神经母细胞瘤细胞Boyden小室体外侵袭模型的建立及意义探讨

Objective To establish a Boyden chamber for invasive human neuroblastoma cell study. Methods We used Boyden chamber in vitro assay to investigate the invasion of four cell lines.100μl four cell suspension of various concentrations (1.5 × 105、 2. 0 × 105 、 2. 5 × 105 、 3. 0 × 105/ml)was added into the upper chamber of the Boyden chamber,and the cells were allowed to penetrate the membrane for 24,36,48 and 60 hours respectively. The invasion rate was assessed by quantifying cells on the under surface of the membrane. Results Boyden chamber invasion models showed that the invasion of four different cell lines was different. The invasive cell quantity was low 24 hours after the incubation, and it increased dramatically at 48 hours. After 48 hours the rate reached a relative plateau. The invasion quantity was high as the cells concentration added increased. The increase slowed down when the cell concentration was above 2. 5 × 105/ml. Under the same conditions, the invasion ability of cells with the capacity of bone marrow metastasis than the cells without ability of bone marrow metastasis.Conclusions We successfully adopted the Boyden chamber for the neuroblastoma cells. It will be crucial for future study of the invasive ability of the neuroblastoma cells.     

神经母细胞瘤细胞Boyden小室体外侵袭模型的建立及意义探讨

Objective To establish a Boyden chamber for invasive human neuroblastoma cell study. Methods We used Boyden chamber in vitro assay to investigate the invasion of four cell lines.100μl four cell suspension of various concentrations (1.5 × 105、 2. 0 × 105 、 2. 5 × 105 、 3. 0 × 105/ml)was added into the upper chamber of the Boyden chamber,and the cells were allowed to penetrate the membrane for 24,36,48 and 60 hours respectively. The invasion rate was assessed by quantifying cells on the under surface of the membrane. Results Boyden chamber invasion models showed that the invasion of four different cell lines was different. The invasive cell quantity was low 24 hours after the incubation, and it increased dramatically at 48 hours. After 48 hours the rate reached a relative plateau. The invasion quantity was high as the cells concentration added increased. The increase slowed down when the cell concentration was above 2. 5 × 105/ml. Under the same conditions, the invasion ability of cells with the capacity of bone marrow metastasis than the cells without ability of bone marrow metastasis.Conclusions We successfully adopted the Boyden chamber for the neuroblastoma cells. It will be crucial for future study of the invasive ability of the neuroblastoma cells.     

siRNA下调AEG-1表达对神经母细胞瘤细胞MMP-9的表达及侵袭能力的影响

目的 观察下调Astrocyte elevated gene-1(AEG-1)基因对神经母细胞瘤细胞中基质金属蛋白酶-9(MMP-9)表达以及细胞侵袭能力的影响.方法 根据RNA干扰原理,设计合成AEG-1 siRNA.通过荧光定量RT-PCR和Western blotting观察神经母细胞瘤细胞中AEG-1基因mRNA和蛋白下调情况;采用Western blotting观察下调AEG-1表达对神经母细胞瘤细胞中MMP-9蛋白表达的影响;通过侵袭实验(Transwell法)观察下调AEG-1表达对神经母细胞瘤细胞侵袭能力的影响.结论 采用AEG-1 siRNA转染神经母细胞瘤细胞,下调细胞内AEG-1的mRNA和蛋白表达(P＜0.05)后MMP-9蛋白表达抑制率分别为63.1%和58.9%.此外下调AEG-1表达使细胞侵袭能力明显减弱(P＜0.05).结论 AEG-1基因下调使神经母细胞瘤细胞中MMP-9的蛋白表达明显减少、活性明显减弱,同时使细胞的侵袭能力降低.由此可见AEG-1在肿瘤细胞的侵袭转移中可能扮演重要角色,将会成为神经母细胞瘤基因治疗的新靶点.

