.72 respectively, with statistical differences from the experimental group (P< 0.001). Conclusion The tumor angiogenesis and growth of NB were significantly inhibited by the TrkA gene. These studies provide a theoretical basis for application of NB antiangiogenesis gene therapy. This work was supported by National Nature Science Foundation (No. 39470739).     

PTEN抑癌基因在晚期神经母细胞瘤中的表达及意义

10号染色体张力蛋白同源物磷酸脂酶(phosphataseandtensinhomologdeletedonchromosometen,PTEN)是迄今为止发现的第一个具有磷酸酯酶活性的抑癌基因,可能有抑制细胞与细胞外基质相互作用的功能[1]。目前研究提示在人类多种肿瘤和遗传性肿瘤易感综合征等疾病中常出现PTEN杂合性缺失(LOH)或突变[2～9],PTEN基因的表达与肿瘤的预后有一定相关。一般认为PTEN变化是肿瘤的早期事件,用来区分良恶性具有一定意义,在国外已有在儿童神经母细胞瘤中的相关报道[10]。本研究运用流式细胞术(flowcytometry,FCM)检测晚期神经母细胞瘤(neuroblasto…     

人血管抑素克隆表达及抑制血管生成的研究

目的　研究并获得重组人血管抑素 (angiostatin) ,探讨其临床应用价值。方法　采用逆转录 -聚合酶链式反应 (RT PCR )方法 ,从人胚胎肝细胞中扩增出人全长血管抑素cDNA片段。用原核细胞表达载体构建 pBV 2 2 0 /angiostatin重组质粒 ,并将其转化大肠杆菌DH 5α进行表达 ,初步纯化。应用鸡胚尿囊膜血管生成实验及脐静脉内皮细胞增殖实验检测其活性。结果　经SDS PAGE分析 ,表达产物相对分子量约 3 8kD ,薄层扫描显示表达产物可达细菌总蛋白的 3 0 %。活性实验显示 ,重组蛋白能够抑制血管形成。结论　重组人Angiostatin在大肠杆菌中实现高效表达 ,并具有很好的生物学活性     

Canstatin基因导入抑制裸鼠肝癌移植瘤的生长及血管生成

目的:探讨Canstatin基因导入对裸鼠人肝癌皮下移植瘤的生长及肿瘤血管形成的作用。方法:建立肝癌HepG2细胞裸鼠皮下移植瘤模型并鉴定,运用Ad-Canstatin/GFP基因治疗,Western blot法检测转染后瘤组织内Canstatin蛋白的表达。治疗期间观察Canstatin对裸鼠及移植瘤生长的影响,用药后HE染色观察肝肾损害,计数外周血白细胞数目;免疫组织化学法检测瘤内微血管密度(microvascular density,MVD)及血管生成拟态(vasculogenic mimicry,VM)的数目。结果:成瘤率100%。Ad-Canstatin转染组Canstatin蛋白表达增多;Ad-Canstatin组裸鼠食量大于两对照组(P<0.05)。同时排泄量、饮水量均少于两对照组(P<0.05)。体重生长曲线显示Ad-Canstatin组体重持续增加,两对照组体重后期出现不增或下降趋势。Ad-Canstatin组移植瘤瘤体积增长缓慢,治疗第10天,瘤体积小于两对照组(P<0.05);Ad-Canstatin组瘤重小于两对照组(P<0.05),抑瘤率:37.50%。Ad-Canstatin组肝、肾、造血系统未见异常。Ad-Canstatin组MVD、VM数目均少于两对照组(P<0.05)。结论:腺病毒介导Canstatin基因可通过抑制瘤内血管生成,从而抑制HepG2肝癌移植瘤的生长,实验期内未见明显不良反应。     

全反式维甲酸诱导神经母细胞瘤分化及TrkA剪接异构体表达的研究

目的：通过对NB细胞系SH-SY5Y进行体外实验,研究ATRA对NB细胞的增殖抑制与诱导分化作用,进而探讨TrkA剪接异构体在不同分化程度的SH-SY5Y细胞中的表达情况。方法：将SH-SY5Y细胞取对数生长期细胞培养分组,利用台盼蓝拒染计数活细胞,倒置相差显微镜观察细胞形态学变化。采用SYBR GreenⅠ实时荧光定量PCR方法检测TrkA三种剪接异构体的表达水平。结果：对照组活细胞数随着培养时间延长而增多,实验组中ATRA对SH-SY5Y的生长抑制作用在（0.1-10）μmol/L浓度范围内有剂量依赖关系。不同浓度的ATRA均可诱导SH-SY5Y细胞产生形态学上的变化,对照组细胞分化百分率随时间延长无明显变化,各时间段间比较,差异不显著（F=2.889,P=0.079）。TrkAⅠ/ⅡmRNA表达水平增加与ATRA浓度及作用时间呈正相关。TrkAⅢmRNA表达与ATRA浓度及作用时间呈负相关。结论：在SH-SY5Y细胞中,ATRA对细胞生长抑制作用呈明显的时间和剂量依赖关系;TrkAⅠ/Ⅱ表达与ATRA作用时间、剂量呈正相关;随着ATRA作用浓度及作用时间的增加,TrkAⅢmRNA表达降低。     

