骨髓基质细胞对神经干细胞分化为神经元的影响

采用细胞共培养方式和免疫化学染色方法 ,研究骨髓基质细胞对神经干细胞分化为神经元、星形胶质细胞和寡突胶质细胞的影响。实验发现 ,体外培养的中脑神经干细胞在与成年大鼠骨髓基质细胞共培养 7d后 ,在神经干细胞后代中神经元比例可达 38.6 %± 10 .8% ,明显高于自然分化组 2 0 % ,提示骨髓基质细胞提供的微环境可明显提高神经干细胞后代中神经元的比例。     

Lymphokine activateed killer (LAK) cell-mediated lysis of murine glioma: trypsinchymotrypsin-sensitive glioma protein is responsible for tumor-selective recognition by LAK cells

The killing of Fischer rat 9L glioma in vitro by lymphokine-activated killer (LAK) cells was studied. LAK cells generated by culturing Fischer spleen cells with recombinant interleukin 2 markedly lysed glioma cells but did not kill syngeneic normal brain tissue in a chromium release microcytotoxicity assay. Susceptibility of glioma to lysis by LAK cells was markedly diminished by pretreating the glioma cells with trypsin or chymotrypsin but was unaffected by pretreatment with neuraminidase, glycosidases, or sodium periodate. These results suggest that LAK cell killing of glioma is probably tumor-selective and that a crucial cell surface determinant on glioma cells responsible for its tumot-selective lysis by LAK is a protein sensitive to trypsin and chymotrypsin.     

Uptake and degradation of tissue plasminogen activator in rat liver

The mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(alpha), and the terminal phase, t1/2 (beta), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells. Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells. Competitive inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types. These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.     

Glial cell transplants that are subsequently rejected can be used to influence regeneration of glial cell environments in the CNS

Transplantation of glial cells into demyelinating lesions in CNS offers an experimental approach which allows investigation of the complex interactions that occur between CNS glia, Schwann cells, and axons during remyelination and repair. Earlier studies have shown that (1) transplanted astrocytes are able to prevent Schwann cells from participating in CNS remyelination, but that they are only able to do so with the cooperation of cells of the oligodendrocyte lineage, and (2) transplanted mouse oligodendrocytes can remyelinate rat axons provided their rejection is controlled by immunosuppression. On the basis of these observations, we have been able to prevent the Schwann cell remyelination that normally follows ethidium bromide demyelination in the rat spinal cord by co-transplanting isogeneic astrocytes with a potentially rejectable population of mouse oligodendrocyte lineage cells. Since male mouse cells were used it was possible to demonstrate their presence in immunosuppressed recipients using a mouse Y-chromosome probe by in situ hydridisation. When myelinating mouse cells were rejected by removal of immunosuppression, the demyelinated axons were remyelinated by host oligodendrocytes rather than Schwann cells, whose entry was prevented by the persistence of the transplanted isogeneic astrocytes. The oligodendrocyte remyelination was extensive and rapid, indicating that the inflammation associated with cell rejection did not impede repair. If this host oligodendrocyte remyelination was prevented by local X-irradiation, the lesion consisted of demyelinated axons surrounded by processes from the transplanted astrocytes. By this approach, it was possible to create an environment which resembled the chronic plaques of multiple sclerosis. Thus, these experiments demonstrate that in appropriate circumstances the temporary presence of a population of glial cells can alter the outcome of damage to the CNS. © 1995 Wiley-Liss, Inc.     

Human peripheral lymphocytes defined by anti-myelin-associated glycoprotein antiserum in healthy individuals and in patients with multiple sclerosis

We investigated both the association between MAG-positive cells and active natural killer cells stained by anti-Leu-11 and the percentage of MAG-positive cells in patients with multiple sclerosis (MS) by indirect immunofluorescence study. Eighty percent of MAG-positive cells were stained with anti-Leu-11. The percentage of MAG-positive cells of 41 healthy individuals was from 5.1 to 16.1% (8.5 +/- 2.7%). The percentage of Leu-7-positive cells (13.8 +/- 4.9%) was always greater than that of MAG-positive cells. In 5 of 17 samples from 14 patients with MS, the percentage of MAG-positive cells was reduced. This finding was not related to disease activity and was not a pathognomonic feature of MS. The percentage of Leu-7-positive cells in patients with MS was not statistically different from that in healthy individuals; however, 4 patients showed a normal number of Leu-7-positive cells and a reduced number of MAG-positive cells. These studies suggest that MAG-positive cells are closely related to active natural killer cells and that "MAG" is a useful marker of human natural killer cells.     