Abstract：

Objective To investigate the effect of astrocyte elevated gene-1 (AEG-1)siRNA induced inhibition of invasion in neuroblastoma cells.Methods A small interference RNA(siRNA) targeting to AEG-1 mRNA(AEG-1 siRNA) was constructed and transfected into neuroblastoma cells with Lipofectamine 2000.A non-specific siRNA(control siRNA) and non-treatment were used as negative control group and blank group.The expression of AEG-1 mRNA was detected by RT-PCR.The proteins of AEG-1 and MMP-9 were detected by Western blotting.Cell invasion after AEG-1 knockdown was observed by Transwell assay.Results Compared with the control group,the expression of AEG-1 mRNA and protein were significantly decreased in the cells transfected with AEG-1 siRNAs(P＜0.05).AEG-1 knockdown by siRNA markedly decreased the expression of MMP-9 protein by 63.1% in neuroblastoma cells and the ability of cell invasion (P＜0.05).Conclusions The expression of AEG-1 mRNA is down-regulated by AEG-1 siRNA in neuroblastoma cells.Moreover,knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibits the expression of MMP-9 protein and cell invasion.Therefore,AEG-1 may play a key role for MMP-9 activity and cell invasion,making it a potential target gene for gene-therapy in neuroblastoma.     

三氧化二砷抑制神经母细胞瘤细胞侵袭性实验研究

目的 探讨不同水平三氧化二砷(As2O3)对神经母细胞瘤(NB)细胞侵袭力的影响,为As2O3用于NB的治疗提供理论依据.方法 1.选取生长良好的NB LA-N-5细胞,分别按As2O3终浓度为0.75、1.50、3.0、6.0 μmol/L加药,作用24 h;2.收集贴壁细胞,计数并重新悬浮,加入到铺有人工基底膜胶的细胞侵袭小室,使每孔约为2×104 个细胞, 孵育24 h;3.取下人工基底膜胶,甲醇固定穿过膜的肿瘤细胞,苏木晶染色;4.光学显微镜观察肿瘤细胞并计数穿过膜的肿瘤细胞数量,了解细胞侵袭力的改变.结果 砷剂作用24 h后,实验组的LA-N-5侵袭细胞数较对照组显著减少(实验组穿过膜的细胞数分别为28.0±4.0,19.33±4.16,6.33±1.53,对照组平均为46.33±6.11)(P=0.013,0.003,0);实验组0.75 μmol/L与1.50 μmol/L组间结果 比较无显著性差异(P=0.06);0.75 μmol/L与3.0 μmol/L、 1.50 μmol/L与3.0 μmol/L组间比较均有显著性差异(P=0,0.007),以3.0 μmol/L组穿过膜的细胞数最少.结论 As2O3能够抑制NB细胞系LA-N-5细胞的侵袭力;3.0 μmol/L组的As2O3抑制作用最强.     

神经母细胞瘤骨及骨髓转移模型的建立

目的建立一种裸鼠神经母细胞瘤骨及骨髓转移模型,探讨其在靶向给药系统中的应用价值。方法 4～6周龄雌性BALB/c裸鼠,从股骨远端向骨髓腔注人神经母细胞瘤LA-N-5细胞悬液5μl(约1×105个细胞)制备神经母细胞瘤骨、骨髓转移模型,观察小鼠生存率、肿瘤局部生长及远处转移情况。结果模型成瘤率100%,LA-N-5细胞进入骨髓腔后抑制正常骨髓细胞生长,向骨皮质侵袭性浸润,形成骨溶解灶,并向淋巴结、肝、肾转移。结论股骨内注射人神经母细胞瘤LA-N-5细胞悬液可以成功制作裸鼠神经母细胞瘤骨、骨髓转移模型,后者能较好模拟人神经母细胞瘤在骨骼微环境中的生长及转归情况,是研究神经母细胞瘤骨髓、骨转移及评价临床前靶向给药的适宜模型。     