全反式维甲酸诱导神经母细胞瘤分化及TrkA剪接异构体表达的研究

目的:通过对NB细胞系SH-SY5Y进行体外实验,研究ATRA对NB细胞的增殖抑制与诱导分化作用,进而探讨TrkA剪接异构体在不同分化程度的SH-SY5Y细胞中的表达情况。方法:将SH-SY5Y细胞取对数生长期细胞培养分组,利用台盼蓝拒染计数活细胞,倒置相差显微镜观察细胞形态学变化。采用SYBR GreenⅠ实时荧光定量PCR方法检测TrkA三种剪接异构体的表达水平。结果:对照组活细胞数随着培养时间延长而增多,实验组中ATRA对SH-SY5Y的生长抑制作用在(0.1-10)μmol/L浓度范围内有剂量依赖关系。不同浓度的ATRA均可诱导SH-SY5Y细胞产生形态学上的变化,对照组细胞分化百分率随时间延长无明显变化,各时间段间比较,差异不显著(F=2.889,P=0.079)。TrkAⅠ/ⅡmRNA表达水平增加与ATRA浓度及作用时间呈正相关。TrkAⅢmRNA表达与ATRA浓度及作用时间呈负相关。结论:在SH-SY5Y细胞中,ATRA对细胞生长抑制作用呈明显的时间和剂量依赖关系;TrkAⅠ/Ⅱ表达与ATRA作用时间、剂量呈正相关;随着ATRA作用浓度及作用时间的增加,TrkAⅢmRNA表达降低。     

干扰异粘蛋白基因的表达抑制乳腺癌血管生成

目的:通过干扰乳腺癌细胞异粘蛋白基因（metadherin,MTDH）的表达水平,观察乳腺癌血管生成的变化,探讨MTDH对乳腺癌血管生成的调控作用及分子机制。方法:构建干扰MTDH表达的质粒,转染乳腺癌MDA-MB-231细胞系,建立低表达MTDH的prp-MTDH细胞系及空载体的prp-control对照组细胞系。收集各组乳腺癌细胞的条件培养基,通过小管形成实验和鸡胚绒毛尿囊膜实验,研究干扰MTDH表达对乳腺癌血管生成的影响;通过qRT-PCR和Western blot技术检测MTDH表达降低对血管生成相关因子ERK1/2、VEGF、MMP2和MMP9的影响。结果:通过qRT-PCR检测MTDH基因的表达,发现prp-MTDH细胞系的MTDH mRNA水平由1.01±0.04降低到0.26±0.02（t=19.86,P＜0.01）,Western blot检测MTDH在蛋白水平亦明显降低。通过小管形成实验和鸡胚绒毛尿囊膜实验,发现干扰MTDH表达后的prp-MTDH细胞系血管生成能力明显下降(t=18.43,P＜0.01;t=10.23,P＜0.05)。通过对血管生成相关因子的检测,发现干扰MTDH表达后,具有活性的p-ERK1/2表达降低,VEGF、MMP2、MMP9均明显降低,差异具有统计学意义（P＜0.05）。结论:通过干扰异粘蛋白基因表达可以抑制乳腺癌的血管生成,异粘蛋白基因可能会成为乳腺癌抗血管生成治疗的新靶点。     

雷公藤红素抑制血管生成的实验研究

目的：探讨雷公藤红素对血管生成的抑制作用。方法：采用MTT法测定雷公藤红素对血管内皮细胞株(ECV)增殖的影响,观察雷公藤红素对ECV迁移实验和小管形成实验的作用,以及对鸡胚尿囊膜血管生成的影响。体内实验采用Matrigel plug方法。结果：雷公藤红素可明显抑制ECV的体外增殖,IC50为1．33μg／ml;可抑制ECV的迁移和小管形成,并且呈明显的剂量依赖性;同时具有抑制鸡胚尿囊膜血管生成和Matrigel plug中的血管新生的作用。结论：雷公藤红素具有明显抑制血管生成的作用,有可能成为有效的血管生成抑制剂。     

