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 :5μＭＡＴＲＡ抑制ＮＢ细胞系ＳＭＳ ＫＣＮＲ细胞增殖 ,而 5ｎＭＡＴＲＡ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 :5μＭＡＴＲＡ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。     

神经母细胞瘤眼部转移与N-myc癌基因扩增的临床意义

目的通过定量研究小儿神经母细胞瘤（ＮＢ）Ｎ－ｍｙｃ基因扩增倍数，观察ＮＢ眼部转移以及与预后的关系。方法选择２５例Ⅳ期ＮＢ患儿（有眼部转移１５例、无眼部转移１０例）做骨穿，有眼部转移者并取转移灶组织，用内参照差示聚合酶链反应（ＰＣＲ）的方法确定ＮＢ细胞的Ｎ－ｍｙｃ拷贝数。结果２５例中有２４例（９６％）出现Ｎ－ｍｙｃ扩增，眼部转移组平均拷贝数（５１．３±３２．４）显著高于无眼部转移组的平均拷贝数（４．３±２．８），Ｐ＜０．０５，且前者两年生存率明显低于后者。结论ＮＢ的疗效及预后与癌基因扩增倍数密切相关，扩增倍数高者预后差。应用差示ＰＣＲ方法可及时判断预后，且操作简便快速，适于临床推广应用。     

差示聚合酶链式反应检测骨髓转移的神经母细胞瘤N－myc扩增及临床意义

测定骨髓转移神经母细胞瘤（ＮＢ）Ｎ－ｍｙｃ扩增情况及评价其与预后的关系。采用差示ＰＣＲ法检测１７例骨髓转移ＮＢ的Ｎ－ｍｙｃ拷贝数，并随访１年。结果，１２例（７０．５９％）证实有Ｎ－ｍｙｃ扩增。Ｎ－ｍｙｃ扩增与ＮＢ分期及患者预后均成显著相关（分别为Ｐ＜０．０５，Ｐ＜０．０１）。有Ｎ－ｍｙｃ扩增者预后差，且扩增倍数越高则预后越差。因此，Ｎ－ｍｙｃ扩增对于ＮＢ分期及预后评估均为有价值的指标，它可能与ｃ－ｍｙｃ协同调控ＮＢ的生物学特性。差示ＰＣＲ作为一种建立在内参照原理上的新方法可定量测定基因拷贝数，比Ｓｏｕｔｈｅｒｎ杂交更简便、快速、灵敏、精确，适于临床应用。尤值一提的是，该法需样品量很少，且无同位素沾染。     

差示聚合酶链式反应检测骨髓转移的神经母细胞瘤N—…

测定骨髓转移神经母细胞（ＮＢ）Ｎ－ｍｙｃ扩增情况及评价其与预后的关系，采用差示ＰＣＲ法检测１７例骨髓转移ＮＢ的Ｎ－ｍｙｃ拷贝数，并随访１年，结果，１２例（７０．５９％）证实有Ｎ－ｍｙｃ扩增，Ｎ－ｍｙｃ扩增与ＮＢ分期及患者预后均成显著相关（分别为Ｐ〈０．０５，Ｐ〈０．０１）。有Ｎ－ｍｙｃ扩增者预后差，且扩增倍数越高则预后越差，因此，Ｎ－ｍｙｃ扩增对于ＮＢ分期及预后评估均为有价值的指标，它可能与ｃ－ｍ     

在神经母细胞瘤中神经营养因子受体TrKA的表达抑制血管生成

在人类神经母细胞瘤（ＮＢ）中血管生成因子的高水平表达与晚期肿瘤分期有关，高血管生成与ＮＢ转移，侵袭性和不良预后相关．该研究旨在说明体内外ＮＢ细胞中重要的血管生成因子如血管内皮生长因子（ＶＥＧＦ）和基本的成纤维细胞生长因子（ｂＦＧＦ）的表达及其生物学活性是否受神经营养因子受体－酪氨酸激酶受体（Ｔｒｋ）Ａ和ＴｒｋＢ表达所影响． 材料和方法进行细胞培养，并将ＳＨ－ＳＹ５Ｙ细胞用全长型 ＴｒｋＡ ｃＤＮＡ Ｋ５３８Ｎ ｃＤＮＡ（为 Ｔｒｋ激酶区缺乏 ＡＴＰ结合带的ＴｒｋＡ变异体）和ＴｒｋＢ　ｃＤＮＡ在常规下稳…     

