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。     

外周血和骨髓微量神经母细胞瘤细胞检测的临床意义

目的 探讨CD45/CD56/GD2单克隆抗体(单抗)流式细胞仪分析法检测周围血和骨髓微量神经母细胞瘤(NB)细胞的临床意义。方法 11例腹部神经母细胞瘤(NB组)和23例其他肿瘤(对照组)患儿,年龄6个月至12岁,用单抗CD45/CD56/GD2流式细胞术检测外周血和骨髓穿刺标本微量NB细胞。结果 NB组入院时血和骨髓微量NB细胞阳性率分别为90.9％(10/11)和72.7％(8/11),对照组血和骨髓均为阴性(P<0.001)。NB组骨髓微量NB细胞检测与涂片细胞学检查阳性率有较好的相关性(P<0.05),但前者阳性率更高,不受骨髓稀释影响。骨髓NB细胞阳性率与临床分期有关,Ⅱ～Ⅳ期明显高于Ⅰ期(P<0.01)。化疗后血和骨髓NB细胞数均明显减少(P<0.05)。术后转移者血NB细胞重新出现阳性。结论 CD45/CD56/GD2单抗流式细胞术检测周围血和骨髓微量NB细胞具有高度敏感性和特异性,有助诊断、鉴别诊断、疗效观察和复发转移监测。     

13-顺维甲酸体外诱导神经母细胞瘤干细胞分化的体外实验

目的 体外观察并鉴定13-顺维甲酸诱导神经母细胞瘤（NB）干细胞的分化作用,为临床用维甲酸治疗NB微小残留病灶提供实验依据。方法 取14例伴骨髓转移的Ⅳ期NB患儿的骨髓液标本,分离出NB细胞,将原代肿瘤细胞接种于无血清干细胞培养基中悬浮培养;在含5 μmol·L-1 13-顺维甲酸的分化培养基中培养神经球细胞,观察细胞的形态变化;RT-PCR法检测神经球细胞诱导前、诱导3和9 d Oct-4表达水平的改变;利用细胞免疫荧光技术比较诱导前及诱导9 d神经球细胞nestin表达的差异。结果 14例骨髓标本中,4例分离到原代NB细胞。无血清培养基中培养4~6 d后,可见原代悬浮神经球形成,传代后成球细胞能再次分裂增殖为神经球。神经球细胞在血清培养基中呈贴壁生长,呈三角形或星形,添加5 μmol·L-1 13-顺维甲酸后,细胞生长速度降低,形态发生明显改变。RT-PCR法检测结果显示,13-顺维甲酸诱导9 d后,神经球细胞Oct-4表达水平逐渐降低;细胞免疫荧光显示诱导9 d后神经球细胞nestin表达减弱。结论 13-顺维甲酸能有效诱导NB干细胞分化。     

细胞色素C1对神经母细胞瘤细胞SH-SY5Y增殖作用的研究

目的分析细胞色素C1(CYC1)在神经母细胞瘤(NB)增殖中的作用,探讨神经母细胞瘤增殖的分子机制。方法首先采用在SH-SY5Y细胞系中慢病毒介导敲减CYC1基因方法,建立神经母细胞瘤CYC1敲减模型,并利用实时定量RT-PCR方法验证敲减效率;随后利用荧光显微镜观察及细胞分析系统连续检测慢病毒感染SH-SY5Y细胞后连续5 d细胞增殖数。结果慢病毒介导的SH-SY5Y细胞中CYC1基因敲减效率达77.0%±4%,具极显著差异(P<0.01);通过细胞分析系统连续检测SH-SY5Y细胞慢病毒感染敲减CYC1后0~5 d细胞数发现细胞增殖速度明显降低,对照组/敲减CYC1组细胞数1~5 d比值分别为(1.00±0.11)、(1.61±0.16)、(1.58±0.17)、(1.97±0.13)、(1.44±0.13),具极显著差异(P<0.01)。结论 CYC1基因沉默可抑制神经母细胞瘤细胞SH-SY5Y增殖,CYC1在调节神经母细胞瘤生长增殖方面发挥重要作用。     

