In vitro properties of a newly established medulloblastoma cell line, MCD-1

Medulloblastomas are poorly differentiated brain tumors believed to arise from primitive pleuripotential stem cells, and tend to express mixed neuronal and glial properties. In the present study, we examined immunohistochemical and neurotransmitter phenotypic properties in a newly established medulloblastoma cell line, MCD-1. MCD-1 cells were immortal, not contact-inhibited, but did not grow in soft agar. Immunohistochemical studies showed positive staining for neurofilament protein (NF), neuron-specific enolase (NSE), synaptophysin, MAP 2, τ, NCAM 180, vimentin, and S-100 protein. The cells expressed specific uptake of glutamate, serotonin, and choline, but not GABA or dopamine. A significant increase in process extension was seen in response to agents that enhance intracellular cyclic AMP, especially 3-isobutyl-1-methylxanthine (IBMX). Process formation induced by IBMX was associated with a decrease in cell proliferation as evidenced by a reduction in numbers of cells incorporating 5-bromo-2-deoxyuridine (BrdU). No increase in process extension was observed following exposure to NGF or retinoic acid. MCD-1 cells were shown to produce transforming growth factor beta (TGFβ), and were immunopositive for mutant p53. Transfection assays with the PG13-Luc reporter plasmid, which contains a p53-responsive enhancer element and a luciferase reporter gene, suggested MCD-1 cells are deficient in wild-type p53 and do not activate p53 on treatment with the anticancer agent adriamycin. The MCD-1 cell line is suggested to represent an abnormally differentiated cell type, which has some properties consistent with a multipotent neuronal phenotype while retaining some properties of immature cells of a glial lineage. The MCD-1 cell line can be used to provide a model of a medulloblastoma cell line that is resistant to growth-controlling and anticancer agents.     

The influence of piracetam on digoxin up-take by different rabbit tissues in vitro

The action of increasing piracetam concentration on the digoxin up-take by various rabbit tissues was investigated in vitro. 125J-digoxin was used and radioactivity was counted in a gamma scintillation counter. Piracetam 100 micrograms/ml has a small increasing action on digoxin up-take by parenchymatous rabbit tissues. This action may be due to the beneficial effect of the substance on tissue microcirculation and cell metabolism. This action is equilibrated or reversed by piracetam 200 micrograms/ml and 400 micrograms/ml. It is suggested that piracetam displays digoxin from its binding-sites but these concentrations are above those ones used for clinical purposes.     

骨髓基质细胞源性神经干细胞体外分化及电生理特性的研究

目的探索骨髓基质细胞(BMSCs)诱导分化为神经干细胞(NSCs),比较血清、维甲酸(RA)、胶质细胞源神经营养因子(GDNF)、脑源性神经营养因子(BDNF)及2-巯基乙醇(2-ME)等不同浓度诱导条件下BMSCs分化情况,以及分化细胞的电生理特性。方法以恒河猴骨髓中分离出的BMSCs为实验对象,利用神经干细胞培养基和RA、GDNF、BDNF、2-ME等生长因子在不同血清浓度下进行培养增殖和诱导分化。Nestin、CD133抗体免疫细胞化学染色鉴定NSCs,NSE、β-tublin鉴定神经元、GFAP鉴定神经胶质细胞。膜片钳检测细胞的电生理特性。结果低浓度血清(2.5%)+RA(0.3mg/L)+GDNF(20μg/L)诱导分化效果较好,且分化的神经元样细胞较未分化细胞的膜特性[静息膜电位(RMP)、膜电容(Cm)、串联电阻值(Rs)]有了显著改变(P﹤0.01)。部分形态成熟的神经元样细胞表现出TTX敏感的快速激活、快速失活的电压依赖性的Na+通道,而未分化细胞却未记录到内向电流;两类细胞均可记录到外向的K+电流,但神经元样细胞的电流峰值强度要显著高于未分化细胞,并且包括两种电流成分:瞬时外向K+电流和延迟整流型的K+电流。结论RA+GDNF及配合使用低浓度血清能够有效诱导骨髓源神经干细胞向成熟神经系细胞分化,且分化的神经元样细胞具有快速激活、快速失活的电压依赖性Na+通道,类似神经细胞的电生理特性。     

In vitro electrophysiological properties of rat dorsal column nuclei neurons.