小儿神经母细胞瘤误诊13例探讨

小儿神经母细胞瘤早期缺乏特异性症状、体征，病灶隐匿，诊断困难，极易误诊误治。现对我院1988年10月～2003年3月，小儿神经母细胞瘤误诊13例情况报道如下。     

神经母细胞瘤裸鼠移植瘤模型的建立及相关应用研究进展

自 196 9年Ｒｙｇａｒｒｄ和Ｐｏｖｌｓｅｎ(Ｃｏｎ ｃｅｒＲｅｓ,1975 )首次将人结肠癌成功移植于裸鼠 ,建立裸鼠移植瘤模型以后。人们已将这一方法应用于许多肿瘤的研究 ,神经母细胞瘤也不例外。 1975年ＬａｗｒｅｎｃｅＨｅｌｓｏｎ第一次成功的将人神经母细胞瘤ＳＫ Ｎ ＳＨ、ＳＫ Ｎ ＭＣ细胞移植于裸鼠皮下建立起移植瘤模型。裸鼠移植瘤模型可以直接模拟人体内肿瘤的自然生长过程 ,与体外细胞培养相比 ,移植瘤模型具有以下优点 :①模型的建立和长期传代相对方便 ,不受成纤维细胞污染 ;②移植瘤的增生受到宿主的营养状况和血供的影响 ,…     

神经母细胞瘤转移动物模型建立的研究进展

神经母细胞瘤(NB)患者多因早期即发生转移而预后不良,研究其转移机制及进行抗肿瘤转移实验治疗有赖于NB转移动物模型的建立。NB转移动物模型建立的难度较大,近年许多通过不同方法建立的转移动物模型是进行NB体内实验研究的有力工具。     

神经母细胞瘤转化为节细胞神经瘤的临床观察

目的：了解神经母细胞瘤转化为良性的节细胞神经瘤的规律,总结经验,提出治疗方法。方法：回顾1981年1月－1998年12月我院收治103例神经母细胞瘤中3例转化为节细胞神经瘤的治疗经过及组织学变化。结果：3例患儿至今生存,分别经过2次以上手术,采用多药联合的强化化疗。组织学已改变成为节细胞神经瘤。结论：对神经母细胞瘤的治疗应 积极,尤其对那些第一次手术不能完整切除肿瘤的患儿更应如此。对带瘤生长时间长,生活质量好的病例应高度怀疑肿瘤有转化为良性肿瘤的可能。     

miR-29b对神经母细胞瘤增殖、侵袭和转移的影响研究

目的检测miR-29b在肾上腺和神经母细胞瘤组织中的表达情况,探讨miR-29b对神经母细胞瘤细胞增殖、侵袭和转移能力的影响。方法收集12例神经母细胞瘤患儿的肿瘤组织和癌旁肾上腺组织标本,实时定量PCR检测miR-29b的表达水平;miR-29相关核苷酸(mimics、inhibitor、NC)瞬时转入SK-N-SH神经母细胞瘤细胞后,CCK-8实验检测miR-29b对该细胞系增殖的抑制作用,Transwell小室、划痕实验进行侵袭、迁移能力实验,Western blot方法检测上皮转化相关蛋白(E-cadherin、Vimentin)的表达水平。结果与癌旁肾上腺组织比较,miR-29b在神经母细胞瘤组织的表达水平明显降低(P<0.05);miR-29相关核苷酸(mimics、inhibitor、NC)转染细胞后,miR-29b mimics组出现抑制细胞增殖的效应(P<0.05);Transwell小室实验显示miR-29b mimics组的穿膜细胞量(36.33±8.08)少于miR-29b inhibitor组(112.30±11.68)和NC组(112.30±11.68);划痕实验显示转染miR-29b mimics后,其愈合率(51.67±4.50)%较miR-29b inhibitor组(87.67±3.79)%和NC组(85.00±3.00)%明显下降,细胞的侵袭、迁移能力明显下降;Western blot实验显示,与NC组相比,miR-29b mimics组E-cadherin表达增高,Vimentin表达降低,而miR-29b inhibitor组中E-cadherin表达降低,Vimentin表达增高(P<0.05)。结论神经母细胞瘤组织中miR-29b表达水平下降,miR-29b过表达可以抑制细胞的增殖、侵袭和迁移能力,这种抑癌作用可能与抑制EMT有关。     

