Schwanns cells were obtained from the distal end of the sciatic nerve following Wallerian degeneration of SD rats. These cells were cultured with the anteriorhorn neuron of spinal cord of 14dayold SD rat fetus. The two kinds of cells were separated by a slice. Through the microscope, the dendrites and the morphology changes at the 24th, 48th, 72th, and 96 th hour after culture were observed. It was demonstrated that the Schwanns cells played the role of maintaining the survival of neuron and promoting the growth of dendrites. It was said that the Schwanns cells could secrete neurotrophic factor which made the body enlarged and caused the dendrites enlonged to several times of the body.
Object ive To summa r i z e the advanc ement of cytoske l e ton and axon outgrowth of neuron. Methods The recent l iterature concerning cytoskeleton and axon outgrowth of neuron was reviewed and summarized. Results The actin filaments and microtubules in neuron were highly polarized and dynamic structures confined to the ti ps of axons and the reci procal interactions between these two major cytoskeletal polymers was also dynamic. Attractive or a repulsive cue whose final common path of action was the growth cone cytoskeleton mediated the growth of axons of neuron by intracellular signaling cascades. Regulating the actin filament and microtubule dynamics as well as their interactions in growth cones played a key role in neurite outgrowth and axon guidance. Rho-GTPases and glycogen synthase kinase 3β (GSK-3β), the two major intracellular signal ing pathways had emerged in recent years as candidates for regulating the dynamics of actin filaments and microtubules. Conclusion The axon outgrowth and guidance depend on well-coordinated cytoskeletal and reciprocal interaction dynamics which also mediate axon regeneration after spinal cord injury. Regulating activity of Rho-GTPases and GSK- 3β simultaneously may acts as key role to regulate the dynamics of cytoskeletal and to determine axon outgrowth.
Objective To observe the ultrastructural characteristics of human retinal progenitor cells cultured in vitro. Methods Six 5-month-old human fetuses（12 eyes）without eye diseases were selected. Retinal progenitor cells from the retina of one eye of each fetus were cultured in vitro，and observed by transmission electronic microscopy（TEM）; while those from the other eye were directly observed by TEM. Results Abundant heterochromatin were found in the karyon of 5-month embryonic retinal neuroepithelial cells，and the figure of the karyons was irregular.A few scattered initial cells were seen in retinal neuroepithelial layer with large karyon，smooth surface，abundant euchromatin，and distinct nucleolus.The human retinal progenitor cells cultured in vitro had the same ultrastructural characteristics as the initial cells:with huge karyon which almost occupied the whole cell，little cytoplasm，distint nucleolus，abundant euchromatin，and little heterochromatin.The cells clung to each other in the neural globoid cell mass.The size of the outer cells was large，and karyokinesis could be found. Conclusion The cultured human retinal progenitor cells are provided with the same ultrastructure characteristics as the initial cells. (Chin J Ocul Fundus Dis, 2006, 22: 185-187)
【Abstract】 Objective To investigate the effectiveness of all-trans-retinoic acid (ATRA) at different concentrationson prol iferation and differentiation of the rat embryonic neural stem cells (NSCs), and to find the optimal concentration of ATRA that promoting the differentiation of NSCs into neurons. Methods NSCs were isolated from cerebral cortex of rat embryos (embryonic day 12-16, average 15 days), and were cultured in serum-free medium (DMEM/F12 medium containing 20 ng/mL bFGF and 20 ng/mL EGF) at the concentration of 1×106 cells/mL. Subcultures were performed 7 days after the primary culture. The cell clusters of the 3rd passage were centrifuged and divided into 5 groups. In the experimental groups (groups A, B, C, D), the ATRA concentration was 0.5, 1.0, 5.0, 10.0 μmol/L in DMEM/F12 complete medium respectively, while in control group (group E), the ATRA concentration was 0 in DMEM/F12 complete medium. The prol iferation rate of each group was analyzedby cell counting