ATRA诱导神经母细胞瘤细胞分化及TrkA表达的研究

目的 观察全反式维甲酸(all-trans retinoic acid,ATRA)体外对神经母细胞瘤(neuroblastoma，NB)细胞增殖的抑制和可能的分子机制及NGF-TrkA信号传导途径在NB细胞分化中的作用。方法 取本院住院2岁男性NB患儿新鲜手术标本，进行原代细胞培养，并对培养细胞进行分离与纯化，建立细胞系，作为细胞模型。通过台盼蓝拒染法计数活细胞；观察ATRA干预前后细胞形态学变化；RT-PCR分析TrkA表达情况。结果 ATRA作用NB细胞后，细胞形态发生显著变化，并可抑制细胞增殖，2d时TrkA表达增加，4d时出现TrkA表达的明显增加。结论 所建立的NB细胞系为可诱导分化型，即N型；≥5μmol／L，ATRA对NB细胞体外增殖有抑制作用并诱导其分化且表现剂量依赖关系；ATRA可诱导NB细胞分化成熟及TrkA mRNA表达水平增高，可能是ATRA体外诱导NB细胞分化逆转的分子生物学机制之一.     

γ-IFN诱导神经母细胞瘤细胞分化及TrKA表达的实验研究

本实验观察不同浓度的γ IFN对NB细胞产生增殖抑制及诱导分化作用时 ,TrkAmRNA表达的变化 ,及其与增殖抑制、分化程度的关系。首先应用 ,常规培养SMS KCNR细胞 ;然后用三种不同浓度的γ IFN处理这些细胞 ;在不同的作用时间 ,通过台盼蓝拒染实验判定γ IFN对NB细胞的的增殖抑制作用及相差显微镜观察NB细胞形态学变化 ;用RNA PCR及SouthernBlot杂交法检测γ IFN对NB细胞的Tr kAmRNA表达的影响。结果表明 :( 1 ) 1 0 0 0IU/ml、2 0 0 0IU/mlγ IFN对人NB细胞的体外增殖有抑制作用。 ( 2 )γ IFN可诱导NB细胞分化成熟及TrkAmRNA表达水平增高 ,其作用随时间延长 ,浓度增加而增强。结论 :( 1 )γ IFN能够抑制NB细胞增殖并诱导其分化 ,同时TrkAmRNA表达增加 ,其作用效应 ,呈量效依赖关系。 ( 2 )TrkAmRNA表达水平的增高可能是γ IFN体外诱导NB细胞分化逆转的分子生物学机制之一     

ATRA诱导神经母细胞瘤细胞分化及TrkB表达的研究

通过对神经母细胞瘤(neuroblastoma，NB)细胞系SMS-KCNR的体外实验，探讨全反式维甲酸(ATRA)对NB细胞增殖抑制的可能分子机制及BDNF(脑源性神经营养因子)-TrkB信号转导途径在NB细胞分化中的作用。通过台盼蓝拒染计数活细胞；用倒置相差显微镜观察加药处理前后细胞形态学变化；通过Northern印迹杂交来分析TrkB基因表达情况的改变。结果：5μM ATRA抑制NB细胞系SMS-KCNR细胞增殖，而5μM ATRA无此作用；ATRA可诱导SMS-KCNR细胞分化成熟；细胞分化过程中伴随TrkB基因表达水平增高。结果显示：5μM ATRA对人NB细胞系MSM-KCNR细胞的体外增殖有抑制作用；ATRA能诱导人NB细胞系SMS-KCNR细胞分化成熟；TrkB基因表达水平的增高可能是ATRA体外诱导NB细胞分化逆转的分子生物学机制之一。     