黄芩甙对大鼠感染性脑水肿NF-κB活性的影响

目的探讨核因子－κＢ（ＮＦ－κＢ）及其抑制蛋白（ＩκＢ）在百日咳菌液所致的大鼠感染性脑水肿模型中的变化及黄芩甙对感染性脑水肿的保护作用是否与抑制ＮＦ－κＢ活化和ＩκＢ降解有关。方法健康ＳＤ大鼠４５只随机分成三组：生理盐水对照组（ＮＳ组）;百日咳菌感染性脑水肿模型组（ＰＢ组）;黄芩甙治疗组（ＢＣ组）。ＢＣ组动物从注菌后 １ｈ起每４ ｈ腹腔注射黄芩甙一次。用电泳迁移率改变法（ＥＭＳＡ）检测各组动物脑组织的 ＮＦ－κＢ与靶基因 ＤＮＡ的结合活性,用Ｗｅｓｔｅｒｎ印迹分析法检测各组动物脑组织的 ＩκＢα表达。结果在 ＮＳ组、ＢＰ１ｈ组 ＮＦ－ κＢ活性较弱,ＢＰ ２ ｈ组 ＮＦ－ κＢ活性增加,以后持续升高,并以 ＰＢ ２４ ｈ组活性最强;ＰＢ ２ ｈ组 ＩκＢα表达开始减少,２４ ｈ降到最低。 ＢＣ ２ ｈ,４ ｈ,８ ｈ,２４ ｈ组 ＮＦ－ κＢ活性低于相应 ＰＢ组。黄芩甙组ＩκＢα表达比相应 ＰＢ组增多。结论百日咳菌所致的大鼠感染性脑水肿模型中ＮＦ－κＢ的活性明显增强,ＮＦ－κＢ活化可能参与了感染性脑水肿发病机制。黄芩甙对感染性脑水肿的保护作用可能是通过抑制ＮＦ－κＢ异常活化和ＩκＢα降解起作用的。     

神经母细胞瘤分子生物学标记物与临床预后判定

神经母细胞瘤（ＮＢ）是儿童时期最为常见的恶性肿瘤之一。目前，对ＮＢ的分子生物学研究已经广泛开展。一批与神经母细胞瘤有关的分子生物学标记物已逐渐被人们所认识。Ｎ－ｍｙｃ癌基因扩增、染色体异常、细胞ＤＮＡ含量、端粒酶活性以及其他原癌基因等都被用来分析ＮＢ的生物学特性，本文综述如下，并对他们在ＮＢ临床预后判定上的应用意义加以总结。     

bcl-2基因调控作用与细胞凋亡在神经母细胞瘤发生发展中的作用

目的：探讨ｂｃｌ－２基因调控作用与细胞凋亡在神经母细胞瘤（ＮＢ）发生发展中的作用。方法：应用ＬＳＡＢ免疫组化及ＴＵＮＥＬ技术检测ＮＢ中ｂｃｌ－２蛋白表达和细胞凋亡。结果：１７例ＮＢ均有ｂｃｌ－２蛋白表达阳性细胞及凋亡细胞，表达指数（ＢＥＩ）９．８％～６１．８％，凋亡指数（ＡＩ）２０‰～１６７‰，二者呈负相关。年龄＜１岁、Ⅰ～Ⅱ及Ⅳ－Ｓ期、ＦＨ型ＮＢ的ＢＥＩ低于年龄≥１岁、Ⅲ～Ⅳ期及ＵＨ型，但差异无显著性意义。ＡＩ在不同分组间差异有显著性意义，且ＡＩ与术后生存时间呈正相关。结论：ｂｃｌ－２基因异常表达可能与神经母细胞或原始神经嵴细胞逃脱凋亡，不能正常成熟或消退，而发展成ＮＢ及ＮＢ的进展有关。ＡＩ高预后好。ＢＥＩ和ＡＩ的检测可为了解ＮＢ的发病机制和判断预后提供实验依据及手段。     