苦参碱对人神经母细胞瘤SH-SY5Y细胞的作用机制

目的 探讨苦参碱对人神经母细胞瘤( NB) SH-SY5Y细胞的作用机制.方法 取对数生长期的细胞,分为对照组(含细胞、无苦参碱)及药物处理组(苦参碱质量浓度为2.0g·L-1,对细胞的作用时间分别为16 h、24 h、32 h)共4组,每组5个复孔.应用四甲基偶氮唑蓝(MTT)比色法检测NB SH-SY5Y细胞增殖抑制率;流式细胞仪(FCM)检测细胞凋亡率;比色法检测Caspase-8的相对活性.采用SPSS 16.0统计软件进行单因素方差分析.结果 MTT检测对照组及各时间点药物处理组增殖抑制率分别为(4.98±1.20)％、(11.01±0.85)％、(15.22±0.71)％和(22.31±1.45)％;FCM法检测对照组及各时间点药物处理组细胞的凋亡率分别为(5.23±1.19)％、(10.74±1.65)％、(14.00±0.98)％和(17.81±1.06)％;比色法测定各组Caspase-8的相对活性分别为(4.25±1.00)％、(10.69±1.10)％、(14.80±1.44)％和(19.80±0.97)％;以上检测各组间比较差异均有统计学意义(Pa＜0.05).结论 苦参碱可抑制人NB SH-SY5Y细胞增殖,并可通过上调Caspase-8的活性诱导NB SH-SY5Y细胞凋亡,其作用随时间的延长逐渐增强.     

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。     

miR-34b-3p靶向FDX1抑制小儿神经母细胞瘤细胞生长和凋亡

目的探究miR-34b-3p在神经母细胞瘤(neuroblastoma, NB)细胞增殖和凋亡过程中的调节作用。方法体外培养人正常背根神经节细胞和NB细胞系, 搜集NB组织和邻近的正常组织。qRT-PCR检测NB组织和细胞中miR-34b-3p和FDX1的表达;CCK-8法、克隆形成实验和流式细胞术检测NB细胞的增殖和凋亡;双荧光素酶报告基因分析FDX1 mRNA 3’’非翻译区(3’’-untranslated region, 3’’-UTR)和miR-34b-3p之间的靶点关系;Western blot检测相关蛋白的表达。采用SPSS 23.0和GraphPad Prism 6.01进行Student’’st-test检验。结果与对照组比较, miR-34b-3p在NB肿瘤组织和癌细胞系(SK-N-BE、SK-N-SH、SH-SY5Y和LAN-6)中均下调(P<0.05)。与对照组比较, miR-34b-3p过表达抑制了癌细胞SK-N-BE和SH-SY5Y的生长, 并诱导凋亡增加(P<0.05)。萤光素酶报告基因证实, miR-34b-3p可以与FDX1 3’’-UTR...     

IL-15对MDS CD34^＋细胞的诱导分化作用探讨

目的 观察IL-15对体外培养的MDS CD34^ 细胞的诱导分化作用，方法 应用MACS免疫磁珠分离系统提取高纯度MDS骨髓CD34^ 细胞，建立以造血生长因子为基础的体外培养体系，用不同浓度的IL-15作用CD34^ 细胞，流式细胞术检测培养后的细胞单克隆抗体分化和细胞周期的表达，细胞化学染色观察细胞形态变化。结果 应用MACS方法成功提取MDS CD34^ 细胞，体外培养MDS CD34^ 细胞生长情况一般，IL-15作用后MDS CD34^ 细胞出现明显分化，细胞形态学显示细胞向成熟转化，实验组的单抗表达水平CD71、CD33、CD19、CD13均较对照组为高，细胞周期分化。结论 IL-15对MDS CD34^ 细胞有一定的诱导分化作用，IL-15对MDS可能有一定的治疗应用前景。     