Attenuation of brain derived neurotrophic factor (BDNF) by ethanol and cytoprotective effect of exogenous BDNF against ethanol damage in neuronal cells

Summary. Ethanol-induced cell damage was investigated using human neuroblastomas SH-SY5Y cells, which can be differentiated by retinoic acid. With 100 mM or more of ethanol, cytotoxicity was significantly higher in undifferentiated cells than in differentiated cells. Thus, a severer effect of ethanol was observed in undifferentiated cells. In differentiated cells it was shown that the secreted amount of brain derived neurotrophic factor (BDNF) and the cyclic AMP responsive element binding protein (CREB) activity were significantly reduced by ethanol. These effects may be involved in ethanol-induced cell damage in differentiated cells. It was reported that neurotrophic factors have protective effects and that the hippocampus exclusively was damaged by ethanol. Since SH-SY5Y cell is a cell line (a neuronal cell model) and similar cytotoxic effect of ethanol was observed in both SH-SY5Y and primary culture neuronal cells, it will be favorable to use primary culture cells to test a protective effect of BDNF. Exogenous BDNF was shown to have a protective effect against ethanol-induced damage in primary culture neurons from rat hippocampi.These authors contributed equally to this work     

Effects of acupuncture on immune response in mice.

The effect of acupuncture points stimulation on the induction of plaque-forming cells (PFC) in spleen cells of BALB/c mice was investigated in vivo and in vitro tests. In in vivo experiment, mice were immunized with 2 x 10(8) sheep red blood cells (SRBC) and the PFC was markedly increased by daily (once a day for 4 days) acupuncture stimulation. The enhancement of PFC by acupuncture was completely blocked by preadministration of procain, hexamethonium, naloxone, propranolol, but not by phentolamine. The enhancement of PFC by acupuncture was also observed in spleen cells of non-immunoized mice when spleen cells of the acupunctured mice were cultured with SRB in vitro. The enhancement of PFC in spleen was observed after stimulation with acupuncture, and a similar effect was also found in bone marrow cells of normal mice, but not in thymic cells. The spleen cells of mice given acupuncture showed no enhancement of PFC after treatment with anti-Thy 1.2 antibody and complement. Furthermore, these helper T cells were found to be not restricted by the H-2 gene complex. These data demonstrate that the helper T cells induced by acupuncture lack the H-2 restriction, and thus suggest that they may be drived from the bone marrow, but not from the thymus. It is therefore concluded that the helper T cells derived from the bone marrow were activated via the sympathetic nervous system stimulated by acupuncture.     

Neuronal death and synapse elimination in the olivocerebellar system. I. Cell counts in the inferior olive of developing rats

A transient multiple innervation of cerebellar Purkinje cells by climbing fibers has been described during postnatal development of the rat. The aim of the present study was to determine if the regression of redundant synapses is related to the loss of presynaptic cells in the inferior olivary nucleus (ION), which is the sole source of climbing fibers in rodents. To this end, the population size of the ION was evaluated by counting healthy cells of the four main subnuclei in rats from birth to adulthood. The cell population at birth was found to be very similar to that of the adult animal (27,655 versus 28,385), but a loss of 25% of the cells occurred in the first five days, presumably through their death since degenerating cells were observed over the same period. Although cell loss was found throughout the whole nucleus, it was more pronounced in the medial accessory olive. A subsequent apparent increase of the cell population was observed so that the adult value was again reached at 15 days. The evolution of the ION population is then characterized by a period of moderate cell death which takes place before the peak of polyneuronal innervation of Purkinje cells by olivary axons is attained. This strongly suggests that the removal of the redundant synaptic contacts established by climbing fibers onto Purkinje cells during development is caused by a progressive reduction of the branching of olivary axons rather than by degeneration of the presynaptic cells.     