差示PCR等方法诊断神经母细胞瘤的N—myc扩增

最近对神经母细胞瘤的Ｎ－ｍｙｃ癌基因扩增研究较深入。Ｎｍｙｃ扩增对ＮＢ的发生、预后有着很重要作用。常规使用的Ｓｏｕｔｈｅｒｎ杂文分析法较为复杂，随着分析生物学的发展，差示聚合酶链反应成为分析基因扩增的手段之一。     

在不同N-myc扩增和表达的神经母细胞瘤株中细胞活力对侵袭力的作用

ｍｙｃ扩增的神经母细胞瘤有极强的侵袭性 ,经常导致远处转移 ,因此预后很差。在向远处转移的过程中 ,局部侵袭是最重要和不可缺少的步骤之一。本文作者研究了细胞活力和神经母细胞瘤细胞侵袭力的相关性 ,并且分析了Ｎ ｍｙｃ表达对细胞活力和神经母细胞瘤细胞侵袭力的影响。选用 6组人类神经母细胞瘤的细胞株 :ＩＭＲ 32、ＧＯＴＯ、ＤＺ、ＳＴ、ＮＢ 6 9、ＳＫ Ｎ ＳＨ。神经母细胞瘤细胞侵袭力的测定采用透过聚碳酸酯过滤膜的细胞计数测定。Ｎ ｍｙｃ扩增和表达通过ＤＮＡ和ＲＮＡ印迹的方法检测。细胞活力的测定是通过对细胞形态图像的…     

N-myc oncogene expression in porcine renal development and oncogenesis

N-myc oncogene expression was characterized in porcine kidneys to investigate the potential role of this gene in normal renal development and oncogenesis. N-myc RNA expression was detected in porcine kidneys from birth until 5 wk of age, which corresponds to the time when glomerular differentiation is completed. Immunohistochemical studies revealed that N-myc protein was selectively expressed in the primordia of renal proximal tubule epithelial cells. These cells were cultured in vitro and continued to express N-myc for a limited time. Comicroinjection of a mutant ras oncogene and N-myc into these cells led to focus formation in soft agar, loss of contact inhibition, and the establishment of an immortalized cell line. These findings support a multistep model of renal oncogenesis that involves overexpression of N-myc.     

诱导分化剂诱导NB4来源的树突状细胞分化的研究

目的研究全反式维A酸(ATRA)联合细胞因子体外诱导NB4细胞来源的树突状细胞(DC)的适宜方法。方法在NB4细胞中,分别加入ATRA、重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF) 重组人白细胞介素-4(rhIL-4)、ATRA rhGM-CSF rhIL-4培养诱导10d。在培养第11天,取适量细胞进行免疫表型分析和形态学观察(所有实验均重复3次)。结果经ATRA rhGM-CSF rhIL-4培养10d,NB4-DC表面,HLA-ABC、CD1a、CD86表达明显增高,而CD33表达明显下降,与诱导前相比,差异有统计学意义(P<0.01),经光镜下观察细胞具有典型的树突状细胞形态学特征;NB4细胞经单独ATRA诱导后向正常髓系细胞分化和成熟。结论通过ATRA、rhGM-CSF及rhIL-4的培养体系可诱导NB4-DC。[临床儿科杂志,2007,25(6):488-491]     

阻断TrkB-BDNF信号传导通路对神经母细胞瘤细胞生长及凋亡的影响

目的：脑源性神经生长因子(BDNF)和其酪氨酸激酶受体B（TrkB）与神经母细胞瘤(NB) 细胞的化疗耐药及恶性预后密切相关。该研究探讨阻断TrkB-BDNF信号传导通路后，NB细胞对化疗药物敏感性的变化。方法：常规培养SH-SY5Y NB细胞，用nM浓度全反式维甲酸（ATRA）诱导TrkB高表达，加入BDNF、化疗药顺铂（CDDP）和特异性酪氨酸酶抑制剂K252a处理。MTT实验方法检测应用K252a前后细胞的存活率的变化；同时应用Western-blot方法检测应用K252a前后TrkB的磷酸化水平的变化；流式细胞仪（FCM）检测细胞凋亡率；透射电镜（TEM）观察凋亡细胞的形态与结构。结果：ATRA+BDNF+CDDP组细胞的存活率及凋亡率与对照组相比，差异无显著性（P>0.05）。而经K252a处理后，细胞对顺铂的敏感性增加，细胞的存活率明显降低，凋亡率明显升高，差异有显著意义。Western-blot分析显示K252a能够阻断TrkB的磷酸化。结论：阻断TrkB-BDNF信号传导通路可提高NB的化疗敏感性，逆转耐药。     