day by day till 7th day, and BrdU positive cell counting 1, 3, 5, 7, 9 days after culture. In addition, collecting the 3rd passage NSCs and divided into 5 groups. In the experimental groups (groups A, B, C, D), the ATRA concentration was 0.5, 1.0, 5.0, 10.0 μmol/L in DMEM/F12 medium containing 5% FBS respectively, while in control group (group E), the ATRA concentration was 0 in DMEM/F12 medium containing 5% FBS. The capacity of NSCs differentiation toward neurons was determined by immunofluorescence double-labell ing and flow cytometry. Results Cell counting 1-7 days after culture in each experimental group (groups A, B, C, D) showed no significant differences (P gt; 0.05). Cell counting at each time point of all the experimental groups were less than those of control group (P lt; 0.05). BrdU positive cells were increased 1, 3, 5, 7, 9 days after culture in each experimental group (groups A, B, C, D), but there was no significant difference between each experimental group(P gt; 0.05). BrdU positive cells at each time point of control groups were more than those of all the experimental groups (P lt;0.05). The differentiation ratio of neurons was enhanced in experimental groups and the optimal ATRA treatment concentration was 1.0 μmol/ L (experimental group B). The differentiation ratio of neurons induced by ATRA in group B was 29.46% ± 0.47%, 47.25% ± 0.46% and 66.81% ± 0.57% respectively after cultured 3, 5 and 7 days, whereas the differentiation ratio of neurons was 11.11% ± 0.59%, 14.10% ± 0.32% and 15.92% ± 0.70% respectively in control group. The majority of NSCs differentiated into astrogl ial phenotypes in control group. By flow cytometry detection, the differentiation ratio of neurons after cultured 3 days and 7 days in experimental groups were more than those in control group (P lt; 0.05). Conclusion ATRA treatment remarkably promoted the differentiation of NSCs into neurons and the optimal concentration was 1.0 μmol/L.
ObjectiveTo systematically review the diagnostic value of neuron specific enolase (NSE) for malignant pleural effusion. MethodsWe comprehensively searched databases including The Cochrane Library (Issue 1, 2012), EMbase, MEDLINE, CBM, CNKI, WanFang Data and VIP from inception to January 2012 to collect studies about the diagnostic value of NSE for malignant pleural effusion. Literature screening according to the inclusion and exclusion criteria, data extraction and methodological quality assessment were completed by two reviewers independently. Then Meta-DiSc software (version 1.4) was used for pooling analysis. ResultsA total of 12 studies were finally included. The results of meta-analysis showed that the value of pooled specificity, sensitivity, positive likelihood radio, negative likelihood radio and diagnostic odds ratio (DOR) were 0.79 (0.76 to 0.84), 0.55 (0.51 to 0.59), 3.2 (1.94 to 5.29), 0.58 (0.45 to 0.74), 7.56 (3.74 to 15.30), respectively; and the area under SROC curve (AUC) was 0.813 1. ConclusionUsing NSE as a maker to diagnose malignant pleural effusion is of certain clinical value, which is used to differentiate benign and malignant pleural effusion.
Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP). Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A), diabetes mellitus (DM) only group (group B), DM + balanced salt solution (BSS) group (group C), DM + hUCMSC group (group D), with 10 rats in each group. DM rats were induced by intraperitoneal injection of streptozotocin. Apoptosis of retinal cells was assayed by dUTP nick end labeling. Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats. Results Compared with group A, large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C, however the apoptotic cells in group D were significantly reduced than group B and C. The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A, throughout the inner plexiform layer (IPL) in group B and C, only distributed in RNFL and GCL in group D. It was obvious that the expression of GFAP in group B and C was higher than group A. Compared with group B and C, the expression of GFAP in group D was significantly reduced. The difference of GFAP expression among the 4 groups was significant (F=79.635, P<0.05). Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats.