miR-137调控人脑胶质瘤细胞增殖侵袭能力的体内外研究

目的探讨miR-137调控人脑胶质瘤细胞的增殖侵袭性生长能力及其机制。方法采用实时PCR分析miR-137在不同品系胶质瘤细胞及不同级别胶质瘤样本中的表达;脂质体介导miR-137模拟物转染胶质瘤细胞,实时PCR检测转染后miR-137的表达;应用MTT法、流式细胞术评价细胞生长和增殖的生物学特征变化;划痕实验、transwell细胞体外迁移实验检测肿瘤细胞迁移、侵袭能力;动物实验评价体内条件下肿瘤生长能力变化;Western blot、免疫组织化学染色检测肿瘤细胞Ki-67、MMP9表达水平。结果实时PCR分析显示：miR-137在胶质瘤中低表达。miR-137模拟物转染LN229和U87细胞后,实时PCR显示：miR-137表达上调;MTT法及流式细胞术显示细胞生长受抑,出现G0/G1期阻滞;划痕实验及transwell实验证实：细胞迁移侵袭能力下降;进一步Western blot、免疫组织化学染色显示：增殖侵袭相关蛋白Ki-67、MMP9表达降低;动物实验反映：肿瘤细胞生长受抑制。结论 miR-137高表达可抑制胶质瘤细胞生长和侵袭能力,提示miR-137可作为基因治疗脑胶质瘤的候选靶点。     

Expression and localization of utrophin in differentiating PC12 cells

To ascertain the role of utrophin in cultured neuronal cells, we investigated its expression and distribution along the NGF-induced differentiation of PC12 cells grown on different substrata. Utrophin mRNA was measured by RT-PCR assay and utrophin protein was quantified by immunoblot analysis. The distribution of utrophin and beta-dystroglycan was analyzed by confocal microscopy. We demonstrate that utrophin protein was increased 4-fold during differentiation of cells grown laminin. Concomitant with this up-regulation, utrophin was enriched at the growth cones in differentiating cells, where it co-localizes with beta-dystroglycan. These data suggest the presence of a utrophin-beta-dystroglycan complex in PC12 cells that participates in the formation and/or stabilization of the growth cone-extracellular matrix adhesion.     

高表达HSP70大鼠C6细胞瘤苗体外抗瘤时靶细胞的超微结构变化

目的 观察高表达热休克蛋白70（HSP70）C6细胞瘤苗擞活的SD大鼠脾细胞杀伤靶细胞的超微结构变化，并初步探讨其可能的抗瘤机制。方法 采用经诱导高表达HSP70的灭活C6细胞作瘤苗．体外刺激大鼠脾细胞进行肿瘤杀伤试验。采用噻唑蓝（MTT）法检测其杀伤活性．流式细胞术（FCM）及电子显微镜观察杀伤瘤细胞的变化。结果 （1）经瘤苗刺激的大鼠脾细胞对C6细胞的杀伤率较直接用灭活C6细胞刺激的脾细胞或未受任何刺激的新鲜脾细胞显著增高。（2）行肿瘤杀伤试验时FCM检测到亚二倍体峰．电镜发现靶细胞受攻击后出现染色质浓聚于核膜边缘．呈境界分明的块状或月形、半月形小体，或整个细胞核固缩成块状物，电子密度高，核膜、胞膜完整，或可见到凋亡小体．部分见髓鞘形成。有些效应细胞坏死。结论介导靶细胞凋亡可能是高表达HSP70的C6细胞瘤苗主动免疫抗瘤效应的一个重要机制。     

