Ionic currents in neuroblastoma clone E-7 cells

Ionic currents were studied in exponentially growing neuroblastoma cells (clone E-7) derived from mouse neuroblastoma C-1300 with the patch-clamp technique in the whole cell mode (Pflügers Arch., 391 (1981) 85-100). In differentiated cells, with one or several processes, an early inactivating inward current approximately equal to 50 microA/cm2 was observed in response to depolarizing steps from the holding potential -60 to -70 mV, which was insensitive to 2 microM tetrodotoxin but readily blocked by Co2+ (6 mM). This inward current was followed by a delayed outward current which was eliminated by 12 mM tetraethylammonium. In the undifferentiated cells, only delayed outward current was observed. It is suggested that, in differentiated cells both Ca2+ and delayed rectifier K+ channels exist, while only the latter are present in undifferentiated cells.     

TBEV对人神经细胞致病性的实验研究

目的 确定蜱传脑炎病毒(Tick-bone encephalitis virus,TBEV)对人神经母瘤细胞的感染性及复制增殖情况.方法 将TBEV感染人神经母瘤细胞SK-N-SH,取不同时间点的细胞培养上清,分别采用real-time RT-PCR方法及空斑测定的方法测定病毒滴度,以确定TBEV在SK-N-SH细胞中的复制增殖情况;同时进行细胞形态观察及免疫荧光检测,以确定TBEV感染后的SK-N-SH细胞形态变化.结果 TBEV感染SK-N-SH细胞后,可进行大量复制增殖,感染后3d,病毒滴度达到峰值,可达2.92×107 PFU/ml,感染细胞产生明显的形态改变,免疫荧光法可检测到细胞内的病毒粒子.结论 TBEV可在人神经母瘤细胞SK-N-SH中大量复制增殖,并可造成细胞明显的形态改变,因此人神经母瘤细胞SK-N-SH可以作为TBEV培养良好的细胞模型.     

Quantification of vesicles in differentiating human SH-SY5Y neuroblastoma cells by automated image analysis

A new automated image analysis method for quantification of fluorescent dots is presented. This method facilitates counting the number of fluorescent puncta in specific locations of individual cells and also enables estimation of the number of cells by detecting the labeled nuclei. The method is here used for counting the AM1-43 labeled fluorescent puncta in human SH-SY5Y neuroblastoma cells induced to differentiate with all-trans retinoic acid (RA), and further stimulated with high potassium (K+) containing solution. The automated quantification results correlate well with the results obtained manually through visual inspection. The manual method has the disadvantage of being slow, labor-intensive, and subjective, and the results may not be reproducible even in the intra-observer case. The automated method, however, has the advantage of allowing fast quantification with explicitly defined methods, with no user intervention. This ensures objectivity of the quantification. In addition to the number of fluorescent dots, further development of the method allows its use for quantification of several other parameters, such as intensity, size, and shape of the puncta, that are difficult to quantify manually.     

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。     

Tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex: a novel catecholaminergic group?

Tyrosine hydroxylase-like immunoreactive (TH-IR) neurons with morphological features of interneurons were found throughout the human cerebral cortex. Quantitative estimates in 14 different cytoarchitectonic areas revealed a specific regional distribution pattern, neurons being less dense in primary cortical areas and denser in higher order associative areas and some limbic related areas. A partial relationship was noted between the density of labeled neurons and that of the known dopaminergic innervation. The role of the cortical TH-IR neurons in catecholaminergic function, however, remains unclear since the presence of other catecholaminergic synthesizing enzymes, dopamine-β-hydroxylase and DOPA decarboxylase, could not be demonstrated at their level. Similar neurons have been observed transiently in the rodent cortex during development; their persistence and topographical extension in the human brain warrants further study on their possible functional role.     

Anti-tumor effect of Huaier extract against neuroblastoma cells in vitro

Huaier extract, the main active constituent proteoglycan, has anti-tumor activity in various experimental and clinical settings. However, the potential anti-neuroblastoma and associated mechanisms have not been investigated. Therefore, in this study, we aimed to elucidate the potential role of Huaier extract in 3 human neuroblastoma cell lines. Our study demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability in a dose-dependent manner. Huaier extract induced cell cycle arrest at G0/G1 phase in neuroblastoma and decreased the cell cycle related protein expression of cyclin D3. Western blotting analysis also showed that Huaier extract induced neuroblastoma cell apoptosis and autophagy. Signaling analysis indicated that Huaier extract suppressed the MEK/ERK and mTOR signaling pathways simultaneously. In conclusion, we verify that Huaier extract causes cell proliferation inhibition, apoptosis, autophagy, and cell cycle arrest in G0/G1 phase via MEK/ERK and mTOR signaling. Huaier extract may act as a complementary agent for treating neuroblastoma.     