紫杉醇抗血管生成作用的实验研究

 目的　探讨紫杉醇的抗血管生成作用。方法　以人脐静脉内皮细胞 (HUVEC)原代培养模型 ,MTT法测定紫杉醇对HUVEC增殖的影响 ;采用鸡胚尿囊膜血管形成模型 (CAM模型 ) ,观察紫杉醇对鸡胚尿囊膜新生血管的抑制作用。结果　紫杉醇在非细胞毒浓度下可在体外抑制HUVEC的增殖 ,在体内抑制鸡胚尿囊膜新生血管形成。结论　紫杉醇在非细胞毒浓度下具有抗血管生成作用。     

南蛇藤素对肿瘤血管的抑制作用

 目的 探讨南蛇藤素对肿瘤血管抑制作用。方法 通过建立动物肿瘤模型，对南蛇藤素予以实验治疗，结合免疫组化，检测其抑瘤率和肿瘤内血管密度，以及瘤体VEGF和bFGF的表达的改变。结果 南蛇藤素具有明显的抑制S180肉瘤作用，其3rag／kg剂量组抑瘤率达46．86％，与5-Fu联用抑瘤率达60．2％，联合给药组和南蛇藤素3mg／kg剂量组的微血管密度、VEGF、bFGF的表达均明显低于对照组间和5-Fu组，差异有显著性（P〈0．01）。结论 南蛇藤素对小鼠S180肉瘤血管有一定的抑制作用，降低VEGF、bFGF的表达可能是其抑制肿瘤血管形成的机制之一。     

Effect of Human Interferons on Morphological Differentiation and Suppression of N-myc Gene Expression in Human Neuroblastoma Cells

The activity of human interferons (HuIFNs) to induce morphological changes and the suppression of N- myc gene expression on human neuroblastoma cells (GOTO and KP-N-RT) was evaluated. Morphological differentiation, characterized as the extension and bifurcation of neurites, the formation of multinucleated giant cells and the formation of neurite networks, was induced by treatment with recombinant HuIFN-γ (rHuIFN-γ and also with natural HuIFN-γ on human neuroblastoma cells (GOTO and KP-N-RT). But recombinant HuIFN-αA and recombinant HuIFN-βdid not induce any changes. The rHuIFN-β and rHuIFN-γ inhibited the growth of GOTO and KP-N-RT cells more strongly than the rHuIFN-αA did. The expression of N- myc gene was suppressed in GOTO cells treated with rHuIFN-γ. The suppressive effect of rHuIFN-γ was dependent on the duration of the treatment. However, rHuIFN-αA and rHuIFN-β did not suppress N- myc gene expression. Moreover, both morphological differentiation and the supprcssive effect on N- myc gene expression by rHuIFN-γ were inhibited in the presence of cycloheximide. These results suggest that the morphological changes and N- myc gene expression in neuroblastoma cells are closely related. Furthermore, this decreased N- myc gene expression during the morphological differentiation may be related to the proteins induced by HuIFN-γ.     

榄香烯对裸鼠胃癌原位移植瘤血管生成的抑制作用

 目的 观察榄香烯对裸鼠胃癌原位移植瘤生长和血管生成的抑制作用。 方法 采用裸小鼠胃癌原位移植模型,随机分为0.9%氯化钠溶液(NS)组、5-Fu组、榄香烯组和联合组 ,腹腔注射给药。比较各组移植瘤瘤重的差异;免疫组织化学法检测肿瘤微血管密度 (Microvessel Density,MVD) 和VEGF、p53蛋白表达,RT-PCR法检测VEGF mRNA表达。 结果 联合组裸鼠胃癌移植瘤的瘤重显著低于NS组(P＜0.05);榄香烯组、联合组瘤组织MVD、VEGF蛋 白、p53蛋白及VEGF mRNA表达亦明显低于NS组(P＜0.05);各组移植瘤的瘤重与瘤组织MVD呈正 相关(r=0.669,P＜0.01)。 结论 榄香烯能抑制裸鼠胃癌原位移植瘤生长和血管生成,其机制可能与抑制裸鼠胃癌组织VEGF和突 变型p53的表达有关。     

长春瑞滨及联合热疗抗血管生成作用的实验

目的探讨长春瑞滨及联合热疗对血管生成的抑制作用。方法以人肺癌A549细胞为对照,采用MTT法观察长春瑞滨及联合热疗对人脐静脉内皮细胞（HUVEC）增殖的影响;通过Transwell趋化实验、小管形成实验及流式细胞术观察长春瑞滨对HUVEC迁移、小管形成及细胞凋亡的影响;利用鸡胚绒毛尿囊膜（CAM）模型,观察长春瑞滨对体内CAM新生血管的影响。结果小剂量（0.1～1 ng/ml）长春瑞滨对HUVEC和A549增殖抑制具有差异细胞毒性（P=0.000）。0.1～1 ng/ml 长春瑞滨呈剂量依赖性抑制HUVEC增殖（r=0.993,P=0.000）,联合热疗具有抑制HUVEC增殖的叠加或协同效应。小剂量长春瑞滨能够抑制HUVEC迁移和小管形成,诱导HUVEC凋亡,遏制CAM新生血管形成。结论小剂量长春瑞滨具有抗血管生成作用,联合热疗具有协同或叠加效应。     