儿童神经母细胞瘤骨髓微小残留病变的临床意义分析

目的探讨儿童神经母细胞瘤(NB)患者骨髓微量NB细胞的动态变化及其与临床特征、治疗转归的关系。方法纳入33例确诊为NB并完善骨髓微量NB细胞监测的患儿,采用流式细胞仪(FCM)检测NB患儿治疗前后骨髓微量NB细胞,结合临床数据,进行Mann-Whitney U tests及Fisher精确概率法分析。结果 (1)初诊时骨髓转移与INRG分期、MYCN扩增状态、治疗疗效及是否复发均无显著相关性。(2)转移期患儿的骨髓转移发生率为69%,提示骨髓是NB肿瘤细胞主要转移地点。(3)治疗后骨髓微小残留病灶(MRD)持续阳性或由阴转阳患儿复发可能性大(P 0.001)。结论本研究初诊转移期患儿转移发生率过半,提示NB侵袭性强,骨髓为主要转移器官。监测骨髓MRD有助于早期发现疾病复发,指导临床治疗。     

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。     

γ干扰素和神经生长因子诱导神经母细胞瘤细胞分化及TrkA的表达

目的　在人神经母细胞瘤 (neuroblastoma ,NB)SMS KCNR细胞系中分别应用γ干扰素(IFN γ)、神经生长因子 (NGF)及联合应用IFN γ和NGF作为诱导剂诱导NB细胞分化 ,观察细胞形态学的变化、细胞增殖情况及TrkAmRNA的表达。方法　常规方法培养人SMS KCNR细胞系细胞 ,收取第 10天培养的细胞 ,提取总RNA ,应用逆转录 聚合酶链反应 (RT PCR)方法检测TrkAmRNA的表达水平 ,应用锥虫兰染色方法计数活细胞数并用倒置相差显微镜观察细胞形态学的改变 ,计数细胞分化率。结果　在KCNR细胞中同时负荷IFN γ和NGF的细胞表现出比单独负荷IFN γ或NGF更加显著的形态学分化 (P <0 0 1)。前者细胞增殖抑制更加明显 (P <0 0 1)。在IFN γ组 ,TrkAmRNA表达增加 (P <0 0 1) ,NGF组TrkAmRNA的表达未见明显改变 (P >0 0 5 ) ,联合用药组TrkAmRNA的表达有明显降低 (P <0 0 1)。结论　在人SMS KCNR细胞中 ,联合应用IFN γ和NGF比单独应用二者有更加显著诱导分化的作用。与IFN γ诱导TrkAmRNA表达增加不同 ,联合用药导致TrkAmRNA表达下调 ,可能存在某种负调节机制。研究结果表明 ,联合应用IFN γ和NGF可能是治疗NB甚至是晚期NB的合理方案     

The effect on cell growth by Wnt1 RNAi in human neuroblastoma SH-SY5Y cell line

Purpose  

We tested the hypothesis that Wnt signaling pathways are critical to neuroblastoma development. Our objective was to explore the novel role that Wnt/β-catenin plays in human neuroblastoma cell line SH-SY5Y, including detection of expression of wnt1 and β-catenin in SH-SY5Y, and the morphological and proliferative changes after Wnt1 RNAi in SH-SY5Y.     