The dorsal column nuclei include the gracile and cuneate nuclei, which receive somatosensory information from the periphery and project to the ventroposterior nucleus of the contralateral thalamus. The aim of this study was to determine the electrophysiological and morphological characteristics of the neurons of the dorsal column nuclei and to identify synaptic events evoked by electrical stimulation of the dorsal column, using an in vitro slice preparation. The results show two types of neurons, termed type I and II. A repolarizing sag distinguished type I cells during hyperpolarizing current injection, suggesting the activation of a Q-current. Moreover, type I cells, but not type II cells, were capable of maintaining spontaneous rhythmic activity at 9-15 Hz. Both types of cells displayed a delay in their return to the resting membrane potential following hyperpolarizing current pulses, indicating the existence of an A-current. Electrical stimuli applied to the dorsal column elicited brief EPSPs and IPSPs in both cell types. EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that they were mediated through non-NMDA receptors. IPSPs were blocked by picrotoxin, implying the activation of GABAA receptors. Intracellular staining with carboxyfluoresceine revealed that type I neurons had elongated somas and primary dendrites that extended radially. Type II cells were smaller and had round somas with few primary dendrites, most of them emerging from one pole of the soma. The axon of many type I neurons was stained and could be followed running ventrally and in rostral direction.     

Piracetam and vinpocetine exert cytoprotective activity and prevent apoptosis of astrocytes in vitro in hypoxia and reoxygenation

The aim of the present study was to establish whether piracetam (2-pyrrolidon-N-acetamide; PIR) and vinpocetine (a vasoactive vinca alkaloid; VINP) are capable of protecting astrocytes against hypoxic injury. Using the model of astrocyte cell culture we observed the cells treated with PIR and VINP during and after in vitro simulated hypoxia. Cell viability was determined by Live/Dead Viability/Cytotoxicity Assay Kit, LDH release assay and MTT conversion test. Apoptotic cell death was distinguished by a method of Hoechst 33342 staining underfluorescence microscope and caspase-3 colorimetric assay. In addition the intracellular levels of ATP and phosphocreatine (PCr) were evaluated by bioluminescence method. Moreover, the effect of the drugs on the DNA synthesis was evaluated by measuring the incorporation of [3H]thymidine into DNA of astrocytes. PIR (0.01 and 1 mM) and VINP (0.1 and 10 microM) were added to the medium both during 24 h normoxia, 24 h hypoxia or 24 h reoxygenation. Administration of 1 mM PIR or 0.1 microM VINP to the cultures during hypoxia significantly decreases the number of dead and apoptotic cells. The antiapoptic effects of drugs in the above mentioned concentrations was also confirmed by their stimulation of mitochondrial function, the increase of intracellular ATP, and the inhibition of the caspase-3 activity. The prevention of apoptosis was accompanied by the increase in ATP and PCr levels and increase in the proliferation of astrocytes exposed to reoxygenation. The higher concentration of VINP (10 microM) was detrimental in hypoxic conditions. Our experiment proved the significant cytoprotective effect of 1 mM PIR and 0.1 microM VINP on astrocytes in vitro.     

Iron chelating, antioxidant and cytoprotective properties of dopamine receptor agonist; apomorphine

There have been many attempts to discover neuroprotective drugs for the treatment of Parkinson's disease (PD). Many of these compounds either do not cross the blood brain barrier or are not very effective in the 6-hydroxydopamine or MPTP (N-methyl-4-phenyl-1,2,3,6-terahydropyridine) models of PD. We have examined several compounds including dopamine receptor agonist bromocritine, lisuride, pergolide and R-apomorphine for their neuroprotective action against the above neurotoxins in PC12 and dopamine neuroblastoma cell lines in culture and in vivo. R-apomorphine exhibited relatively potent neuroprotective action in vitro, cell culture and in vivo as a radical scavenger and iron chelator, because of its catechol structure. The recent clinical trials with apomorphine, where parkinsonian subjects can be weaned off L-dopa would suggest that this drug either exerts a neuroprotective action or that continuous sustained stimulation of dopamine receptor may be responsible for its unusual pharmacological activity. Apomorphine has a far more broad neuroprotective activity in the various models as compared with 1-selegiline and may therefore be an ideal drug to study neuroprotection in parkinsonian subjects with the use of PET or SPECT.     

