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。     

儿童肾上腺肿瘤104例临床分析

目的分析儿童肾上腺肿瘤的临床特征,提高对儿童肾上腺肿瘤诊断和处理的认识。方法回顾分析1991—2002年12年间中山大学附属第一医院收治的104例儿童肾上腺肿瘤的临床特点、诊断和病理的关系。结果发病年龄0～14岁,男63例,女41例。神经母细胞瘤75例;肾上腺皮质肿瘤22例,其中癌及腺瘤各11例;嗜铬细胞瘤2例;其他5例。神经母细胞瘤患儿以发热、腹痛、腹部包块为主要表现,尿香草杏仁酸(VMA)可不升高;肾上腺皮质肿瘤患儿中外周性性早熟的临床及内分泌激素改变多于库欣综合征表现。血乳酸脱氢酶(LDH)水平对鉴别良/恶性肿瘤有提示意义。结论儿童肾上腺肿瘤以神经母细胞瘤及肾上腺皮质肿瘤最为常见,临床表现异于成人,应结合临床及影像学特征综合诊断。     

Intraoperative radiation therapy for advanced neuroblastoma: the problem of securing the IORT field

The purpose of this study is to evaluate the efficacy of intraoperative radiation therapy (IORT) and the problem of securing the IORT field in advanced pediatric neuroblastoma. Between 1996 and 2005, 12 children received IORT for advanced pediatric neuroblastoma patients. Electron beam energies ranged from 10 to 12 MeV and median dose was 10 Gy (8-12 Gy). All of them had surgery with IORT against the primary tumor site and the abdominal aorta surroundings. A gross total resection (GTR) was achieved in 10 patients and subtotal resection (STR) was two patients. All of 12 patients were classified as high risk. Nine patients were alive 17-120 (mean 48 months) after diagnosis. Local tumor control was achieved in 100% of patients, of whom one experienced local recurrence outside the IORT field. At the operation, it was difficult to secure the IORT field because of the angle of the radiation cylinder in three patients. One of the three of these patients experienced local recurrence outside of the IORT field in the upper side of superior mesenteric artery and two of three patients had an external beam radiation after surgery, and there was no local recurrence. One patient had a postoperative ileus, and one patient had transient diarrhea and hydronephrosis. For advanced neuroblastoma patients, IORT produced excellent local control after surgery. However, there is a problem of securing the IORT field. For local control, it is necessary to add an external beam radiation after IORT when it is difficult to secure the IORT field.     

Complex constitutional subtelomeric 1p36.3 deletion/duplication in a mentally retarded child with neonatal neuroblastoma

Monosomy 1p36 is one of the most frequent subtelomeric microdeletion syndromes characterized by distinct craniofacial features and developmental delay/mental retardation. Other common symptoms include hypotonia, seizures, brain abnormalities, visual, auditory and heart defects. Neuroblastoma is a rare feature since to our knowledge only two patients with “pure” 1p36 deletion have been described. We report on a child with developmental delay and facial dysmorphy who developed neuroblastoma at 1 month of age. No primary site outside of the liver could be demonstrated and the tumour regressed spontaneously. Standard karyotyping was normal while subtelomeric screening using Multiplex Ligation-dependent Probe Amplification (MLPA) method revealed a constitutional de novo subtelomeric 1p36 deletion. Subsequent Agilent 244K oligonucleotide array-based comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis showed a complex 1p36.3 deletion/duplication rearrangement. Among the best candidate genes predisposing to the development of neuroblastoma located in 1p36, the AJAP1 gene is the only gene present in the duplication while CHD5, TNFRSF25 and CAMTA1 are located outside of the rearrangement. Therefore, a gene-dosage effect involving a gene located in the duplication including AJAP1 might explain the neuroblastoma observed in our patient. The rearrangement might equally interfere with the expression of a gene located outside of it (including CHD5 located 1 Mb away from the rearrangement) playing a role in the tumorigenesis. In conclusion, this study illustrates the complexity of such rearrangement characterized by array CGH and strengthens that constitutional 1p36.3 rearrangement predisposes to the development of neuroblastoma.     

反义寡核苷酸抑制神经母细胞瘤LA-N-5细胞血管内皮生长因子mRNA表达及其生物效应的研究

目的研究血管内皮生长因子(VEGF)反义寡核苷酸(ASODN)转染对神经母细胞瘤LA-N-5细胞VEGF mRNA表达的影响以及对肿瘤细胞增殖、分化的影响。方法用LipofectamineTM2000介导的VEGF ASODN和错义寡核苷酸(MSODN)转染LA-N-5细胞,半定量RT-PCR检测各组细胞VEGF165和VEGF121 mRNA转染前后不同时间表达的变化;MTT法测定转染后各组细胞的生长曲线及抑制率。结果半定量RT-PCR检测结果显示,在转染后72 h VEGF165和VEGF121 mRNA的表达:ASODN组为0.346±0.029和0.227±0.036,ASODN+LipofectamineTM2000组为0.275±0.035和0.165±0.017。ASODN组和ASODN+LipofectamineTM2000组均显著抑制VEGF mRNA的表达,ASODN+LipofectamineTM2000组抑制作用较ASODN组更强(P<0.05);转染后ASODN组和ASODN+LipofectamineTM2000组细胞增殖显著受抑,在48 h时抑制率最高,分别为(39.92±2.7...     