N-myc oncogene amplification in a patient with IV-S neuroblastoma

Neuroblastoma (NB) is a tumor usually arising in children and young adults showing different degrees of malignancy. Recently, the presence of N-myc amplification in neuroblasts has been associated with a poor outcome in the late stages of disease (Evans's Stage IV). Until now no amplification of N-myc gene has been observed in Stage IV-S, usually considered to have a favorable prognosis. In this paper we report a case of a child affected by NB Stage IV-S showing mild N-myc gene amplification. The finding of N-myc amplification in our patient shows that such alteration of the N-myc oncogene is not necessarily correlated with a poor prognosis; in this light the role of N-myc amplification in the neoplastic process should be reconsidered.     

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。     

正常骨髓基质细胞对全反式维甲酸诱导分化HL-60细胞的作用

目的研究BMSC对ATRA抑制HL-60细胞增殖、诱导其分化的作用。方法分别将融合的基质细胞层(SC)、2%戊二醛固定的基质细胞层(FSC)和基质细胞,条件培养液(SCM)与HL-60细胞共同培养。SC、FSC、SCM组分别加或不加ATRA为实验组,另设阴性对照和ATRA对照组。通过细胞计数观察细胞增殖情况及通过细胞形态观察,NBT还原试验,分析细胞分化状态。结果细胞计数示SC、FSC、SCM各组均能抑制悬浮生长的HL-60细胞的增殖;SC和FSC能增强ATRA对HL-60细胞的抑制增殖作用(P<0·01)。SC、FSC、SCM各组的诱导分化率、NBT还原率等分化指标均与阴性对照组无差异(P>0·05);SC和FSC可促进ATRA对HL-60细胞的诱导分化作用(P<0·01)。结论BMSC可增强ATRA对悬浮生长的HL-60细胞的抑制增殖和诱导分化作用,这可能是通过BMSC与HL-60细胞之间的相互作用调节的。     

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.     

Retinoic-acid-resistant neuroblastoma cell lines show altered MYC regulation and high sensitivity to fenretinide

BACKGROUND: High-dose, pulse-13-cis-retinoic acid (13-cis-RA) given after intensive cytotoxic therapy improves event-free survival for high-risk neuroblastoma (NB), but more than 50% of patients have tumor recurrence. PROCEDURE: We conducted multistep selection for resistance to all-trans-retinoic acid (ATRA) in NB cell lines with (SMS-KCNR and LA-N-5) or without (SMS-LHN) MYCN genomic amplification. RESULTS: After 12 exposures to 10 microM ATRA, the two MYCN-amplified cell lines (KCNR 12X RR and LA-N-5 12X RR) showed partial resistance to the cytostatic/differentiation effects of ATRA; complete resistance was seen in LHN 12X RR. ATRA-selected cells showed general RA resistance (cross-resistance to 13-cis-RA). Transient (KCNR 12 X RR, LA-N-5 12X RR) or sustained (LHN 12X RR) novel overexpression of c-myc was associated with RA resistance. RA-insensitive overexpression of MYCN by transduction in SMS-LHN also conferred RA resistance. Both parental and RA-resistant lines showed 2-4 logs of cell kill in response to N-(4-hydroxyphenyl)retinamide (4- HPR, fenretinide). Compared to parental lines, 4-HPR achieved 1-3 log greater cell kills in RA-resistant LHN 12X RR, LA-N-5 12X RR, KCNR 12X RR, and MYCN-transduced SMS-LHN or SK-N-RA. NB cell lines (n = 26) from 21 different patients showed that 16 of 26 (62%) were sensitive to 4-HPR (LC(90) < 10 microM), including lines established at relapse after myeloablative and/or 13-cis-RA therapy. CONCLUSION: Thus, RA-resistant NB cell lines can be sensitive (and in some cases collaterally hypersensitive) to 4-HPR.