NF-κB通路在神经母细胞瘤侵袭转移中的作用及机制

目的 以自主建立的有骨髓转移的神经母细胞瘤细胞系(QDDQNM1)为对象,初步探讨NF-κB通路在神经母细胞瘤(NB)转移过程中的作用及其机制.方法 本实验将细胞系(QDDQ-NM1)分为3组,即:肿瘤坏死因子干预组(Ⅰ组)、正常对照组(Ⅱ组)和PDTC干预组(Ⅲ组).利用RT PCR检测NF-κB通路主要蛋白P65 mRNA及其下游蛋白趋化因子受体4(CXCR4) mRNA的表达;Western-blot法检测P65蛋白及CXCR4蛋白的表达;Transwell小室检测各组间瘤细胞的转移侵袭能力.结果 Ⅰ、Ⅱ、Ⅲ组P65 mRNA相对含量分别为0.7211±0.2233、0.6002±0.1822和0.5553±0.0904,CXCR4 mRNA相对含量分别为0.4128±0.0176、0.3045±0.1910和0.1829±0.0091,各组间差异有统计学意义(P＜0.05).Ⅰ、Ⅱ、Ⅲ组P65蛋白相对含量分别为0.9026±0.0375、0.8340±0.0379和0.8007±0.0793,CXCR4蛋白相对含量分别为0.9472±0.0193、0.9091±0.0216和0.6025±0.0384,各组间差异有统计学意义(P＜0.05).瘤细胞培养48 h后,Ⅰ、Ⅱ、Ⅲ组中穿过ECM胶的细胞数分别为55.8000±2.7749、46.6000±5.9414和30.2000±7.4967,各组间差异有统计学意义(P＜0.05).结论 NF-κB通路通过调节其下游蛋白CXCR4的表达来增强NB细胞的侵袭能力.通过抑制NF-κB通路来降低NB细胞的侵袭转移能力,有望成为治疗NB的新途径.     

Ezrin蛋白、CD44、 E-钙粘素与神经母细胞瘤转移的关系

目的 探讨Ezrin蛋白、CD44、E-钙粘素在神经母细胞瘤中的表达及其与神经母细胞瘤转移的关系.方法 应用免疫组织化学二步法检测40例神经母细胞瘤标本中Ezrin蛋白、CD44、E-钙粘素的表达情况,并应用统计学方法比较其在局灶期、进展期的病例、FH型(组织结构良好型)和UFH型(组织结构不良型)及不同年龄性别病例中的表达程度.结论 Ezrin蛋白在进展期患儿中的表达为20例,明显高于局灶期病例的15例,且有统计学意义(H=6.124,P=0.013).而在进展期病例中CD44的表达为6例,明显低于局灶期的12例,E-钙粘素的表达5例则明显低于局灶期的11例,并且均有统计学意义(H=4.950,P=0.026;H=5.455,P=0.020).Ezrin蛋白和CD44在FH型和UFH型表达的差异具有统计学意义(H=5.182,P=0.023;H=4.496,P=0.034).而 E-钙粘素在FH 中表达为阳性的有11例.在UFH型中为5例,经统计学检验两者之间差异无明显统计学意义(H=2.754,P=0.097).Ezrin蛋白在不同年龄组中的差异具有统计学意义,而CD44和E-钙粘素没有,三者在不同性别组中的表达无明显统计学差异.经统计学分析Ezrin蛋白的表达与CD44的表达呈负相关(χ2=4.675,P=0.031,r=-0.323).Ezrin蛋白的表达与E-钙粘素蛋白的表达呈负相关(χ2=6.077,P=0.014,r=-0.123).E-钙粘素蛋白的表达与CD44的表达呈正相关(χ2=6.077,P=0.014,r=-0.363).结论 Ezrin蛋白、CD44和E-钙粘素的表达与NB的转移有关,其中Ezrin蛋白可促进肿瘤的转移,而CD44、E-钙粘素则对肿瘤的转移有抑制作用.年龄是影响Ezrin蛋白表达的因素之一,但不影响CD44和E-钙粘素的表达,而性别对Ezrin蛋白、CD44和E-钙粘素的表达没有影响.在NB的发生、发展过程中Ezrin蛋白与CD44之间,Ezrin蛋白与E-钙粘素之间可能存在负协同效应,而CD44与E-钙粘素之间可能存在正协同效应.