9-顺式维甲酸协同γ-干扰素诱导神经母细胞瘤分化、凋亡及生长抑制的实验研究

目的 观察9-顺式维甲酸（9-cis retinoic acid，9-cis RA）联合γ干扰素（gammm interferon，γ-IFN）在体外诱导神经母细胞瘤（neuroblastoma，NB）SK-N-SH细胞分化、凋亡过程中细胞形态、周期的变化，探讨其协同作用的效果及其机制。方法 将细胞分为A（对照组）、B（9-cisRA用药组）、C（γ-IFN用药组）和D（9-cisRA和γ-IFN联合用药组）4组，在处理后适当的时间分别观察细胞形态学变化，进行分化神经节细胞计数，用Hoechst-PI荧光染色法观察细胞的凋亡和死亡，四甲基偶氮唑盐（MTT）法测定NB细胞抑制率，流式细胞仪（flow cytometry，FCM）检测细胞周期分布及其凋亡和死亡。结果 联合用药组细胞形态学分化明显，神经节细胞分化率显著高于其他各组（P〈0．01）；MTT法测定显示联合用药能显著提高NB细胞抑制率并明显优于单独用药；Hoechst-PI荧光染色及FCM钙依赖性磷脂结合蛋白V和碘化丙啶（annexin V／PI）染色法检测一致显示联合用药对于诱导NB细胞早期凋亡和坏死具有显著的协同作用；FCM细胞周期动力学分析发现联合用药组C0-G1期细胞比率增加，S期细胞比率减少。结论 9-cisRA联合γ-IFN在体外对NB细胞的分化、凋亡和生长抑制能产生明显的协同作用，为其联合应用于临床提供理论依据和指导。     

γ—IFN诱导神经母细胞瘤细胞分化及TrKA表达的实验研究

本实验观察不同浓度的γ－IFN对NB细胞产生增殖抑制及诱导分化作用时,TrkA MRNA表达的变化,及其与增殖抑制,分化程度的关系。首先应用,常规培养SMS－KCNR细胞;然后用三种不同浓度的γ－IFN处理这些细胞,在不同的作用时间,通过台盼蓝拒染实验判定γ－INF对NB细胞的增殖抑制作用及相差显微镜观察NB细胞形态学变化;用RNA－PCR及Southern Blot杂交法检测γ－IFN对NB细胞的Tr-kA mRNA表达的影响。结果表明：（1）1000IU／ml,2000IU/ml,γ-INF对人NB细胞的体外增殖有抑制作用。（2）γ－IFN可诱导NB细胞分化成熟及TrkA mRNA表达水平增高,其作用随时间延长,浓度增加而增强。结论：（1）γ－IFN能够抑制NB细胞增殖并诱导其分化,同时TrkA mRNA表达增加,其作用效应,呈量效依赖关系。（2）TrkA mRNA表达水平的增高可能γ－IFN体外诱导NB细胞分化逆转的分子生物学机制之一。是     

转神经生长因子基因诱导神经母细胞瘤分化的研究

目的观察转染神经生长因子（nerve growth factor，NGF）基因诱导神经母细胞瘤（neuroblastoma，NB）细胞系的分化，探讨NGF在NB细胞分化中的作用。方法取本院住院NB患儿新鲜手术标本，进行原代细胞培养并分离纯化，建立细胞系，作为细胞模型。通过脂质体法介导含有NGF基因的载体质粒转染NB细胞。倒置相差显微镜观察转染前后细胞的形态学变化；MTT法及有丝分裂指数测定细胞增殖的改变。结果转染后的NB细胞表达较高水平的NGF，细胞增殖受到抑制并发生形态学改变。结论所建立的NB为可诱导型，即N型；转染NGF基因的NB细胞表达较高水平的NGF；转染NGF基因的NB细胞系表现为增殖抑制和分化。     

TrkA基因在神经母细胞瘤增殖及形态分化中的作用

目的 观察酪氨酸激酶受体A(TrkA)基因抑制神经母细胞瘤生长、诱导其分化的可行性。方法 利用脂质体转染法将TrkA cDNA重组质粒转染入人神经母细胞瘤(NB)SH-SY5Y细胞系中,应用RT-PCR技术鉴定转染后基因的表达,比较转染前后SH-SY5Y细胞的增殖率及细胞的分化程度。结果 成功转染TrkA基因并在SH-SY5Y细胞中稳定表达,转染后SH-SY5Y细胞的增殖率较亲代细胞明显下降(P<0.01),细胞的分化程度增加(P<0.01)。结论 TrkA基因的高表达能有效抑制神经母细胞瘤的生长,并能促其向良性分化,为神经母细胞瘤的基因治疗提供理论依据。     

神经生长因子受体与神经母细胞瘤

神经母细胞瘤 (ｎｅｕｒｏｂｌａｓｔｏｍａ,ＮＢ)是小儿常见的恶性肿瘤之一 ,Ⅰ、Ⅱ期的ＮＢ预后较好 ,而进展期ＮＢ虽经手术、化疗、放疗甚至骨髓移植等综合治疗 ,仍有很高的复发率 ( 60 .0 %左右 ) ,但ＮＢ却有特殊的肿瘤生物学特性 ,可在出生几个月内自然消退或转变为良性神经节细胞瘤 ,ＩＶ Ｓ期的ＮＢ虽然已经发生远处转移 (肝、骨髓等 ) ,却预后良好 ,且有较高的自消率[1] 。近年的研究表明 ,神经生长因子(ｎｅｒｖｅｇｒｏｗｔｈｆａｃｔｏｒＮＧＦ)可以诱导ＮＢ的分化 ,神经生长因子高亲和力受体(ＴｒＫＡ)、低亲和力受体 (Ｐ75Ｎ…     