Objective Telomerase reverse transcriptase (TERT) is the key factor to determine cell growth and l ifespan. Meanwhile, it is tightly related to resistance of cell to stress and apoptosis. However, up till now l ittle is known about the role TERT plays in nervous system. To investigate the effect of conditioned medium from astrocytes (AS) transfected with TERT on neurons subjected to hypoxia-ischemia-reperfusion (HI-RP) through construction of in vitro HI-RP model of neurons. Methods An eukaryote expression plasmids containing rat full length TERT gene was constructed as pcDNA3-TERT. Twenty newborn rats at age of 3 days were sacrificed and their cerebral cortex were collected for isolation and cultivationof AS. Then AS were transfected with pcDNA3-TERT through l iposomes mediation, and positive clones were selected by G418 and expanded for continuous culture to establ ish the plamid pcDNA3-TERT transfection group. Meanwhile, the empty plasmid pcDNA3 transfection group and the non-transfection group were establ ished as control. The expression of gl ial fibrillary acidic protein (GFAP), which was the specific marker of the AS, was detected by immunocytochemistry, as well as the expression of TERT. Astrocyte conditioned medium (ACM) of the plamid pcDNA3-TERT transfection group was collected as TERT-ACM, while the ACM of the empty plasmid pcDNA3 transfection group and the non-transfection group were collected respectively as p-ACM and ACM. Next, 60 rats at age of 1 day were sacrificed and their cerebral cortex were collected for isolation and cultivation of neurons. The neurons were randomly divided into experimental group and normal group, the experimental group were further divided into 4 groups including control group, ACM group, p-ACM group, and TERT-ACM group. The neurons of control group were subjected to HI damage in serum-free DMEM, and the neurons of ACM group, p-ACM group, and TERTACM group were subjected to HI damage in different medium which contained ACM, p-ACM, and TERT-ACM, respectively. After duration of HI for 3 hours under the environment with 5%CO2, 1%O2, and 94%N2; the neurons of experimental groups were placed in CO2 incubator to imitate RP for 3, 6, 18, 24, and 36 hours in vitro. The neurons of normal group were not subjected to HI and RP treatment. During the treatment of HI-RP, the survival ratio of neurons was detected by means of MTT, the lactate dehydrogenase (LDH) activity of neuron medium with LDH detection kit, and the neuronal apoptosis by means of TUNEL. Results The percentages of GFAP positive cells were 98%, 99%, and 98% in non-transfection group, plasmid pcDNA3-TERT transfection group, and plasmid pcDNA3 transfection group, respectively. There was no expression of TERT in no-transfection group and plasmid pcDNA3 transfection group, and the percentage of TERT positive cells in plasmid pcDNA3- TERT transfection group was 98%. Compared with normal group, the survival ratio of ......（余见正文）
Objective To investigate the glutamate toxicity on inner stratum retinal neurons(ISRN) and the neurotoxicity quantity-efficacy relation. Method Retinal explants obtained from 30 neonatal mices were implanted into two pieces of 24-well culture plates (48 wells). The 48 wells were divided into three groups: control group, glutamate exposure 24 h group, and glutamate exposure with further lasting 6 h group. The retinal explants were sectioned, and then stained with HE after 24 h in vitro. The cells in retinal ganglion cells (RGCs) layer and inner nuclear layer (INL) were analyzed by light microscope at 1 000times; magnification , and the number of normal morphological cells was counted under three 1 000times; magnificat ion fields. Results Some cells in ISRN (include RGCs and INL c ells) showed pykno tic nuclei and necrosis after 24 h in control culture. Glutamate exposure 24 h group:at the 2 mmol and 4 mmol concentrations of glutamate, the situation of the normal morphological cells in ISRN had no difference from that of the control group (Pgt;0.05). At the concentration of glutamate more than or equal to 6 mmol, the number of normal morphological cells in ISRN was significantly less than that of the control group (Plt;0.05), and with the increase of glutamate concentration, the number of normal morphological cells was reduced. Glutamate exposure with fur ther lasting 6 h group: at the concentration of glutamate equal to 6 mmol, the n umber of normal morphological cells in INL was significantly less than that of the control group (Plt;0.05), while the number of normal morphological cells in RGCs layer had no difference between two groups (Pgt;0.05). At the concentration of glutamate more than or equal to 8 mmol, the number of normal morphological cels in RGC s layer and INL was significantly less than that of the control group (Plt;0.05 ). Conclusion Glutamate has the neurotoxicity for ISRN in vitro, and the effect is dose-dependant. (Chin J Ocul Fundus Dis, 2001,17:311-314)