The role of dendritic cells in neuroblastoma: Implications for immunotherapy

Neuroblastoma is a solid tumor, which is originated from some neural tissues. The immune system including the innate and adaptive immune system fights against this tumor. Dendritic cells (DCs) play an important role in this way by recognizing tumor antigens and activating specific types of T cells. These cells are derived from monocytes that are induced by inflammatory factors secreted by different cells in the tumor microenvironment (TME). There are different types of DCs, including monocyte-derived DCs (moDC), plasmacytoid DCs (pDC), conventional DCs type 1 and 2 (cDC1 and cDC2), and Langerhans cells. DCs connect the innate and the adaptive part of the immune system and have an important role in anti-tumor immunity. There are some vaccines that involve specific types of DCs, which can be used to prevent neuroblastoma. Also, we can use the combination of inflammatory factors and DCs as a substitute for chemotherapy.     

神经母细胞瘤细胞中MYCN调控miRNA的研究进展

转录因子MYCN是MYC癌家族的一员,与人类多种肿瘤有关,最常见的为神经母细胞瘤(neuroblastoma,NB).MYCN通过激活和抑制靶基因的转录,以调节细胞的生命活动和肿瘤发生.近年来,越来越多的研究证实,在神经母细胞瘤中MYCN介导的致癌作用是通过调控一些与肿瘤相关的miRNAs来实现的.     

Interaction of 4-aminopyridine with normal and chloramine-T-modified K channels of neuroblastoma cells

The steady-state effects and rate of action of 4-aminopyridine (4-AP) on normal and chloramine-T (CL-T)-modified voltage-dependent potassium (K) currents were studied in neuroblastoma cells with the whole-cell voltage-clamp current recording technique. 4-AP apparently slows both the activation and inactivation of the normal current but does not modify the time course of the CL-T-modified current. These differential effects of 4-AP are interpreted as resulting from the existence of two types of K channels with different 4-AP sensitivities under normal conditions and similar 4-AP sensitivities after CL-T, which furthermore slows their inactivation [8, 9]. While the onset of 4-AP action on the normal current is delayed and can be described by the difference of two exponentials, the onset of 4-AP action on CL-T-modified current starts immediately after the external application of the drug and can be described by the sum of two exponentials. The 4-AP-induced block of the normal current exhibits use-dependent features and is relieved by long conditioning depolarizations. In contrast, the block of the CL-T-modified current is not use-dependent. At high 4-AP concentrations (1–10 mM), the steady-state block of the normal current reaches a saturating value of 95%, while the steady-state block of the CL-T-modified current and the unblocked normal current only reaches a saturating value of 35%. The results suggest that CL-T inhibits a channel or membrane constituent which contributes to the inactivation of channels and increases their apparent affinity for 4-AP when they are in closed or open states. Both the steady-states and rates of the effects of 4-AP can be quantitatively modelled with the assumption of the existence of two receptor sites for 4-AP: a blocking site whose occupancy occludes the channel and an allosteric site whose occupancy inhibits the binding of 4-AP to the blocking site.     

Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells

(1) The kinetic properties of single sodium channels modified by the pyrethroid fenvalerate have been analyzed by patch clamp techniques using the cultured mouse neuroblastoma cells. (2) Fenvalerate drastically prolonged the open time of single sodium channels from the normal value of 5 ms to several hundred milliseconds during a depolarizing pulse. The channels remained open after termination of a depolarizing pulse for as long as several seconds. (3) The channel lifetime varied with the membrane potential, attained a maximum at –70 mV, and decreased with hyperpolarization and depolarization from –70 mV. (4) Prolonged openings of the modified channels allowed a current-voltage curve for a single channel to be plotted by sweeping a ramp pulse. The single channel conductance had a value of 11 pS and was linear over potentials ranging from 0 to –100 mV. (5) Power density spectral analysis of the open channel current noise indicated a single Lorentzian curve with a cut-off frequency at 90 Hz, indicating that the increase in noise during channel opening resulted from a relatively slow kinetic process. (6) The probability of the channel being modified by fenvalerate was independent of the length of time during which the channel was opened. This observation suggests that channel modification had taken place before the channel opened. This study of the prolonged opening at the single channel level provides a new insight into open channel properties and the kinetics of channel modification.     