神经母细胞瘤细胞分化逆转与癌基因表达调控

神经母细胞瘤（NB）是小儿常见的实体肿瘤，恶性度极高。自行消退与体外诱导后分化成熟是其特点之一，分化疗法是临床治疗的一个新方向。本文综述了近年来NB细胞分化机制的分子生物学研究进展，提示诱导分化是一个多种癌基因参与的复杂过程。     

神经母细胞瘤细胞系血细胞分化抗原的表达

神经母细胞瘤(NB)是儿童最常见的颅外实体瘤，预后相对较差。NB细胞系是由形态和生化特性多样的细胞群组成。NB细胞系从形态可分为三种类型的细胞：神经母细胞型(N型)、许旺细胞型(S型)和中间型(I型)。     

Neuronal differentiation in human neuroblastoma cells by nerve growth factor following TrkA up-regulation by interferon-gamma

BACKGROUND: TrkA mRNA expression has been reported to be related to favorable outcome of neuroblastoma (NB). Previously, we found that interferon-gamma (IFN-gamma) can enhance TrkA mRNA expression in NB cell lines. In the present study, we examined the effect of nerve growth factor (NGF) on IFN-gamma-induced TrkA protein to clarify the relationship between TrkA and cell differentiation of NB. PROCEDURE: The effect of IFN-gamma on the TrkA mRNA expression was screened in six human NB cell lines and a freshly prepared sample, SK-rib, from a stage IV patient. Using two of them, we examined their morphological change during simultaneous loading of NGF and IFN-gamma. Tyrosine phosphorylation pattern after 5 min of NGF stimulation was also examined in immunoblot analysis with anti-gp140(trkA) antibody and antiphospho tyrosine antibody. RESULTS: After a 4-day treatment with 500 IU/ml IFN-gamma, TrkA mRNA increased in five cell lines and SK-rib cells in association with growth inhibition. Although the degree of morphological differentiation did not increase in proportion to the TrkA expression induced by IFN-gamma, continuous loading of both IFN-gamma and NGF caused marked morphological differentiation in a cultured KP-N-RT cell line and SK-rib cells during 10 days. Moreover, 5 min of NGF stimulation after IFN-gamma treatment caused the phosphorylation of TrkA protein and downstream proteins. CONCLUSIONS: IFN-gamma could induce the functional NGF receptor even in the aggressive phenotype of NB.     

ATRA诱导神经母细胞瘤SMS-KCNR分化与N-myc癌基因表达

目的 观察全反式维甲酸（ＡＴＲＡ）诱导神经母细胞瘤（ＮＢ）细胞分化时Ｎ－ｍｙｃ癌基因的变化以及与时间的关系。方法 用不同浓度的ＡＴＲＡ处理ＮＢ细胞系ＳＭＳ－ＫＣＮＲ,用相差显微镜观察细胞形态学改变。采用Ｎｏｒｔｈｅｒｎ印迹杂交方法检测ＮＢ细胞分化前后Ｎ－ｍｙｃ癌基因的ｍＲＮＡ表达水平。结果 用１０＾－６ｍｏｌ／Ｌ的ＡＴＲＡ处理２４～１２０ｈ,ＮＢ细胞的胞体减小,神经突起延长。在加ＡＴＲＡ２４ｈ后,     

p75 mediated apoptosis in neuroblastoma cells is inhibited by expression of TrkA

BACKGROUND: Neurotrophins mediate their effects by binding to members of the Trk family of receptor tyrosine kinases and to the low-affinity nerve growth factor receptor p75. Nerve growth factor (NGF) has been demonstrated to support survival and differentiation of neuroblastoma (NB) cells by activation of the TrkA receptor. The p75 receptor belongs to the tumor necrosis factor (TNF) family of death receptors and has been suggested as a receptor that mediates apoptosis in neuronal and NB cells. PROCEDURE: To investigate the effect of p75 expression in NB, we transfected the p75 cDNA into SH-SY5Y cells, an NB cell line lacking expression of both p75 and TrkA. RESULTS: Cell clones expressing elevated levels of p75 showed a high degree of apoptosis even in 10% serum-supplemented medium. Apoptotic signaling by p75 was ligand-independent and only partly caspase-dependent. The level of apoptosis correlated directly with the expression level of the receptor, indicating that p75 may activate the cell death program directly. However, additional transfection of TrkA into SY5Y-p75 cells resulted in a significantly reduced rate of apoptosis even in the absence of NGF. CONCLUSIONS: Thus, expression of the TrkA receptor itself inhibits p75 mediated apoptosis in NB cells.     