In vitro studies on the fibrinolytic, thrombolytic and fibrinogenolytic properties of a tissue plasminogen activator from guinea pig keratocytes

The fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37 degrees C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (less than 50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3-6 hr with the concentration of GPK activator in the range of 1-5 micrograms/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.     

In vitro growth, differentiation and biological characteristics of neural stem cells

INTRODUCTIONNeural stem cells (NSCs) [1-3] are defined as undifferentiated neuralcells that are characterized by their high proliferative potential whileretaining the capacity for self-renewal and multipotency. HumanNSCs can be derived from embryonic stem…     

Morphological, immunophenotypical and electrophysiological properties of resting microglia in vitro

Morphological, immunophenotypical and electrophysiological properties were investigated in isolated cultured murine microglia before and after exposure to astrocyte-conditioned medium (ACM). Following application of ACM, microglial cells underwent a dramatic shape transformation from an amoeboid appearance to a ramified morphology. In parallel to morphological changes, a downregulation of macrophage surface antigens was observed in microglia exposed to ACM. Staining intensities for major histocompatibility complex (MHC) class II molecules and for the adhesion molecules leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) were significantly decreased in ramified microglia 5 days after exposure to ACM. In microglial cells treated daily with ACM over a period of 5 days, the smallest staining intensities for all surface antigens as well as the smallest ramification index as a measure for the highest degree of ramification were determined. In addition, upregulation of delayed rectifier K + currents was observed in microglia exposed to ACM for 1 day or treated daily with ACM for 5 days. In contrast, untreated amoeboid microglia or ramified microglia analysed 5 days after exposure to ACM did not express delayed rectifier K + currents. Analyses of the resting membrane potential and expression levels and properties of inward rectifier K + currents did not reveal any differences between untreated and ACM-treated microglia. It is suggested that electrophysiological properties of microglia do not strongly correlate with the morphology or the immunophenotype of microglial cells.     

成骨细胞的体外培养与鉴定☆

背景：成骨细胞的体外培养方法均存在一定的局限性。目的：对体外培养成骨细胞的来源,成骨细胞的培养方法及培养条件,鉴定成骨细胞的标志进行总结。方法：以“Osteoblast, cell culture, identification”为检索词,检索PubMed数据库(2000/2010),以“成骨细胞、细胞培养、鉴定”为检索词,检索万方数据库(2000/2010),文献检索语种限制为英文和中文。纳入与成骨细胞的体外培养及鉴定密切相关的研究,排除重复性研究和Meta分析后对文献进行综述。结果与结论：随着体外细胞培养技术的发展,人们已经从许多动物的骨、骨膜、骨髓及骨外组织中成功培养了成骨细胞,经鉴定具有典型成骨细胞的特征及良好的生物学特性,目前体外培养成骨细胞为常用的体外实验模型,成为研究骨生理、病理及修复的重要手段,也成为研究成骨细胞生长代谢及骨组织工程的基础。     

In vitro oligodendrogliotrophic properties of cell adhesion molecules in the immunoglobulin superfamily: Myelin-associated glycoprotein and N-CAM