Abstract：

Objective To study Ezrin protein,CD44 and E-cadherin expressions and their association to metastasis of neuroblastoma(NB).Methods The expressions of Ezrin Protein,CD44 and E-cadherin in 40 NB patients were detected by two-step immunohistochemistry staining,and the expression levels of these proteins were analyzed according to local stage and advanced stage,favor able histology(FH) and the unfavorable histology(UFH),age and gender.Results Strong expression of Ezrin Protein was detected only in 15 patients in local stage tumors,and in 20 advanced stage tumors(compared local stage tumors,H=6.124,P=0.013).Strong CD44 staining was found in 12 in local stage tumors,and in 6 advanced stage tumors(H=4.950,P=0.026).Strong E-cadherin staining was found in11 local stage tumors,and in 5 advanced stage tumors(H=5.455,P=0.020).The difference between Ezrin Protein expression in the type of FH and UFH type was significant(H=5.182,P=0.023),and so did the CD44(H=4.496,P=0.034).The expression of Ezrin protein was related to the age.There was negative correlation between the expression of Ezrin and the CD44(χ2=4.675,P=0.031,r=-0.323),and the expression of Ezrin and the E-cadherin(χ2=6.077,P=0.014,r=-0.123).While positive correlation between the CD44 and E-cadherin(χ2=6.077,P=0.014,r=-0.363) was noted.Conclusions Ezrin Protein,CD44 and E-cadherin expressions may be related to metastasis and progression of NB.While Ezrin protein can stimulate tumor metastasis,CD44 and E-cadherin may have an inhibitory effect.Age is one of factors influence the expression of Ezrin protein in neuroblastoma.     

神经母细胞瘤并骨转移患儿临床特征及预后相关因素的单中心分析CSCD

目的分析伴骨转移神经母细胞瘤(neuroblastoma,NB)患儿的临床特征及预后相关因素,总结临床诊疗经验,以提高伴骨转移NB患儿的生存率。方法以2013年12月至2020年12月重庆医科大学附属儿童医院肿瘤外科收治的伴骨转移NB患儿为研究对象,收集并总结患儿临床资料及预后情况。随访时间截至2021年3月31日。结果共收集97例NB患儿,男68例,女29例,男女比例为2.4∶1;中位年龄为49.4个月。首发症状:发热47例(48.5%),骨痛38例(39.2%),腹痛或腹胀29例(29.9%),咳嗽15例(15.5%);伴骨转移的NB患儿存在多类骨转移(62.9%),且合并骨髓转移(73.2%),整体预后差,1年生存率为93.6%,5年生存率仅20.2%。经生存分析发现,女性、伴多类骨转移、肿瘤位于腹部、首诊时LDH测定值大于660 U/L以及术中肿瘤残留是预后不佳的影响因素,其中手术切除范围是独立预后影响因素。结论伴骨转移的NB患儿临床表现多样,其预后受诸多因素影响,肿瘤复发或进展是主要的致死原因。对于此类患儿,建议术中尽可能完全切除肿瘤,以改善预后。     

神经母细胞瘤并骨转移患儿临床特征及预后相关因素的单中心分析

目的 分析伴骨转移神经母细胞瘤(neuroblastoma,NB)患儿的临床特征及预后相关因素,总结临床诊疗经验,以提高伴骨转移NB患儿的生存率.方法 以2013年12月至2020年12月重庆医科大学附属儿童医院肿瘤外科收治的伴骨转移NB患儿为研究对象,收集并总结患儿临床资料及预后情况.随访时间截至2021年3月31日...     

Case selection in minimally invasive surgical treatment of neuroblastoma

PURPOSE: The experience with minimally invasive surgery (MIS) in the treatment of neuroblastoma (NB) is anecdotal. The purpose of this study was to evaluate a retrospective cohort of NB patients who underwent MIS resection of their primary tumors. METHODS: A retrospective study of NB patients who underwent MIS resection of their primary tumors over a 3-year period was undertaken. Study outcomes included complications, completeness of resection, and event-free and overall short-term survival. RESULTS: Of a total of 21 children who underwent surgical resection for NB during the period of study, 8 (38%) underwent selected MIS resection. Six of the eight (75%) tumors were adrenal in origin and the remainder were located in the posterior mediastinum. Distribution by International Neuroblastoma Staging System (INSS) stage was: stage 1 (3), stage 2 (2), and stage 4 (3). One stage 4 tumor was N-myc amplified. All stage 4 patients experienced a >50% tumor volume cytoreduction in response to preoperative chemotherapy. All MIS resections were performed without need for blood transfusion, or conversion to open procedure, and there were no perioperative complications. All eight patients were alive and disease-free at a median 18-month follow-up. CONCLUSIONS: With appropriate preoperative case selection based on anatomic features, MIS tumor resection in patients with NB can be performed safely and effectively.     