Objective To investigate the possibility of theadipose tissue-derived stromal cells(ADSCs) to differentiate into the neuron-like cells and to explore a new cell source for the transplantation related to the central nervous system. Methods Adipose was digested by collagenase, cultured in the fetal bovine serum containing a medium. Trypse was used to digest the cells and the cell passage was performed. The 3rd to the 9th passage ADSCs were used to make an induction. Isobutylmethylxanthine, indomethacin, insulin, and dexamethasone were used to induce the ADSCs to differentiate into the neuron-like cells and adipocytes. Sudan black B and immunocytochemistry were used to identify the cells. Results A population of the ADSCs could be isolated from the adult human adipose tissue, they were processed to obtain a fibroblast-like population of the cells and could be maintained in vitro for an extendedperiod with the stable population doubling, and they were expanded as the undifferentiated cells in culture for more than 20 passages, which indicated their proliferative capacity. They expressed vimentin and nestin, and characteristics of the neuron precursor stem cells at an early stage of differentiation. And the majority of the ADSCs also expressed the neuron-specific enolase and βⅢ-tubulin, characteristics of the neurons. Isobutyl-methyxanthine, indomethacin, insulin, and dexamethasone induced 40%-50% of ADSCs to differentiate into adipocytes and 0.1%0.2% of ADSCs into neuron-like cells. The neuron-like cells had a complicated morphology of the neurons, and they exhibited a neuron phenotype, expressed nestin, vimentin, neuron-specific enolase and βⅢ-tubulin, but some neuron-like cells also expressed thesmooth muscle actin (SMA), and the characteristics of the smooth muscle cells; however, the neurons from the central nervous system were never reported to express this kind of protein. Therefore, the neuron-like cells from the ADSCs could be regarded as functional neurons. Conclusion Ourresults support the hypothesis that the adult adipose tissue contains the stem cells capable of differentiating into the neuron-like cells, and they can overcome their mesenchymal commitment, which represents an alternative autologous stemcell source for transplantation related to the central nervous system.
ObjectiveTo investigate the effect of electroacupuncture on the apoptosis of hippocampal neurons in C57BL/6J mice with status epilepticus by observing the changes of hippocampal subtle neuron pathology and apoptosis.MethodsMale C57BL/6J mice were used to prepare epileptic status models of lithium-pilocarpine mice, and then 7-day electroacupuncture stimulation (Baihui, Fengfu) were given to the mice model. Open field experiment and new object recognition experiment were performed to observe the changes of cognitive abilities. The pathological changes of hippocampal neurons were detected by HE staining. Hippocampal apoptosis protein (Caspase-3) and microtubule-associated protein (MAP-2) were detected by immunohistochemistry. Effect of electroacupuncture on apoptosis of hippocampal neurons in C57BL/6J mice with status epilepticus were recorded.Results① Compared with the control group, the vertical movement, modification times, and number of crossings of the model group all decreased significantly (P<0.000 1,P<0.000 1,P<0.000 1), and their cognitive ability decreased significantly (P<0.01). Compared with the model group, vertical movements, modification times, and number of crossings were increased in the electroacupuncture (EA) group (P<0.01,P<0.05,P<0.05), and the cognitive ability of new objects was increased (P<0.01). ② HE staining showed that the model group had significant damage to the hippocampal neurons of mice, and the cells swelled, nuclear collapsed and vacuoles appeared. In the EA group, the injury of hippocampal neurons was alleviated, and cell edema and vacuolization were alleviated. ③ Immunohistochemistry showed that compared with the control group, the IOD of the Caspase-3 positive cells in the hippocampus of the model group increased significantly (P<0.000 1), and the IOD of the MAP-2 positive cells decreased significantly (P<0.01); Compared with the electroacupuncture, the IOD of the Caspase-3 positive cells in the hippocampus of the mice decreased (P<0.05), and the IOD of the MAP-2 positive cells increased (P<0.05).ConclusionsElectroacupuncture can improve the pathological changes of hippocampal neurons in C57BL/6J mice with status epilepticus, promote cytoskeletal repair, reduce neuronal apoptosis in hippocampus, and antagonize the damage of hippocampal neurons induced by status epilepticus.