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.     

DKK-1在神经母细胞瘤中的表达及其意义

目的 研究Dickkopf-1 (DKK-1)在儿童神经母细胞瘤(NB)中的表达及其意义.方法 运用免疫组织化学方法检测DKK-1在42例NB,19例神经节细胞瘤及15例正常肾上腺组织中的表达水平.运用实时荧光定量PCR(qRT-PCR)、Western blotting检测DKK-1在分化程度依次增高的3株NB细胞株IMR-32、SK-N-SH和SH-SY5Y中的mRNA及蛋白表达水平.结果 免疫组织化学研究发现DKK-1在正常肾上腺组织中呈强阳性表达(免疫组织化学评分为1.912±0.047),在神经节细胞瘤组织中呈弱阳性表达(免疫组织化学评分为1.214±0.095),在NB组织中呈阴性表达(免疫组织化学评分为0.371±0.293).神经节细胞瘤和NB中DKK-1的表达量分别与正常对照组相比较,差异均有统计学意义(all P＜0.001).在3种组织中,不同组间DKK-1表达量的差异有统计学意义(F=211.375,P＜0.001).qRT-PCR研究发现DKK-1在IMR-3、SK-N-SH和SH-SY5Y中平均相对表达量分别为(25.323±8.651)％、(58.852±10.223)％和(75.385±9.381)％,均呈低表达水平,不同组间DKK-1表达差异有统计学意义(F=57.628,P＜0.001).Western blotting研究发现在IMR-32、SK-N-SH和SH-SY5Y中DKK-1蛋白表达相对灰度值依次增高,分别为0.093±0.016、0.397±0.051和0.411±0.062,不同组间蛋白表达差异有统计学意义(F=156.783,P＜0.001).结论 DKK-1在NB组织及细胞株中均呈低表达水平,且DKK-1在NB中的表达可能与该肿瘤的分化密切相关,提示其或许可以作为一个特殊的分子标记物用于NB的早期诊断及预后监测.     

HA117调节DPF3表达对神经母细胞瘤分化的影响

目的 探讨HA117及DPF3在儿童神经母细胞瘤(NB)中的表达及意义,及其对NB分化的影响和可能机制.方法 运用实时荧光定量PCR (qRT-PCR)、免疫组化法检测HA117、DPF3在35例NB、8例节细胞神经母细胞瘤(GNB)和10例神经节细胞瘤(GN)中的表达水平.构建HA117过表达和空载慢病毒,分别转染SH-SY5Y细胞株,构建HA117过表达组和对照组.采用qRT-PCR和Western Blot检测HA117和DPF3的RNA和蛋白质表达水平.采用10 μmol/L维甲酸诱导HA117过表达组和对照组细胞,普通光学显微镜观察细胞形态,免疫荧光法检测神经特异性标志物MAP2蛋白质表达,Western Blot检测HA117过表达组和对照组细胞MAP2、NSE蛋白质的表达情况.结果 HA117的RNA在NB中的相对表达量为30.14±15.96,明显高于GNB/GN的1.03±0.37(P＜0.05),DPF3的mRNA在NB中的表达量为0,81±0.17,明显低于GNB/GN的3.14±1.09(P＜0.05),与HA117的表达趋势相反.免疫组化检测发现DPF3在GNB/GN中呈强阳性表达,在分化中的NB中呈弱阳性表达,在未分化/分化差的NB中呈阴性表达.过表达HA117可在mRNA和蛋白水平明显下调SH-SY5Y中DPF3的表达.经10 μmol/L维甲酸诱导7d后,对照组细胞生长明显减慢,细胞长出突起,发生神经元样分化;HA117过表达组SH-SY5Y的MAP2、NSE的表达水平明显低于对照组(P＜0.05).结论 HA117和DPF3的表达可能与NB的分化相关,HA117可能通过下调DPF3的表达抑制NB的分化.     

SNHG7-miR-653-5p-STAT2反馈通路在神经母细胞瘤进展中的调节作用研究

目的 研究lncRNA SNHG7在神经母细胞瘤(neuroblastoma,NB)进展中的作用,以及SNHG7-miR-653-5p-STAT2反馈通路在神经母细胞瘤进展中的调节作用.方法 从青岛大学附属医院收治的NB患儿体内获得92对NB组织及相邻非肿瘤组织.采用qRT-PCR检测SNHG7在神经母细胞瘤肿瘤组织及...     

