Eg5基因在神经母细胞瘤细胞增殖和凋亡中的作用

目的探讨Eg5基因对神经母细胞瘤(NB)细胞增殖和凋亡的影响。方法取2株NB细胞株SHSY-5Y和NBL-S,分别转染针对Eg5基因的小干扰RNA(siRNA),分为细胞对照组(用等体积的水代替siRNA,只含有脂质体,未转染细胞)、siRNA对照组(转染siRNA对照)和siRNA干扰组(转染Eg5 siRNA),每组6孔,每孔1×10~6个细胞。使用实时定量聚合酶链式反应(real-time PCR)检测Eg5 mRNA表达水平的变化;使用5-乙炔基-2’-脱氧尿苷(EdU)掺入实验和克隆形成实验检测下调Eg5表达对NB细胞增殖的影响;使用流式细胞术检测下调Eg5表达对NB细胞凋亡和细胞周期的影响。结果荧光定量PCR检测结果显示,siRNA干扰组Eg5 mRNA表达水平与细胞对照组和siRNA对照组相比显著减少,差异有统计学意义(P<0.05);EdU掺入实验和克隆形成结果表明,siRNA干扰组细胞中DNA的合成量和细胞克隆数量低于细胞对照和siRNA对照组,差异有统计学意义(P<0.05);流式细胞术检测结果显示,细胞对照组和siRNA对照组细胞的凋亡比率低于siRNA干扰组细胞的凋亡比率,差异有统计学意义(P<0.05);细胞周期的检测结果显示,细胞对照组和siRNA对照组细胞中G0/G1期所占比率<50%,而siRNA干扰组细胞中G0/G1期所占比率>70%,差异有统计学意义(P<0.05)。结论在NB细胞中使用siRNA下调Eg5表达,能够抑制NB细胞增殖,促进细胞凋亡。     

外源性Bax基因过表达对神经母细胞瘤SH-SY5Y细胞株Smac蛋白表达的影响

目的观察Bcl-2相关X蛋白（Bax）基因过表达对人神经母细胞瘤SH-SY5Y细胞第二个线粒体来源的半胱氨酸酶的激活剂（Smac）蛋白表达的影响。方法采用脂质体Lipofectamine 2000将携带人Bax基因的过表达质粒转入SH-SY5Y细胞（转染组）,同时设空载体组和未转染组。通过G-418筛选后获得转入目的基因的细胞克隆。Hoechst33258染色观察凋亡细胞,免疫印迹法法检测细胞中Bax、Smac蛋白表达,免疫荧光染色检测Bax、Smac蛋白在细胞中的分布。结果转染后用G-418持续筛选2周可获得G418抗性的细胞克隆。Smac主要分布于胞质,Bax主要位于细胞核。转染组SH-SY5Y细胞Bax蛋白表达水平（1.067±0.036）较空载体组（0.436±0.035）和未转染组（0.444±0.046）显著增高（P〈0.05）;转染组Smac蛋白表达水平（1.408±0.044）相比空载体组（0.708±0.021）和未转染组（0.748±0.041）显著增高（P〈0.05）;空载体和未转染组细胞Bax和Snac蛋白表达均无显著差异（P〉0.05）。转染组较未转染组细胞凋亡率显著增加（P〈0.05）。结论外源性Bax基因转入人神经母细胞瘤细胞中并稳定过表达,能够上调Smac蛋白表达,促进细胞凋亡。     

Internalization and signal transduction of PrP106−126 in neuronal cells

Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood steps in the pathogenesis of prion diseases. We have thus analyzed the internalization and signal transduction of the neurotoxic fragment of the prion protein PrP_106–126 in the rat neuroblastoma cell line B104 by fluorescence microscopy and quantification by ELISA and in primary neuronal cells from mice. Phospholipase D (PLD) is known to be an enzyme involved in the regulation of secretion, endocytosis and receptor signalling. We determined the PLD activity using a transphosphatidylation assay and could show that PLD is involved in PrP_106–126 internalization. The determination of receptor activity via quantification of ERK1/2 phosphorylation and cAMP level measurement verified the PrP_106–126-induced signal transduction in B104 cells and primary neuronal cells. PrP_106−126-induced a decrease in cAMP level in neuronal cells. These studies indicate the involvement of PLD in PrP_106–126-endocytosis and mediated cellular signalling by an unidentified inhibitory G-protein-coupled receptor and may allow the development of therapeutic agents interfering with prion uptake and/or PLD function using PLD as a possible pharmaceutical target.     