RNAi抑制Wnt1信号通路对人神经母细胞瘤SH-SY5Y细胞增殖抑制的实验研究

目的 检测人神经母细胞瘤细胞株SH-SYSY中Wnt1的表达情况,并通过抑制Wnt1基因的表达来干预Wnt1信号通路,观察其对神经母细胞瘤增殖的影响,阐明Wnt1信号通路与神经母细胞瘤之间的相关性.方法 检测人神绛母细胞瘤株SH-SY5Y中Wnt1在基因和蛋白水平的表达及细胞内定位情况;将SH-SY5Y细胞分为四组,分别是阴性对照组、空白载体组、非特异性siRNA组和Wnt1-siRNA组.用针对Wnt1的特异性siRNA阻断该细胞中Wnt1信号通路的传递,分别检测上述四组细胞中Wnt信号通路中Wnt1及β-catenin浓度的变化;显微镜下观察细胞形态、数量的改变;用MTT方法检测肿瘤细胞增殖情况.结果 SH-SY5Y中的Wnt1在mRNA转录水平以及蛋白水平均有表达,且位于细胞浆内.采用Wnt1的特异性siRNA技术干预细胞后,Wnt1特异性siR-NA干预组中Wnt1蛋白的表达较阴性对照组、空白载体组及非特异性siRNA组明显下降(P<0.01),其下游通路中的关键蛋白β-catenin的表达同样明显下降(P<0.01);光学显微镜下发现特异性干预组中细胞明显较其他三组少,死亡漂浮的细胞较多,细胞突触减少;MTT结果提示,Wnt1基因表达被抑制后,SH-SY5Y细胞的增殖率明显下降(P<0.01).结论 Wnt1在人神经母细胞瘤细胞株SH-SY5Y中呈阳性表达;阻断Wnt1信号通路可以抑制人神经母细胞瘤细胞株SH-SY5Y的增殖.Wnt1信号通路可能是影响神经母细胞瘤增殖的重要通路.     

Lymphokine-activated killer cytotoxicity in neonatal mononuclear cells: in vitro responses to tumor cell lines from pediatric solid tumors

The presence of neonatal (cord) lymphokine-activated killer (LAK) cell activity toward natural killer cell resistant Raji and Daudi cell lines has recently been reported from our laboratory. We investigated the future therapeutic use of LAK adoptive immunotherapy by examining LAK in vitro cytotoxicity from both neonatal and adult mononuclear cells against solid tumor cell lines of relevance to pediatric oncology: SH-SY5Y (neuroblastoma), SK-NM-C (neuroblastoma-neuroepithelioma), NEP-1 (Wilms' tumor), SK-ES-1 (Ewing's sarcoma), and A-204 (rhabdomyosarcoma). Cord and adult mononuclear cells were activated by recombinant IL-2 (100 mu/ml) for 5-7 days and added in an effector:target ratio of 40:1 to 51Cr-labeled target cells. Specific cell lysis was determined after a 4-h incubation. There was a significantly high level of cord and adult LAK cytotoxicity against Wilms' (76.4 +/- 9.8 versus 77.3 +/- 6.8%) and Ewing's (84.2 +/- 5.5 versus 71.1 +/- 6.5%) cell lines and significant but moderate LAK activity against neuroepithelioma (52.0 +/- 6.6 versus 55.4 +/- 4.5%) and rhabdomyosarcoma (46.6 +/- 5.7 versus 43.9 +/- 5.2%) cell lines. There was no difference between cord and adult LAK activity toward these targets. However, a differential response toward the more classical neuroblastoma cell line, SH-SY5Y, was noted with significantly more LAK cytotoxicity from cord mononuclear cells than adult mononuclear cells (51.2 +/- 6.9 versus 28.5 +/- 8.2%) (p less than or equal to 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of retinoic acid on proliferation,apoptosis, cytotoxicity,migration, and invasion of neuroblastoma cells