siRNA沉默CD99基因表达对髓母细胞瘤DAOY细胞增殖和凋亡的影响

目的 探讨siRNA沉默CD99基因表达对髓母细胞瘤细胞系DAOY细胞增殖和凋亡的影响。方法 利用CD99 siRNA转染髓母细胞瘤DAOY细胞,细胞分为沉默1组（转染CD99 siRNA-1#）、沉默2组（转染CD99 siRNA-2#）、对照组（转染siRNA阴性对照）,采用实时荧光定量PCR和免疫印迹法检测沉默效果。利用CCK-8细胞增殖实验、平板克隆形成实验检测细胞增殖能力,采用流式细胞术检测细胞周期和细胞凋亡。结果 沉默1组和沉默2组CD99蛋白和mRNA表达水平均明显低于对照组（P<0.05）,而沉默2组又明显低于沉默1组（P<0.01）;因此,选用CD99 siRNA-2#进行后续实验。沉默CD99基因表达后3、4、5 d,沉默2组细胞增殖水平较对照组明显降低（P<0.05）;平板克隆形成实验结果显示,沉默2组细胞克隆集落数明显减少（P<0.05）。沉默2组G0/G1期细胞百分比较对照组明显增加（P<0.05）,S期细胞、G2/M期细胞百分比明显低于对照组（P<0.05）。沉默2组细胞早期凋亡率（8.75%±1.47%）显著高于对照组（3.42%±1.21%;P<0.05）。结论 CD99基因在髓母细胞瘤DAOY细胞的增殖和凋亡中发挥重要作用,推测CD99基因可能成为治疗髓母细胞瘤的一个新靶点。     

TNF alpha increases activity of gamma-glutamyl transpeptidase in cultured rat astroglial cells

To investigate the presence of gamma-glutamyl transpeptidase (gammaGT) in brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells were studied. Astroglial cultures contained a specific gammaGT activity of 2.3 +/- 0.9 nmol/min/mg protein. A similar specific gammaGT activity was measured for oligodendroglial cultures, whereas microglial cells and neurons contained less than 30% of the specific gammaGT activity of astroglial cultures. The activity of gammaGT in astroglial cultures was elevated strongly by the presence of tumor necrosis factor-alpha (TNFalpha) in a time- and concentration-dependent manner. Maximal activity of gammaGT was observed after incubation of astroglial cultures for 3 days with 30 ng/mL TNFalpha. Under these conditions the specific gammaGT activity was increased by threefold compared to controls. Presence of the gammaGT-inhibitor acivicin completely inhibited gammaGT activity both in TNFalpha-treated and in control cells. In addition, the increase in astroglial gammaGT activity after application of TNFalpha was prevented completely by the presence of the protein synthesis inhibitor cycloheximide. gammaGT is involved in extracellular processing of glutathione (GSH) that is exported by astroglial cells. After TNFalpha-treatment the concentration of GSH in the medium of astroglial cells was reduced significantly compared to control cells. In conclusion, the data presented demonstrate that TNFalpha stimulates gammaGT synthesis in astroglial cells and thereby improves the capacity to process GSH exported by these cells.     

Subtype-dependent postnatal development of taste receptor cells in mouse fungiform taste buds

Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ～60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed.     

Trophic Factors Produced by Retinal Cells Increase the Survival of Retinal Ganglion Cells In Vitro

The naturally occurring neuron death of normal development has been shown to depend on trophic factors produced and released by target cells. It has also been shown that the afferent supply and local interactions play a role in the control of this degenerative phenomenon. We studied the effect of trophic factors produced by intrinsic retinal cells on the survival of retinal ganglion cells in vitro. Retinae of newborn hooded rats were retrogradely labelled with horseradish peroxidase injected into the superior colliculus to permit the identification of retinal ganglion cells in culture. We tested the effect of conditioned media either from aggregates or from explants of retinal cells from neonatal rats on the survival of ganglion cells in vitro. Our results showed that both conditioned media increased the survival of these cells. The trophic activity was dose-dependent, was maintained after dialysis against a 12 kDa membrane, was abolished by heating at 56°C for 30 min, and was not found in conditioned medium from cerebral cortical explants. Conditioned medium obtained without fetal calf serum presented the same trophic effect. These results suggest that the local control of developmental neuron death by intrinsic retinal cells may be mediated by neurotrophic factors.     