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)     

神经母细胞瘤及胚胎肾上腺组织端粒酶基因表达的研究

目的 观察神经母细胞瘤和胚胎肾上腺组织中端粒酶基因（hTR/hTRT)的表达,以及神经母细胞瘤细胞在药物诱导分化时端粒酶基因表达的变化,探讨端粒酶在神经母细胞瘤形成和发展中的作用。方法 体外转录法制备生物素标记的hTR、hTRTcRNA探针。神经母细胞瘤存档石蜡标本20例,2-5个月胎儿肾上腺组织石蜡标本12例,应用原位杂交组织化学（ISHH）方法检测hTR及hTRT表达,用维甲酸类似物R9158诱导人神经母细胞瘤细胞系SH-SY-5Y及SK-N-SH,细胞（90.0%)阳性。各月龄胎儿肾上腺组织均有hTR及hTRT表达,神经嵴细胞和继发皮质细胞内信号强于原发皮质细胞,神经嵴细胞形态极似神经母细胞瘤细胞,维甲酸敏感细胞SH-SY-5Y在R9158作用5d后出现明显形态学变化,表达hTR的细胞明显减少,维甲酸耐受细胞SK-N-SH玩明显变化。结论 端粒酶基因在神经母细胞瘤和胚胎肾上腺组织中高表达,神经母细胞瘤细胞在药物诱导下分化时端粒酶基因hTR表达明显降低。端粒酶在神经母细胞瘤形成和发展中有重要作用。     

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。     

神经母细胞瘤骨髓转移细胞体外原代培养技术的实验研究

目的：根据神经母细胞瘤(neuroblastoma，NB)易发生早期骨髓转移的特点，建立NB骨髓转移细胞体外原代培养的实验技术。方法：对27例伴骨髓转移的NB患者进行NB骨髓转移细胞体外原代培养，并与已建立的SMS-KCNR和SH-SY5Y细胞系进行比较，在倒置相差显微镜下观察细胞生长情况及形态学特点，同时应用单克隆抗体F3系列检测原代培养的NB骨髓转移细胞。结果：在27例NB骨髓转移细胞体外原代培养中，培养成功9例，其细胞生长情况及形态学特点与SMS-KCNR和SH-SY5Y细胞系基本一致，单克隆抗体F3系列检测均阳性。结论：NB骨髓转移细胞体外原代培养技术的成功建立为NB体外诱导逆转及多药耐药等复杂机制研究提供更接近体内生物学性状的细胞来源。     

5-氮杂胞苷增强阿霉素对神经母细胞瘤细胞的抗瘤活性

目的：许多神经母细胞瘤细胞系及肿瘤组织标本中caspase-8表达缺失, caspase-8基因沉寂的主要原因是其启动子区域高度甲基化。该文探讨去甲基化药物5-氮杂胞苷对caspase-8表达及对化疗药阿霉素抗人神经母细胞瘤细胞作用的影响及其影响机制。方法：应用RT-PCR方法检测5-氮杂胞苷作用前后SH-SY5Y细胞中caspase-8 mRNＡ水平变化。MTT分析研究5-氮杂胞苷与化疗药阿霉素联合应用前后人神经母细胞瘤细胞SH-SY5Y的存活率,以及加入caspase-8活性抑制剂后存活率的变化。结果：RT-PCR方法发现SH-SY5Y细胞株不表达caspase-8 mRNA, 5-氮杂胞苷作用3 d后可检测到 caspase-8 mRNA表达,5 d后表达较第3天增加; 5-氮杂胞苷与不同浓度阿霉素(0.05, 0.1, 0.25, 0.5 μg/mL)联合应用后SH-SY5Y细胞存活率为(77.61±7.30)%,（57.35±6.64）%,（46.25±4.46）%,（35.59±5.12）%,同一浓度单独应用阿霉素组细胞存活率为（94.89±4.15）%,（80.60±8.50）%,（64.48±4.92）%,（52.32±6.71）%,5-氮杂胞苷与阿霉素联用组细胞存活率明显低于单用阿霉素组,差异均有显著性。Caspase-8抑制剂组与同一浓度的5-氮杂胞苷+阿霉素组比较,细胞存活率明显增加,依次为（92.95±3.48）%,（78.39±4.28）%,（62.31±6.50）%,（49.92±5.77）%,差异均有显著性。结论：阿霉素对神经母细胞瘤细胞SH-SY5Y具有增殖抑制作用,5-氮杂胞苷可以增强阿霉素的抗瘤活性。其发生机制可能是通过上调caspase-8 mRNA的表达而实现的。［中国当代儿科杂志,2007,9（6）：577-579］     