乳腺癌转移抑制基因1对卵巢癌细胞血管生成的影响及其机制探讨

目的探讨乳腺癌转移抑制基因1(breast cancer metastasis suppressor 1,BRMS1)对人卵巢癌细胞OVCAR3诱导血管内皮细胞形成管腔能力的影响及可能的机制。方法应用脂质体介导的方法将BRMS1 shRNA重组质粒转染人卵巢癌细胞OVCAR3,经G418筛选获得稳定转染株。荧光定量PCR和Western blot法检测BRMS1 mRNA和蛋白的表达水平;体外血管形成实验检测OVCAR3诱导人脐静脉内皮细胞(HUVECs)的血管形成能力;Western blot法检测生长抑制因子4(ING4)和白细胞介素-6(IL-6)的蛋白表达情况。结果稳定转染BRMS1 shRNA后,OVCAR3细胞BRMS1的表达在 mRNA和蛋白水平均受到明显抑制。体外血管形成实验提示,BRMS1表达下调后能显著促进卵巢癌细胞诱导的HUVECs形成管腔样结构的能力;Western blot法显示干扰组中ING4蛋白表达量较对照组下降30%,而IL-6蛋白表达上调,是空白对照组的1.5倍,差异有统计学意义(P<0.05)。结论干扰BRMS1基因的表达可促进卵巢癌细胞诱导血管形成能力,其机制可能与调节下游基因ING4和IL-6的表达有关。     

Signal Transduction Pathways through TRK-A and TRK-B Receptors in Human Neuroblastoma Cells

Little is known about the signal transduction pathways of TRK family receptors in neuroblastoma (NB) cells. In this study, an NB cell line, designated MP‐N‐TS, was established from an adrenal tumor taken from a 2‐year‐old boy. This cell line expressed both TRK‐A and TRK‐B receptors, which is rare in a single NB cell line. Therefore, the MP‐N‐TS cell line was used to determine whether the signal transduction through these constitutive receptors is functional. Three neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/ 5 (NT‐4/5), induced tyrosine phosphorylation of panTRK, and BDNF and NT‐4/5 induced tyrosine phosphorylation of TRK‐B. Tyrosine phosphorylation of panTRK and/or TRK‐B by the neurotro‐phins was inhibited in the presence of a tyrosine kinase inhibitor K252a. Tyrosine phosphorylation of Src homologous and collagen (She), extracellular signal‐regulated kinase (ERK)‐l and ERK‐2, and phospholipase C‐γl (PLC‐γl) was increased by the three neurotrophins and the increase was inhibited in the presence of K252a. Activation of Ras, detected as the GTP‐bound form of Ras, was induced by the three neurotrophins. The neurotrophins did not modulate the expressions of TRK‐A or TRK‐B mRNA, but they did induce the expression of c‐fos mRNA. Exogenous NGF induced weak neurite outgrowth, whereas exogenous BDNF and NT‐4/5 induced distinct neurite outgrowth. Exogenous BDNF and NT‐4/5 increased the number of viable cells, while NGF did not. Our results demonstrate that the signal transduction pathways through TRK‐A and TRK‐B in MP‐N‐TS cells are functional and similar, and the main downstream signaling pathways from the three neurotrophins are mitogen‐activated protein kinase (MAPK) cascades through She, activated Ras, ERK‐1 and ERK‐2, and the transduction pathway through PLC‐γl. Further, BDNF and NT‐4/5 increased cell viability. The MP‐N‐TS cell line should be useful for clarifying the TRK‐A and TRK‐B signaling pathways responsible for the different prognoses in patients with NB.     

Increased Expression of trk Proto-oncogene by γ-Interferon in Human Neuroblastoma Cell Lines

Three human neuroblastoma cell lines were examined to determine the effect of recombinant γ-interferon (IFN-γ) treatment on the expression of trk proto-oncogene. Increased levels of trk proto-oncogene mRNA were observed in two neuroblastoma cell lines (KP-N-RT and KP-N-SI(FA)) after IFN-γ treatment. The levels of trk mRNA increased with growth inhibition and morphological change in a time- and dose-dependent manner. The decreased level of N- myc mRNA after IFN-γ-treatment in KP-N-RT was inversely correlated with trk mRNA. Our results suggest that IFN-γ can modulate the signal transduction of nerve growth factor in human neuroblastoma cells.