整合素α2对神经母细胞瘤细胞粘附于胶原蛋白的调节作用

目的：探讨两价阳离子和针对整合素α2的 特异性阻断性单克隆抗体对神经母细胞瘤细胞株SK-N-SH细胞粘附于胶原蛋白（Col）的影响，探讨整合素α2β1对该细胞粘附反应的调节作用。方法:将含有不同M g2+、Ca2+浓度和有或无抗α2单克隆抗体6F1的SK-N-SH细胞混悬液。加入96孔 平底非细胞培养板，此反应板已包被Ⅰ型大鼠尾Col（4 μg/well），所粘附的细胞用BCA方 法测定，以吸光度（A570）代表粘附细胞的数量。结果:Mg 2+能明显增加SK-N-SH细胞Ⅰ型Col的粘附反应；Mg2+浓度为1 mmol/L时，细胞 粘附的反应已接近高峰,与对照组相比A570分别为0.59±0.03和0.2 5±0.0 1 (P＜0.01），而Ca2+对细胞的粘附无明显影响；抗α2单克隆抗体6F1（10 mL /L）能显著阻断SK-N-SH细胞粘附于胶原蛋白(A=0.27±0.01)，其粘附水平接近无Mg 2+的粘附反应水平（A=0.32±0.01）。 结论:整合素α2介导 SK-N-SH细胞对胶原蛋白粘附反应，提示整合素α2可能参与调节神经母细胞瘤的转移。     

LRIG3基因过表达对人神经母细胞瘤细胞系SK-N-MC细胞生物学行为的影响

目的 探讨富含亮氨酸重复序列免疫球蛋白样蛋白3（LRIG3）基因过表达对人神经母细胞瘤细胞系SK-N-MC细胞生物学行为的影响。方法 体外培养人神经母细胞瘤细胞系SK-N-MC细胞,分成空白对照组（A组,不转染任何载体或质粒）、载体组（B组,转染载体）和LRIG3过表达组（C组,转染含LRIG3基因过表达质粒）。 荧光定量PCR和免疫印迹法检测LRIG3、Caspase-3和Caspase-9的mRNA和蛋白表达水平。Transwell试剂盒检测细胞侵袭能力,AV-PI试剂盒检测细胞凋亡水平,CCK-8试剂盒检测细胞增殖能力。结果 C组Caspase-3和Caspase-9的mRNA和蛋白表达水平以及细胞凋亡率明显高于A组和B组（P<0.05）,细胞增殖率和细胞侵袭能力明显低于A组和B组（P<0.05）,而A组和B组之间均无统计学差异（P>0.05）。结论 LRIG3过表达可以抑制人神经母细胞瘤细胞系SK-N-MC细胞增殖和侵袭,促进其凋亡,其机制可能与促进Caspase-3和Caspase-9表达有关     

Involvement of K+ channels in the quercetin-induced inhibition of neuroblastoma cell growth

The effects of the flavonoid quercetin on cell proliferation and voltage-dependent K⁺ current were studied on mouse neuroblastoma×glioma hybrid cells. In the presence of 1 % fetal calf serum, quercetin inhibited both cell proliferation and K⁺ current with effective doses inducing half-maximum effects of 10 M and 70 M respectively. Valinomycin (1 nM) antagonized 80 % of the growth-inhibitory effects of 10 M quercetin. The results suggest that at least a part of the antiproliferative effect of quercetin is mediated by a K⁺ channel blockade. They further confirm a role for K⁺ channels in mitogenesis and cell proliferation.     

The mechanism of the actions of oxaliplatin on ion currents and action potentials in differentiated NG108-15 neuronal cells

Oxaliplatin (OXAL) is a platinum-based chemotherapeutic agent which is effective against advanced or metastatic gastrointestinal cancer. However, the mechanisms responsible for the development of the neuropathy induced by this agent remain unclear. In this study, we attempted to evaluate the possible effects of OXAL on ion currents and action potentials (APs) in NG108-15 cells differentiated with dibutyryl cyclic-AMP. Application of OXAL decreased the peak amplitude of voltage-gated Na⁺ current (I_Na) with no change in the overall current–voltage relations of the currents. This agent also produced a concentration-dependent slowing of I_Na inactivation. A further application of ranolazine reversed OXAL-induced slowing of I_Na inactivation. Unlike ranolazine or riluzole, OXAL had no effect on persistent I_Na elicited by long ramp pulses. OXAL (100 μM) also had little or no effect on the peak amplitude of L-type Ca²⁺ currents in NG108-15 cells, while it suppressed delayed-rectifier K⁺ current. In current-clamp recordings, OXAL alone reduced the amplitude of APs; however, it did not alter the duration of APs. However, after application of tefluthrin, OXAL did increase the duration of APs. Moreover, OXAL decreased the peak amplitude of I_Na with a concomitant reduction of current inactivation in HEK293T cells expressing SCN5A. The effects of OXAL on ion currents presented here may contribute to its neurotoxic actions in vivo.