BACKGROUND: Because of the known property of less aggressiveness of differentiated cells compared to immatured cells all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. We are interested in examining the influence of retinoic acid (RA) on proliferation, apoptosis, cytotoxicity, migration, and invasion in dependence of the differentiation of neuroblastoma cells classified into N-type (SK-N-FI, SH-SY5Y), I-type (SK-PN-DW), and S-type (SK-N-LO, SK-N-MC) cells. PROCEDURE: Neuroblastoma cells were exposed to 10(-5) M RA and 200 ng/ml camptothecin (CAM) (control substance for apoptosis). Proliferation, apoptosis, and cytotoxicity were quantified by photometric assays. The influence on migration and invasion of neuroblastoma cells was examined by a scratch-test and by the measurement of the invasion through matrigel coated chamber inserts. RESULTS: In general, RA treatment induced proliferation inhibition predominantly in the cell lines SK-PN-DW (16%, P < 0.05) and SK-N-MC (8%, (P < 0.001), respectively. In the N-type cell lines SK-N-FI (P > 0.05) and SH-SY5Y (P < 0.001) no proliferation inhibition was determined conforming with no detection of apoptosis. CAM confirmed its capability to induce apoptosis in the cell lines SH-SY5Y (43.6%, P < 0.05), SK-PN-DW (54.8%, P > 0.05), and SK-N-MC (28.9%, P < 0.0 01) except for SK-N-FI with only 9.3% (P > 0.05), but after 24 hr of treatment. Minor signs of restricted migration were observed, while RA treatment reduced significantly the invasion rate through Matrigel of SK-N-FI to 13.3% (P < 0.01), SH-SY5Y to 19.2% (P < 0.05), SK-N-MC to 27.8% (P < 0.05), and SK-N-LO to 17.7% (P < 0.01). CONCLUSIONS: It is demonstrated that RA treatment can interfere with cell growth and in invasion by inducing neuronal differentiation in N-type and apoptosis in S-type neuroblastoma cell lines.     

miR-144通过靶定ITGβ1调控神经母细胞瘤的侵袭转移

目的 本研究旨在探讨miR-144对神经母细胞瘤的侵袭和转移的影响,并探索miR-144是否通过靶定ITGβ1调节神经母细胞瘤的侵袭和转移.方法 利用生物信息学技术预测miR-144的靶基因ITGβ1,应用荧光报告基因检测以及荧光定量PCR、Western Blot实验对靶基因进行验证.在神经母细胞瘤SH-SY5Y和SK-N-BE分别瞬时转染化学合成的成熟双链miR-144和靶基因敲降质粒及对照质粒,建立过表达miR-144细胞模型和靶基因敲降细胞模型.通过 Transwell侵袭实验和划痕实验检测细胞的体外侵袭和转移能力.结果 Transwell实验结果表明,在神经母细胞瘤细胞SH-SY5Y和SK-N-BE中过表达miR-144后,细胞的侵袭数目分别为41±2和55±2,与对照组相比差异有统计学意义(P<0.01).抑制ITGβ1基因的表达后,体外划痕实验结果表明,过表达miR-144后,与对照组相比,SH-SY5Y和SK-N-BE细胞的转移能力分别下降了65.45%和75.41%(P<0.01).荧光报告基因检测结果显示过表达miR-144后,含有结合位点的荧光报告质粒的相对荧光量降低了37.11%(P<0.01),而将结合位点进行基因突变后miR-144对靶基因的抑制作用消失.过表达miR-144后,与对照组相比,SH-SY5Y和SK-N-BE细胞内的ITGβ1 mRNA水平分别降低了40%和43%,Western Blot检测结果显示,ITGβ1蛋白水平分别降低了39%和40%.结论 miR-144通过靶定ITGβ1基因抑制神经母细胞瘤的侵袭和转移过程.     

Cytotoxicity of lymphokine-activated killer cells against human neuroblastoma cells: modulation by neuroblast differentiation

The cytolytic activity of lymphokine-activated killer (LAK) cells against human neuroblastoma (NB) cells was investigated using the continuous NB cell lines, IMR-32, Kelly, and two subclones of SK-N-SH, SH-SY5Y (neuroblastic phenotype), and SH-EP (non-neuronal phenotype). NB cells were found to be sensitive targets of LAK. Of the SK-N-SH subclones, the neuroblasts, SH-SY5Y, were more susceptible to LAK killing than were the non-neuronal cells, SH-EP. Pretreatment of the targets SH-SY5Y and SH-EP with the differentiating agents, retinoic acid (RA, 10 microM), herbimycin A (236 nM), or nerve growth factor (10 ng/ml), did not substantially alter LAK killing. Furthermore, these differentiating agents did not measurably affect LAK activity during the cytolysis assay or with 1-h preincubation of the LAK effectors. However, co-incubation of the LAK cultures over the 3-day activation period with RA (1 microM) or PGE2 (1 microM) inhibited cytolysis by 80%, suggesting that these agents interfere with an early activation step of LAK. These results support the potential use of LAK treatment for neuroblastoma, in combination with differentiation agents that do not affect neuroblastoma sensitivities toward LAK cells. However, some differentiation agents, (e.g., RA) and endogenous prostaglandins (e.g., PGE2) may interfere with LAK activation.     