Different effects of TrkA expression in neuroblastoma cell lines with or without MYCN amplification

BACKGROUND: Expression of the neurotrophin receptor TrkA is associated with a favorable prognosis in neuroblastoma (NB) and promotes growth inhibition and neuronal differentiation. Aggressive, MYCN-amplified NB tumors express little or no TrkA mRNA, suggesting that MYCN overexpression may inhibit TrkA expression. PROCEDURE: To study the interactions of TrkA expression and MYCN amplification in NB, we stably expressed the TrkA receptor in the MYCN single copy cell lines SH-SY5Y and NB69 as well as in the MYCN amplified cell lines CHP134 and IMR5. RESULTS: All four transfected cell lines demonstrated high TrkA expression and similar activation of the TrkA receptor and of mitogen-activated protein kinases as well as induction of immediate-early genes in response to nerve growth factor (NGF). Introduction of TrkA restored NGF responsiveness of SH-SY5Y and NB69 cells, as demonstrated by morphologic differentiation, growth inhibition, and enhanced survival in serum-free medium. However, no morphologic, growth, or survival responses to NGF were detected in MYCN-amplified CHP134 and IMR5 TrkA transfectants. CONCLUSIONS: Thus, transfection of TrkA into MYCN amplified NB cell lines only partly restored the TrkA/NGF signaling pathway, suggesting additional inhibitory effects of MYCN overexpression on TrkA signaling.     

宫内发育迟缓对大鼠脑内NGF及TrkA表达影响的研究

目的：观察神经生长因子（NGF）及酪氨酸激酶受体A（TrkA）在宫内发育迟缓(IUGR)大鼠脑不同发育阶段的变化,探讨IUGR大鼠脑发育迟缓的机制。方法：32只孕鼠随机分成 IUGR 组和正常对照组。采用孕期全程低蛋白饮食的方法建立IUGR大鼠模型。各组仔鼠于生后 0 d、7 d、14 d、21 d断头取脑,免疫组织化学及 Western blot 方法检测脑中 NGF 和 TrkA 蛋白的表达情况。结果：免疫组化及Western blot结果均显示低蛋白饮食组0 d、7 d、14 d、21 d仔鼠脑内 NGF 及 TrkA 蛋白的表达均较正常组显著降低,差异有统计学意义（P<0.01）。结论：脑内NGF 及TrkA蛋白表达的降低可能是IUGR大鼠脑发育迟缓发生的机制之一。     

Prognostic and biological role of neurotrophin-receptor TrkA and TrkB in neuroblastoma

Expression of different neurotrophin receptors of the tyrosine kinase (Trk) family plays an important role in the biology and clinical behavior of neuroblastomas (NB). Observations from several independent studies suggest that high expression of TrkA is present in NB with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive NB with MYCN-amplification. To determine expression of Trk receptors and ligands in primary NB, we developed a reliable semiquantitative duplex RT-PCR protocol, that requires only 1 microgram RNA per tumor sample. Activation of TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events and promotes neuronal differentiation in vitro. Activation of TrkB by its ligand brain derived neurotrophic factor (BDNF) has been associated with proliferation and survival of NB cells. To study Trk signal transduction pathways and their biological effects in NB, we stably expressed TrkA and TrkB cDNA in the human NB cell line SH-SY5Y. Introduction of TrkA and TrkB restored responsiveness of SH-SY5Y cells to the ligands NGF and BDNF, respectively, and resulted in morphological differentiation. Expression of TrkA resulted in growth inhibition of the transfectants compared to parental cells, whereas TrkB transfectants demonstrated an increased proliferation rate. Further insight into the differences of TrkA and TrkB signaling may suggest new options for the treatment of NB. As expression of TrkA is a strong prognostic factor especially in MYCN non-amplified NB, a prospective study of Trk receptor expression using RT-PCR should be performed for German neuroblastoma patients.