不同转移潜能人神经母细胞瘤细胞系趋化因子受体CXCR4的表达及对瘤细胞趋化作用的影响

目的 以自建的人神经母细胞瘤转移瘤(QDDQ-NM1)及原位瘤(QDDQ-NM2)细胞系的细胞为研究对象,探讨CXCR4在不同转移潜能人神经母细胞瘤细胞系细胞中的表达情况及其对瘤细胞趋化作用的影响.方法 采用RT-PCR检测两神经母细胞瘤细胞系细胞CXCR4 mRNA的表达;流式细胞仪直接免疫荧光法检测两细胞系细胞膜表面CXCR4蛋白的表达;通过趋化和趋化抑制实验观察CXCR4特异性配体CXCL12对QDDQ-NM1和QDDQ-NM2细胞系瘤细胞的趋化作用.结果 RT-PCR显示QDDQ-NM1细胞系CXCR4mRNA的相对含量为0.57±0.08,QDDQ-NM2细胞系为0.29±0.05,两者比较差异有统计学意义(P=0.005＜0.05),流式细胞仪直接免疫荧光法检测两细胞系细胞膜表面CXCR4蛋白表达的阳性率分别为(64.59±1.57)%和(36.72±0.57)%,两者的差异有统计学意义(P=0.00＜0.05),CXCR4特异性配体CXCL12可在一定范围内呈浓度依赖性的趋化QDDQ-NM1和QDDQ-NM2细胞系细胞的迁移,以100 ng/ml的效果较为明显,两细胞系迁移到聚碳酸酯膜下的细胞数差异有统计学意义(P＜0.05).CXCR4特异性拮抗剂AMD3100能有效抑制这种趋化作用(P＜0.05).结论 QDDQ-NM1细胞系的细胞功能性高表达趋化因子受体CXCR4,可能与人神经母细胞瘤QDDQ-NM1细胞系细胞的体外高转移潜能有关.

Abstract：

Objective To study the expression of functional chemokine receptor CXCR4 and its effects on the metastatic potential of human neuroblastoma.Methods Two human neuroblastoma cell lines were used in this study,including QDDQ-NM1 with high metastatic potential and QDDQ-NM2 with low metastatic potential.The expression of CXCR4 was explored at mRNA level using RT-PCR,and the protein level by flow cytometry.Chemotaxis assay was also performed to study the migratory response of QDDQ-NM1 and QDDQ-NM2 to CXCR4 ligand CXCL12.Results The mRNA of CXCR4 was higher in QDDQ-NM1 group than that in QDDQ-NM2 group (0.57±0.08 vs 0.29±0.05,P =0.005).The expression of CXCR4 on QDDQ-NM1 group was also higher than that in QDDQ-NM2 group [(64.59 ±1.57) % vs (36.72 ± 0.57) %,P＜0.05] The QDDQ-NM1 cells exhibited stronger migratory response to CXCL12 in a concentration dependent manner (P＜0.05),and the response peaked to CXCL12 at 100 ng/ml.AMD3100,a specific CXCR4 antagonist,could reverse the tumor's migratory response to CXCL12.Conclusions The expression of CXCR4 is associated with the metastatic potential of human neuroblastoma.     

Olfactory neuroblastoma as a second malignant neoplasm in a patient previously treated for childhood acute leukemia

Various kinds of second malignant neoplasms after successful treatment for childhood acute leukemia have been reported. The authors describe an unusual case of an olfactory neuroblastoma in a patient previously treated for childhood acute leukemia including autologous bone marrow transplantation. The prophylactic cranial irradiation and the total body irradiation during autologous bone marrow transplantation may have induced the development of their patient's olfactory neuroblastoma. Although a second primary olfactory neuroblastoma is rare, it should be added to the list of second malignant neoplasmsin the sinonasal region.