Stromal cells and human malignant neuroblasts derived from bone marrow metastasis may share common karyotypic abnormalities: the case of the IGR-N-91 cell line

BACKGROUND: Stage IV neuroblastoma is characterized by tumor invasion and metastatic dissemination. Cell lines derived from such neuroblastomas have a high in vitro proliferation capacity. PROCEDURE: We established three neuroblastoma cell lines derived from involved bone marrow of three patients with stage IV neuroblastoma and performed a cytogenetic study. RESULTS: Various culture conditions allowed us to distinguish two cell subpopulations: malignant neuroblasts (Nb-type) and substrate-adherent stromal cells (Str-type). Karyotypic analyses revealed two specific chromosomal abnormalities in diploid malignant IGR-N-331 neuroblasts, der(1)t(1;7)(p22;q11) and der(5)t(5;17)(q35;q21), one unbalanced translocation der(1)t(1;17)(p35;q21)x2 in hyperdiploid malignant IGR-N-337 neuroblasts, and a normal karyotype in both corresponding stromal subpopulations. In contrast, in the IGR-N-91 model, both cell types shared two unbalanced translocations, t(1;4)(q12;p15) and t(2;10)(p14;q11), suggesting that stromal cells and malignant neuroblasts originate from a common stem cell. CONCLUSIONS: Based on our findings, we postulate that genetically modified stromal cells may influence the metastatic potential of malignant neuroblasts.     

雷帕霉素对人神经母细胞瘤细胞株生长抑制及诱导凋亡的研究

目的 研究雷帕霉素对人神经母细胞瘤细胞株SH-SY5Y生长抑制及诱导凋亡的作用.方法 雷帕霉素处理SH-SY5Y细胞,并设立对照组,以CCK-8检测细胞生长活性,流式细胞法(FCM)检测细胞周期,DNA梯度条带(DNA Ladder)和罗丹明123-碘化丙啶(Rhodamine 123/PI)染色法分析细胞凋亡.结果 雷帕霉素处理后,神经母细胞瘤细胞呈浓度依赖性生长抑制.20μM雷帕霉素处理12 h后,细胞周期出现G2/M期、S期阻滞,G2/M期(24.22±2.89)%、S期(22.05±0.96)%的细胞百分比明显高于对照组.罗丹明123-碘化丙啶检测细胞凋亡率(54.35±5.39)%也明显高于对照组,并有明显的凋亡条带出现.结论 雷帕霉素具有抑制神经母细胞瘤细胞株SH-SY5Y生长、诱导其凋亡的作用.     

TrkA signal transduction pathways in neuroblastoma

BACKGROUND: Favorable neuroblastomas frequently express high levels of the TrkA receptor, and these tumors have a propensity to either differentiate or regress, but the mechanisms responsible for these two fates are unclear. PROCEDURE: To study TrkA signal transduction in neuroblastoma (nb), we stably expressed wild-type TrkA and five TrkA mutants in the human nb cell line SH-SY5Y. Resulting single cell clones were characterized by TrkA mRNA and protein expression and by autophosphorylation of the receptor. RESULTS: Introduction of TrkA restored nerve growth factor (NGF) responsiveness of SH-SY5Y cells, demonstrated by morphological differentiation and induction of immediate-early genes. TrkA overexpression leads to growth inhibition in the absence of NGF, whereas NGF treatment results in increased proliferation. CONCLUSIONS: Analysis of downstream signaling elements in mutated TrkA receptors indicates that NGF-induced differentiation is dependent on TrkA kinase activity, but several redundant pathways seem to be used farther downstream. This suggests differences from TrkA pathways identified in PC12 cells.