Analysis of the function of GABA(B) receptors on inhibitory afferent neurons of Purkinje cells in the cerebellar cortex of the rat

Purkinje cells, the output neurons of the cerebellar cortex, receive inhibitory input from basket, stellate and neighbouring Purkinje cells. The aim of the present study was to clarify the role of GABAB receptors on neurons giving inhibitory input to Purkinje cells. In sagittal slices prepared from the cerebellar vermis of the rat, the GABAB receptor agonist baclofen lowered the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded in Purkinje cells. These effects were prevented by the GABAB receptor antagonist CGP 55845. Two mechanisms were involved in the depression of the inhibitory input to Purkinje cells. The first mechanism was suppression of the firing of basket, stellate and Purkinje cells. The second mechanism was presynaptic inhibition of GABA release from terminals of the afferent axons. This was indicated by the finding that baclofen decreased the amplitude of IPSCs occurring in Purkinje cells synchronously with action potentials recorded in basket cells. A further support for the presynaptic inhibition is the observation that baclofen decreased the amplitude of autoreceptor currents which are due to activation of GABAA autoreceptors at axon terminals of basket cells by synaptically released GABA. The presynaptic inhibition was partly due to direct inhibition of the vesicular release mechanism, because baclofen lowered the frequency of miniature IPSCs recorded in Purkinje cells in the presence of cadmium and in the presence of tetrodotoxin plus ionomycin. The results show that activation of GABAB receptors decreased GABAA receptor-mediated synaptic input to cerebellar Purkinje cells both by lowering the firing rate of the inhibitory input neurons and by inhibiting GABA release from their axon terminals with a presynaptic mechanism.     

Role of low-affinity p75 receptor in nerve growth factor-inducible growth arrest of PC12 cells

Mutant PC12 cell clones (PC84 cells) were obtained by transfection with nerve growth factor (NGF) cDNA. These cells secreted active NGF, extended short processes, and proliferated faster than the parental PC12 cells. These features are of great interest because the parental PC12 cells cease proliferation and extend long processes when transfected with NGF cDNA. PC84 cells expressed a high level of acetylcholinesterase activity and neurofilament M, which indicates that PC84 cells were differentiated. The inhibition of TrkA by K252a diminished the short processes of PC84 cells but had no effect on their fast proliferation. The expression level of TrkA in PC84 cells was comparable to that in PC12 cells; whereas that of another NGF receptor, p75, was significantly lower. These data suggest that the decrease of p75 contributed to the continuous growth of PC84 cells, which was confirmed by suppressing p75 activity of PC12 cells with the antisense oligonucleotide of p75 or with anti-p75 neutralizing antibody. The treated cells did not cease proliferation in the presence of NGF and extended short processes. Our results suggest that NGF signaling via TrkA affects the differentiation characteristics of PC12 cells but that an additional signaling via p75 is necessary for the growth arrest of the cells.     

表达VASH1基因的人脑胶质瘤U-87MG细胞对化疗药物敏感性的变化

目的 探讨表达人血管生成抑制因子１（VASH1）的人脑胶质瘤U-87MG细胞对化疗药物的敏感性变化。方法 构建针对VASH1的慢病毒载体pGCL-GFP-VASH1,经测序鉴定后转染293T细胞,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞,荧光显微镜下检测转染效率;通过RT-PCR和Western blot分析U-87MG细胞VASH1 mRNA和蛋白表达水平;用CCK-8法检测U-87MG细胞在化疗药物顺铂和替莫唑胺作用下的存活率。流式细胞仪检测U-87MG细胞凋亡。结果 成功构建pGCL-GFP-VASH1慢病毒载体,并成功转染U-87MG细胞,转染率达70％以上;RT-PCR和Western blot结果证实转染VASH1慢病毒载体的U-87MG细胞表达VASH1 mRNA和蛋白。在顺铂或替莫唑胺作用下,表达VASH1的U-87MG细胞存活率均较未表达VASH1的U-87MG细胞明显降低（P<0.01）,而且U-87MG细胞凋亡率明显增加（P<0.01）。结论 VASH1慢病毒载体转染U-87MG细胞可使其稳定表达VASH1,并提高人脑胶质瘤U-87MG细胞对化疗药物敏感性、增加细胞凋亡率。     

Tissue factor activity in HeLa cells measured with a continuous chromogenic assay and ELISA reader

Tissue factor activity expressed by Hela cells cultured in 96-well plates has been quantitated in situ using a continuous spectrophotometric assay. Following the assay, cells assayed without physical disruption remained as viable as cells not subjected to the assay. Very little (or no) tissue factor was expressed in nondisrupted cells relative to that available in cells disrupted by freeze-thawing and sonication. Total tissue factor activity (that available in disrupted cells) decreased not as a simple function of time after subculturing, but was inversely related to cell density.