To determine if cell recognition molecules interact trophically with oligodendrocytes (OCs), their effect as growth substrates for differentiating oligodendroblasts was studied in primary culture. Oligodendroblasts purified from postnatal rat cerebrum by immunopanning were plated on substratum-bound cell adhesion molecules or extracellular matrix glycoproteins in chemically defined medium in which OCs terminally differentiate but survive poorly. Growth on myelin-associated glycoprotein (MAG) and neural cell adhesion molecule (N-CAM) selectively increased the number of viable cells per culture 2 weeks after plating as much as tenfold and sixfold, respectively, over background survival on an albumin substrate, whereas L1, tenascin-R, tenascin-C, fibronectin, and laminin were ineffective. Neither MAG nor N-CAM stimulated bromodeoxyuridine incorporation into cultures, indicating that enhanced proliferation did not contribute to better survival. Compared to growth on polyornithine alone, oligodendroblast differentiation in the added presence of MAG or N-CAM was qualitatively unchanged; >90% of surviving cells developed into OCs that matured further by immunocytochemical and morphological criteria. A striking difference, however, was the quantitative effect of MAG and N-CAM substrates on oligodendrite outgrowth, increasing myelin-like membrane formation two- to threefold (>8 × 10³ μm²/cell). These findings support the concept that autotypic or heterotypic cell contact-mediated signaling by recognition molecules at the OC surface contributes trophic support of myelinogenesis. © 1996 Wiley-Liss, Inc.     

In vitro characterization of agonist, antagonist, inverse agonist and agonist/antagonist benzodiazepines

1. The efficacy of coupling between the benzodiazepine receptor and chloride channel as well as the coupling to the GABA receptor is differentially affected by different benzodiazepine ligands.

2. In general, the order of efficacy with regard to allosteric effects of benzodiazepine ligands on the chloride channel ([³⁵S]TBPS) and GABA receptor ([³H]muscimol), is: agonist > agonist/antagonist > partial agonist > antagonist; with inverse agonists acting in a manner opposite to the classical benzodiazepine agonists.

3. The chloride ionophore is allosterically modulated both by benzodiazepine and GABA receptors.     

星形胶质瘤细胞的分离培养及生物学特性观察

目的建立人脑胶质瘤细胞体外分离培养体系,观察不同病理分级胶质瘤细胞的基本生物学特性,探讨病理分级与胶质瘤细胞生物学特性之间的关系。方法采集手术切除新鲜胶质瘤标本,进行体外分离培养,运用免疫细胞化学染色、流式细胞仪和染色体核型分析等技术对胶质瘤细胞形态学、生长特性和染色体核型变化情况进行观察和比较分析。结果①原代培养胶质瘤细胞主要呈现圆形、星形和梭形三种细胞形态。②星形细胞瘤细胞生长速度与病理分级相关,恶性程度越高,细胞增殖越快。③染色体核型分析结果可见胶质瘤细胞存在染色体数目与结构异常。结论星形胶质瘤细胞的分离培养试验提示:星形细胞瘤细胞生长速度与病理分级呈正相关,恶性程度越高,增殖速度越快。     

大鼠骨髓基质干细胞体外分离、培养及分化的相关实验研究

目的探索大鼠骨髓基质干细胞(BMSC)体外分离、培养及纯化的合适实验条件,并探讨其在体外定向诱导分化为神经元样细胞的可行性。方法通过密度梯度离心法从成年大鼠骨髓中分离出BMSC,而后通过贴壁培养法培养及纯化,观察其生长特性;对纯化后的BMSC使用碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)进行定向诱导分化,并进行免疫细胞化学鉴定。结果体外培养的BMSC传4代后,纯度最高,可达(95.23±3.06)%;其诱导分化7 d后,(75.43±7.63)%的细胞表现为-βTubulinⅢ阳性的神经元样细胞,(33.01±6.73)%的细胞则为GFAP阳性的胶质细胞。结论BMSC在体外培养条件下生长良好,经bFGF和EGF诱导后可大量分化为神经元样细胞和神经胶质细胞。     

In vitro characterization of intrinsic properties and local synaptic inputs to pyramidal neurons in macaque primary motor cortex