Early ontogeny of catecholaminergic structures in the sheep brain

Summary The localization of tyrosine hydroxylase was studied in the brain of sheep foetus during early ontogeny using immunohistochemistry. The first immunoreactive neurons appeared very early since they were found on day 30 of pregnancy in the medioventral part of the mesencephalic flexure. The distribution of the different catecholaminergic groups of neurons was similar to the adult's after 75 days of pregnancy. The latest group to appear was the A₁₂ group.Comparison of the development of the sheep foetus with rodents or primates, more commonly studied, is difficult because of its different development. It seems, however, that catecholaminergic structures appear earlier in sheep and rodents than in human. Considering the early appearance of these transmitters in the central nervous system, their role on brain development has to be studied in the futureAbbreviations used in the text ACTH adrenocorticotropin hormone - BSA bovine serum albumin - FLM fascicules longitudinales mediales - FITC fluorescein isothiocyanate - FSH follicle stimulating hormone - LH luteinizing hormone - LPH lipotropin hormone - PBS phosphate buffered saline - TH tyrosine hydroxylase - TH-IR tyrosine hydroxylase immunoreactive Abbreviations used in the figures AI adhesio interthalamica - BO bulbus olfactorius - CA commissura anterior - Cer cerebellum - cf cervical flexure - ChO chiasma opticum - CM corpus mamillare - COL colliculi - CP commissura posterior - Ep epiphysis (pineal gland) - FMT fasciculus mamillothalamicus - FR fasciculus retroflexus - Hyp hypophysis;mf, mesencephalic flexure - NO nervus opticus - P pons - pf pontic flexure;tg, tongue - I-II lateral ventricles - III third ventricle - IV fourth ventricle     

Hypoxia and reoxygenation increased BACE1 mRNA and protein levels in human neuroblastoma SH-SY5Y cells

Ischemic cerebrovascular diseases, usually involved in hypoxia and reoxygenation, have been reported to increase the risk of dementia such as Alzheimer's disease (AD). β-site amyloid protein precursor (APP)-cleaving enzymes (BACE1) have been identified to participate in the secretion of β-amyloid peptides (Aβ), and its expressive alteration would contribute to the AD neuropathology. We have investigated the effect of hypoxia (0% O₂, 24 h) and reoxygenation (0 h, 12 h and 24 h after 24 h hypoxia) on BACE1 mRNA and protein levels in human neuroblastoma SH-SY5Y cells. At the same time, we also examined the effect of hypoxia and reoxygenation on APP mRNA and protein levels. We demonstrated that hypoxia and reoxygenation did not alter APP mRNA and protein level, However compared to those of controls, BACE1 mRNA levels were up-regulated by 31.5% (P = 0.028) and 35.1% (P = 0.005) at 12 h and 24 h and the protein levels increased to 22%(P = 0.021), 42% (P = 0.000) and 51.5% (P = 0.000) at 0 h, 12 h and 24 h after reoxygenation, respectively. Thus by up-regulating of BACE1 mRNA and protein level in the neuronal cell, hypoxia may be a linkage in the pathophysiology between cerebravascular diseases and AD.     

Modification of K+ channel properties induced by fatty acids in neuroblastoma cells

The effects of fatty acids on voltage-dependent potassium (K⁺) channels in neuroblastoma cells were studied using the whole-cell current recording technique. At a concentration of 5 M, unsaturated and medium chain length (C₁₀–C₁₄) saturated fatty acids accelerated the apparent inactivation of the K⁺ current. This effect was reversed by albumin. In the absence of exogenous fatty acids, albumin slowed the inactivation of the K⁺ current. The acceleration of the K⁺ current inactivation induced by unsaturated fatty acids was associated with an increase in the sensitivity of K⁺ channels to 4-amino-pyridine. It is concluded that kinetic and pharmacological properties of K⁺ channels are, in part, controlled by membrane fatty acids which, in this way, should contribute to an apparent diversity of K⁺ channels and the modulation of cell excitability.     