神经母细胞瘤的辨治

探讨中医对神经母细胞瘤的病因病机、辨证论治的认识。     

大豆苷元对神经母细胞瘤细胞增殖的抑制作用

目的探讨大豆苷元对神经母细胞瘤SH—SY5Y细胞凋亡和增殖的影响。方法采用细胞生长抑制实验（MTT法）、流式细胞仪检测等方法观察SH—SY5Y细胞的形态变化、细胞凋亡率及细胞周期变化。结果10．0～80．0μg／ml的大豆苷元明显抑制细胞的增殖，且有剂量依赖性；与对照组相比，大豆苷元处理24h后，各组SH—SY5Y细胞均出现亚二倍体峰（Sub—G1），细胞凋亡率与大豆苷元呈剂量依赖性。结论大豆苷元可诱导神经母细胞瘤SH—SY5Y细胞凋亡，对SH—SY5Y细胞增殖有明显的抑制作用。     

Renal artery thrombosis — an unusual complication following removal of a neuroblastoma

A 2-month-old female infant with a retroperitoneal neuroblastoma on the left side was admitted to Niigata University Hospital. Her systolic blood pressure, as high as 220 mmHg, was attributed to renovascular hypertension because the plasma renin activity was increased up to 208 ng/ml per hour. Excision of the tumor, including the left kidney, and periaortic lymphnode dissection were performed. For safety during the lymphadenectomy, Nelaton catheters were placed around the inferior vena cava, aorta, superior mesenteric artery, and right renal artery. On the 2nd postoperative day anuria was noted, and aortography was performed revealing obstruction of the right renal artery, inferior mesenteric artery, and left common iliac artery. Thrombectomy via aortic incision was performed immediately to restore the blood flow. The patient developed chronic renal failure, however, and still requires hemodialysis or peritoneal dialysis. Pediatric surgeons should bear in mind that extensive periaortic lymphadenectomy may cause postoperative arterial thrombosis.     

Mother's Attitudes towards the Mass Screening of Neuroblastoma

Two hundred and two mothers whose, infants participated in mass screening of neuroblastoma in Sapporo City, and 200 mothers whose infants did not, were interviewed about their attitudes towards the mass screening. Only a few nonparticipants (3.5%) thought the mass screening was unnecessary. More than half of non-participations (56.0%) were due to mothers' forgetfulness or to loss of the "Neuroblastoma Screening Examination Set" during the two months' interval between the delivery of the set and the sample submission. Mailing of the set at six months for the purpose of eliminating this interval would greatly reduce such neglect of the mass screening. On the other hand, not many infants would participate in re-screening for detection of false negative cases, since the percentage of mothers who were willing to make their children take part in rescreening for a fee of 1,000 yen (about 7 U.S. dollars, which would be administratively necessary) was less than 60%. ( Acta Paediatr Jpn 1989; 31: 166–170)     

Increased Expression of trk Proto-oncogene by γ-Interferon in Human Neuroblastoma Cell Lines

Three human neuroblastoma cell lines were examined to determine the effect of recombinant γ-interferon (IFN-γ) treatment on the expression of trk proto-oncogene. Increased levels of trk proto-oncogene mRNA were observed in two neuroblastoma cell lines (KP-N-RT and KP-N-SI(FA)) after IFN-γ treatment. The levels of trk mRNA increased with growth inhibition and morphological change in a time- and dose-dependent manner. The decreased level of N- myc mRNA after IFN-γ-treatment in KP-N-RT was inversely correlated with trk mRNA. Our results suggest that IFN-γ can modulate the signal transduction of nerve growth factor in human neuroblastoma cells.     

神经母细胞瘤S－100蛋白免疫组化研究及图象分析DNA倍体的预后价值

本文对３２例病理确诊的神经母细胞瘤进行回顾性研究，对所有病理标本重新进行组织学分型，同时进行ＮＳＥ及Ｓ－１００蛋白免疫组化染色，并利用全自动图象分析仪对其中２８例进行了瘤细胞ＤＮＡ定量测定及倍体分析。结果表明：ＮＳＥ及Ｓ－１００蛋白免疫组化染色可反映神经母细胞瘤分化程度，Ｓ－１００蛋白阳性细胞的出现是该肿瘤分化成熟及预后良好的表现，ＤＮＡ异倍体是预后良好的可靠标志。