Primates (including humans) have a highly developed corticospinal tract, and specialized motor cortical areas which differ in key ways from rodents. Much work on motor cortex has therefore used macaque monkeys as a good animal model for human motor control. However, there is a paucity of data describing the fundamental functional architecture of primate primary motor cortex, which is best addressed with in vitro approaches. In this study we examined the cellular properties and the micro‐circuitry of the adult macaque primary motor cortex by carrying out in‐vitro intracellular recordings. We aimed to characterize the basic properties of the cortical circuitry by studying the intrinsic properties of its pyramidal neurons and their physiological interconnectivity. We studied the passive and active electrophysiological properties of pyramidal neurons in both superficial and deep cortical layers. Both superficial and deep pyramidal neurons exhibited bursting behaviour that could act as powerful excitation for downstream targets. Synaptic connections were lamina specific. Neurons in the deep layers had convergent excitatory inputs from all cortical layers whereas superficial neurons had only significant inputs from superficial layers. This sheds light on the functional architecture of the primate primary motor cortex and how its output is shaped. We also took the unique opportunity in our recording technique to characterize the relationship between intracellular and extracellular spike waveforms, with implications for cell‐type identification in studies in awake behaving monkey. Our results will aid the interpretation of primate studies into motor control involving extracellular spike recordings and electrical stimulation in primary motor cortex.     

In vitro analysis of the origin,migratory behavior,and maturation of cortical pyramidal cells

In cerebral cortex of rat and monkey, the neuropeptide somatostatin (SOM) marks a population of nonpyramidal cells (McDonald et al. [1982] J. Neurocytol. 11:809-824; Hendry et al. [1984] J. Neurosci. 4:2497:2517; Laemle and Feldman [1985] J. Comp. Neurol. 233:452-462; Meineke and Peters [1986] J. Neurocytol. 15:121-136; DeLima and Morrison [1989] J. Comp. Neurol. 283:212-227) that represent a distinct type of gamma-aminobutyric acid (GABA) -ergic neuron (Gonchar and Burkhalter [1997] Cereb. Cortex 7:347-358; Kawaguchi and Kubota [1997] Cereb. Cortex 7:476-486) whose synaptic connections are incompletely understood. The organization of inhibitory inputs to the axon initial segment are of particular interest because of their role in the suppression of action potentials (Miles et al. [1996] Neuron 16:815:823). Synapses on axon initial segments are morphologically heterogeneous (Peters and Harriman [1990] J. Neurocytol. 19:154-174), and some terminals lack parvalbumin (PV) and contain calbindin (Del Rio and DeFelipe [1997] J. Comp. Neurol. 342:389-408), that is also expressed by many SOM-immunoreactive neurons (Kubota et al. [1994] Brain Res. 649:159-173; Gonchar and Burkhalter [1997] Cereb. Cortex 7:347-358). We studied the innervation of pyramidal neurons by SOM neurons in rat and monkey visual cortex and examined putative contacts by confocal microscopy and determined synaptic connections in the electron microscope. Through the confocal microscope, SOM-positive boutons were observed to form close appositions with somata, dendrites, and spines of intracortically projecting pyramidal neurons of rat area 17 and pyramidal cells in monkey striate cortex. In addition, in rat and monkey, SOM boutons were found to be associated with axon initial segments of pyramidal neurons. SOM axon terminals that were apposed to axon initial segments of pyramidal neurons lacked PV, which was shown previously to label axo-axonic terminals provided by chandelier cells (DeFelipe et al. [1989] Proc. Natl. Acad. Sci. USA 86:2093-2097; Gonchar and Burkhalter [1999a] J. Comp. Neurol. 406:346:360). Electron microscopic examination directly demonstrated that SOM axon terminals form symmetric synapses with the initial segments of pyramidal cells in supragranular layers of rat and monkey primary visual cortex. These SOM synapses differed ultrastructurally from the more numerous unlabeled symmetric synapses found on initial segments. Postembedding immunostaining revealed that all SOM axon terminals contained GABA. Unlike PV-expressing chandelier cell axons that innervate exclusively initial segments of pyramidal cell axons, SOM-immunoreactive neurons innervate somata, dendrites, spines, and initial segments, that are just one of their targets. Thus, SOM neurons may influence synaptic excitation of pyramidal neurons at the level of synaptic inputs to dendrites as well as at the initiation site of action potential output.