ROLE OF CASPASE 8 AS A DETERMINANT IN TRAIL SENSITIVITY OF NEUROBLASTOMA CELL LINES

Objective: Resistance of neuroblastoma (NB) cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis is thought to be caused by loss of caspase 8 expression. The aim of this study is to investigate if induction of caspase 8 by γ-interferon (IFNγ) renders NB cells sensitive to TRAIL. Methods: Caspase 8 expression was monitored by RT-PCR and Western blot analysis. The effects of IFNγ, TRAIL, IFNγ + TRAIL, and caspase 8 inhibitor + TRAIL on the growth and apoptosis of NB cells were detected with the methods of reduction rate of Alamar blue assay and flow cytometry. The relative caspase 8 activity was measured with colorimetric assay. Results: An increased expression of caspase 8 mRNA and protein was found after treatment with IFNγ. IFNγ in combination with TRAIL decreased proliferation of NB cell line SH-SY5Y cells. The killing effect of TRAIL on NB cells expressing caspase 8 was depressed by caspase 8 inhibitor. The relative caspase 8 activity of NB cells expressing caspase 8 increased with the prolongation of TRAIL action time. Conclusions: TRAIL induced apoptosis in NB cells expressing caspase 8.     

顺铂及依托泊苷对神经母细胞瘤细胞中Trk基因家族表达的影响及临床意义

目的神经母细胞瘤(NB)是儿童常见的实体瘤,而三种不同Trk基因的表达变化与NB的预后及转归关系密切;为研究如何优化化疗药物疗效以提高NB患儿的生存率,本文检测了顺铂(DDP)及依托泊苷(VP16)对人NB的SK-N-SH细胞系的杀伤作用及对细胞中Trk基因家族表达的影响。方法通过CCK-8药物诱导细胞毒性实验检测不同浓度梯度的DDP及VP16对SKN-SH细胞的杀伤作用以及半数杀伤浓度(IC50);用qRT-PCR方法检测DDP及VP16在不同浓度下分别作用24 h及48 h后,对SK-N-SH细胞中TrkA、TrkB和TrkC基因表达变化。结果(1)IDDP及VP16药物浓度的增加与其对SK-N-SH细胞的杀伤作用呈线性关系;且48 h比24 h杀伤作用更明显。(2)SK-N-SH细胞中TrkA及TrkC的表达均随着DDP浓度和作用时间的增加而上调,尤其在10μg/mL浓度、24h最明显;而TrkB的表达在24 h时随着DDP浓度增加而上调,48 h时该促进作用不明显;(3)SK-N-SH细胞中TrkA、TrkB及TrkC的表达亦随着VP16浓度和作用时间的增加而上调。结论 DDP及VP16对NB细胞SK-N-SH有明显杀伤作用并对其Trk基因家族的表达有影响。     

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.     

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.     

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.     

Caspase-8在TRAIL诱导神经母细胞瘤细胞凋亡中的作用

目的：应用干扰素(IFNγ)诱导神经母细胞瘤（neuroblastoma, NB）细胞caspase 8的表达并观察其是否可以恢复NB细胞对肿瘤坏死因子相关凋亡诱导配体（TRAIL）的敏感性。方法：应用RT PCR方法检测IFNγ作用前后NB细胞caspase-8 mRNA的表达;应用Alamar Blue法及流式细胞术检测IFNγ、TRAIL、IFNγ+TRAIL、IFNγ+caspase-8抑制剂zIETD-FMK+TRAIL对NB细胞生长及凋亡的影响;应用比色法测定NB细胞caspase-8相对活性。结果：对TRAIL敏感的CHP212细胞表达caspase-8,且经IFNγ处理后caspase-8 表达水平逐步增加;对TRAIL不敏感的SY5Y细胞不表达caspase-8,IFNγ作用后其caspase-8 mRNA表达明显增加。IFNγ与TRAIL联用对SY5Y细胞有明显诱导凋亡作用。CHP212细胞caspase-8相对活性随TRAIL作用时间的延长逐步升高;IFNγ与TRAIL联合作用的SY5Y细胞caspase-8相对活性明显高于未加药物处理的对照组、IFNγ组、TRAIL组及抑制剂组。结论：表达caspase-8的NB细胞对TRAIL的诱导凋亡作用敏感,TRAIL诱导NB细胞凋亡过程中伴随caspase-8活性的增加。［中国当代儿科杂志,2010,12(11)：902-907］     