Differentiation of human B lymphocyte subpopulations induced by an alloreactive helper T-cell clone

We have used cloned alloreactive helper T cells to determine if direct T cell-B cell interaction can induce differentiation of human peripheral blood B cells which do not respond to pokeweed mitogen (PWM). T-cell clone 2F8 was derived from a one-way mixed lymphocyte reaction. 2F8 cells are T3⁺T4⁺T8^–IL-2R⁺ and proliferate in response to irradiated stimulator cells, but not autologous cells, in the absence of exogenous interleukin-2. 2F8 cells provide allospecific help for polyclonal proliferation and differentiation of B cells in the absence of any other stimulus. The magnitude of this response is comparable to that of the response of the same B cells to PWM and fresh autologous T cells. 2F8 cells could also provide nonspecific help for unrelated donor B cells in the presence of PWM, with no requirement for costimulation by irradiated stimulator cells. Allospecific stimulation of B cells was completely inhibited by antibodies to class II major histocompatibility complex (MHC) framework determinants and was abrogated by 1000-rad irradiation. Cloned 2F8 T cells stimulated differentiation of both small, high-density B cells and larger B cells, generating up to 30% plasma cells with either fraction. B cells forming rosettes with mouse erythrocytes were also induced to differentiate by the helper T cell clone. As found previously, neither small, high-density B cells nor mouse rosette⁺ B cells responded well to PWM. Direct interaction with allospecific T cells induces differentiation of a broader spectrum of B cells than soluble growth and differentiation factors in conjunction with polyclonal activators such as PWM and protein A containing staphylococci.     

TCRVβ基因谱系分析在AIDS疾病T细胞克隆性研究中的应用

对于T淋巴细胞TCRVB基因家族以及T细胞的克隆性的研究是近年的热点问题.通过对艾滋病人(模型)的TCRVβ基因谱系分析,测定特定CDR3长度及序列出现的频率能反映T细胞克隆扩增的程度和功能状态,进而有助于对MDS的临床诊断、免疫机制、药物或疫苗的治疗效果等的理解.     

Modification of electrophysiological and pharmacological properties of K channels in neuroblastoma cells induced by the oxidant chloramine-T

The effects of chloramine-T (CL-T) on voltage-dependent potassium channels in neuroblastoma cells were analysed using the whole-cell current recording technique. CL-T irreversibly decreased the peak whole — cell K current, considerably slowed its inactivation and shifted its activation-voltage curve towards positive voltages by 6 mV. Under control conditions, the inactivation of the whole-cell K current could be described by the sum of two exponentials, F and S, whose time constants at +50 mV were _F=1.00±0.15 s and _S=5.72±0.47 s respectively. After CL-T, it could be described by the sum of two (S₁ and S₂) or three (F, S₁ and S₂) exponentials whose time constants at +50 mV were: _F=0.81±0.22 s, _S1=6.46±0.60 s and _S2=48.56±3.64 s. Under control conditions, F and S inactivating components of the whole-cell K current were blocked by 4-aminopyridine, with a Hill coefficient of 1 and apparent dissociation constants of 0.04 and 0.7 mM respectively. After CL-T, both S₁ and S₂ components were equally blocked by 4-aminopyridine with a Hill coefficient of 0.25, being reduced to 64% of their control values by 10 mM. CL-T is known to slow the inactivation of sodium channels and to oxidize sulphydryl amino acids and unsaturated lipids. It is concluded that the inactivation gates of voltage-dependent sodium and potassium channels are either constituted of the same amino acid residues or are controlled by unsaturated lipids surrounding or bound to the channel proteins.     

Effect on memory processes of anti-vasopressin serum microinjected into the dorsal raphe nucleus: The role of catecholaminergic neurotransmission

Summary Anti-arginine⁸-vasopressin serum was microinjected into the mesencephalic dorsal raphe nucleus immediately after the learning trial, in a one-trial learning passive avoidance reaction. The treatment attenuated passive avoidance behavior 24 h after treatment, suggesting a role of the endogenous vasopressin of this area in memory processes. On the other hand, the antiserum did not influence passive avoidance behavior if 6-hydroxydopamine was microinjected into the raphe region. The data suggest that the antiserum may have primarily interacted with catecholaminergic terminals, which enter the dorsal raphe nucleus.This work was supported by the Scientific Research Council, Ministry of Health, Hungary [Grant No. 4-08-0302-03-0(T)]     

Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells

Neuroblastoma is a solid tumor that occurs mainly in children. Malignantneuroblastomas have a poor prognosis because conventional chemotherapeutic agents arenot very effective. Survivin, a member of the inhibitor of the apoptosis proteinfamily, plays a significant role in cell division, inhibition of apoptosis, andpromotion of cell proliferation and invasion. Previous studies found that survivin ishighly expressed in some malignant neuroblastomas and is correlated with poorprognosis. The aim of this study was to investigate whether survivin could serve as apotential therapeutic target of human neuroblastoma. We employed RNA interference toreduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzedthe effect of RNA interference on cell proliferation and invasion invitro and in vivo. RNA interference of survivin led to asignificant decrease in invasiveness and proliferation and increased apoptosis inSH-SY5Y cells in vitro. RNA interference of survivin inhibited tumorgrowth in vivo by 68±13% (P=0.002) and increased the number ofapoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA(siRNA) treatment controls. Moreover, RNA interference of survivin inhibited theformation of lung metastases by 92% (P=0.002) and reduced microvascular density by60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivinmRNA and protein expression both in vitro and invivo compared with negative siRNA treatment controls. RNA interference ofsurvivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasisformation. These results support further clinical development of RNA interference ofsurvivin as a treatment of neuroblastoma and other cancer types.     

嗅神经母细胞瘤的光镜、电镜和免疫组织化学观察

报道了１１例嗅神经母细胞瘤的光镜、免疫组化及其中２例超微结构观察结果。１１例嗅神经母细胞瘤中１０例可见瘤细胞排列成大小不等的巢状或条索状；８例有Ｈｏｍｅｒ－Ｗｒｉｇｈｔ菊形团，５例见Ｆｌｅｎｅｒ菊形团（其中４例同时伴有Ｈｏｍｅｒ－Ｗｒｉｇｈｔ菊形团）；８例可见瘤细胞之间由细胞突起形成的嗜酸性纤维样背景。电镜下１例瘤细胞内见神经原纤维，２例均有神经内分泌颗粒，并对其颗粒进行了形态测量。多种抗体免疫组化染色：ＮＳＥ１１例均为阳性，Ｖｉｍｅｎｔｉｎ，Ｓ－１００、Ｋｅｒａｔｉｎ各有４例、３例和１例阳性，ＮＦ，ＥＭＡ，ＣＥＡ，ＬＣＡ均为阴性。文中对嗅神经母细胞瘤的病理形态及免疫组化特征在诊断中的意义进行了讨论。     

儿童腹膜后神经母细胞瘤56例临床分析

目的总结儿童腹膜后神经母细胞瘤（NB）的Ⅰ临床特征和预后,提高对儿童腹膜后NB的认识。方法回顾性分析首都医科大学附属北京儿童医院血液中心2008年1月至2011年1月收治的腹膜后NB患儿的临床表现、实验室检查、治疗和随访等资料,评价近期和远期疗效,评估2年无病生存率（EFS）。结果56例确诊腹膜后NB患儿纳入分析,男34例,女22例。发病中位年龄41．5（7～147）个月。病程中位数为1．7个月（6d至60个月）。Ⅱ期1例,Ⅲ期2例,Ⅳ期52例,1Vs期1例。高危53例,中危2例,低危1例。①起病时发热24例（42．8％）,肢体疼痛18例（32．1％）,腹部包块14例（25．0％）,腹痛12例（21．4％）,下肢无力3例（5．4％）。（2）49／56例（87．5％）LDH〉240U·L^-1,47／51例（92．2％）NSE〉16．3ng·mL^-1,44／53例（83．0％）24h尿VMA〉30μmol,14／30例（46．7％）SF〉1500ng·mL^-1。③肿瘤原发于肾上腺23例（41．1％）,原发于腹膜后25例（44．6％）,同时原发于肾上腺和腹膜后2例（3．6％）,胸腹联合6例（10．7％）。肿瘤包绕腹部大血管29／49例（59．2％）。27／53例（50．9％）瘤灶直径〉10cm,瘤灶包绕腹部大血管的发生率显著增高（P=0．002）。LDH在瘤灶包绕腹部大血管患儿中显著升高（P=0．021）,在瘤灶直径〉10cm患儿中亦显著升高（P=0．017）。④47例（83．9％）伴骨髓受累,46例（82．1％）有不同程度的骨骼转移,6例（10．7％）伴有腹腔其他脏器转移,3例（5．4％）伴有远处淋巴结受累。⑤40例行原发或转移瘤病理活检,其中神经节母细胞瘤10例（25．0％）,NB30例（75．0％）。⑥1例Ⅳs随诊观察,13例失访,42例采用BCH-NB高危方案规律化疗,16例治疗中肿瘤进展,其余26例完全缓解,继续随访中,中位随访时间19．5（3—52）个月,2年EFS为57．4％。结论儿童腹膜后NB发病年龄较大,临床多表现为发热、肢体疼痛和腹部包块。LDH在巨大瘤灶及瘤灶包绕腹部大血管的患儿中明显升高。病理学检查以NB多见,2年EFS为57．4％。