苦参碱对人神经母细胞瘤SH-SY5Y细胞的作用及机制

目的 探讨苦参碱(matrine,Ma)对人神经母细胞瘤(neuroblastoma,NB) SH-SY5Y细胞的作用及机制。方法 取对数生长期的细胞,分为对照组(含细胞无Ma)及4个浓度组（Ma的作用浓度）分别为0.5 mg/ml、1.0 mg/ml、2.0 mg/ml和3.0mg/ml,每组5个复孔。应用四甲基偶氮唑蓝(MTT)比色法检测神经母细胞瘤SH-SY5Y细胞的增殖抑制率;流式细胞仪(FCM)检测凋亡率;RT-PCR方法检测TrkA mRNA的表达。最后采用SPSS 16.(0统计软件进行单因素方差分析,P＜0.05为差异有统计学意义。结果 MTT比色法检测对照组及各浓度组增殖抑制率分别为(7.25±1.55)％、(12.56±1.94)％、(17.22±0.90)％、(22.39±0.78)％和(25.35±1.38)％;流式细胞仪检测对照组及各浓度组凋亡率分别为(5.30±0.60)％、(10.04±1.18)％、(14.34±1.79)％、(21.36±0.96)％和(24.80±1.53)％;RT-PCR方法检测TrkA mRNA的表达,对照组及各浓度组的灰度值分别为：0.502±0.043、0.584±0.032、0.644±0.052、0.836±0.056和0.948±0.030。以上检测各组间比较差异均有统计学意义(P＜0.05)。结论 苦参碱在体外环境下作用神经母细胞瘤SH-SY5Y细胞,可以有效抑制该细胞的增殖、诱导调亡并促进TrkA mRNA的表达,这为临床上神经母细胞瘤的治疗开拓了新的思路。     

苦参碱联合顺铂对SH-SY5Y细胞药物敏感性及TrkB表达的影响

目的探讨不同浓度苦参碱对神经母细胞瘤SH-SY5Y细胞存活及凋亡的影响,及苦参碱降低顺铂化疗耐药性的作用及可能机制。方法分别以全反式维甲酸(ATRA)、脑源性神经生长因子(BDNF)、顺铂、苦参碱处理SH-SY5Y细胞,实验分对照组、ATRA诱导的顺铂干预组、顺铂干预组、苦参碱干预组、苦参碱联合顺铂干预组。应用四甲基偶氮蓝比色法检测苦参碱与顺铂对SH-SY5Y细胞存活率的影响,流式细胞仪检测苦参碱与顺铂对SH-SY5Y细胞凋亡率的影响,Western-blot方法检测SH-SY5Y细胞的p-TrkB表达。结果除ATRA诱导的顺铂干预组外,其余各组SH-SY5Y细胞的存活率及凋亡率与对照组比较,差异均有统计学意义(P<0.05);顺铂干预组与ATRA诱导的顺铂干预组比较,SH-SY5Y细胞的存活率及凋亡率差异有统计学意义(P<0.05);相同浓度的苦参碱干预组与苦参碱联合顺铂干预组比较,SH-SY5Y细胞的存活率及凋亡率差异有统计学意义(P<0.05);苦参碱联合顺铂组与顺铂干预组比较,SH-SY5Y细胞的存活率及凋亡率差异有统计学意义(P<0.05)。苦参碱0.5 mg/ml组与顺铂干预组比较,SH-SY5Y细胞的存活率及凋亡率差异无统计学意义(P>0.05)。各组SH-SY5Y细胞间,p-TrkB的表达水平差异无统计学意义(P>0.05)。结论苦参碱可以抑制SH-SY5Y细胞的增殖并诱导凋亡,从而提高顺铂化疗的敏感性,该作用与TrkB/BDNF信号通路(间)无相关性。