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Objective:To analyze the differential expression of neural precursor cell-expressed developmentally downregulated 8（NEDD8） protein in nasal polyp tissues of patients with different pathological types of chronic rhinorhinosinusitis with nasal polyps（CRSwNP）. Methods:All specimens were obtained from the specimen library of Beijing Tongren Hospital, and were all patients who underwent nasal endoscopic surgery for chronic rhinosinusitis in Beijing Tongren Hospital. Hematoxylin-eosin staining（HE） was used to detect the number of eosinophils in nasal polyps, and CRSwNP patients were grouped according to the number of eosinophils in nasal polyps, immunohistochemistry was used to detect and analyze the expression level of NEDD8 protein in nasal polyps. Results:The expression level of NEDD8 protein in nasal polyps of patients with eosinophilic chronic rhinorhinosinusitis with nasal polyps was significantly higher than that of patients with non-eosinophilic chronic rhinosinusitis and nasal polyps（P<0.05）. In addition, there was a significant positive correlation between the expression level of NEDD8 protein and the number of eosinophils in nasal polyp tissue（r=0.79, P=0.02）. Conclusion:There are differences in the expression of NEDD8 protein in patients with chronic rhinosinusitis and nasal polyps of different pathological types.
Subject(s)
Humans , Nasal Polyps/metabolism , Rhinitis/diagnosis , NEDD8 Protein/metabolism , Sinusitis/diagnosis , Eosinophils/metabolism , Chronic DiseaseABSTRACT
Objective:To explore the effect of miR-1249-5p on the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer.Methods:The relationship between the expression level of miR-1249-5p and the overall survival of prostate cancer patients was analyzed using OncoMir Cancer Database (OMCD). The human prostate cancer cell line PC-3 was divided into two groups: miR-1249-5p group and negative control group. Mediated by Lipofectamine 2000, miR-1249-5p mimics liposome complex or negative miRNA liposome complex were transfected into PC-3 cell at logarithmic growth stage. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-1249-5p in PC-3 cell of two groups. Colony formation assay was used to detect the changes of the proliferation ability of PC-3 cell in the two groups. Transwell experiment was used to detect the changes of PC-3 cell invasion in the two groups, and the cell cycle changes of the two groups of PC-3 were detected by flow cytometry. The miRNA prediction software miRGator was used to predict the target gene of miR-1249-5p. RT-qPCR and Western blotting were used to detect the target gene expression of miR-1249-5p. Measurement data were expressed as mean±standard deviation ( ± s), and t-test was used for comparison between two groups. Results:Compared with prostate cancer patients with low miR-1249-5p expression, prostate cancer patients with higher miR-1249-5p expression had longer overall survival, and the difference was statistically significant ( P<0.01). The expression level of miR-1249-5p in the miR-1249-5p group (10.74±1.19) was significantly higher than that of the negative control group (1.56±0.27), the difference was statistically significant ( P<0.01). The number of colonies formed in the miR-1249-5p group (35.86±6.94) was significantly less than that in the negative control group (88.94±11.66), and the difference was statistically significant ( P<0.01). The number of transmembrane cells [(25.01±6.83)/high power field of view] in the miR-1249-5p group was significantly less than that of the negative control group [(82.76±8.35)/high power field of view], and the difference was statistically significant ( P<0.01). The proportion of cells in the G 0-G 1 phase in the miR-1249-5p group [(50.79±6.61)%] was significantly higher than that in the negative control group [(27.09±2.30)%], the difference was statistically significant ( P<0.01), and PC-3 cell were inhibited in the G 0-G 1 phase. Neural precursor cell expressed developmentally down-regulated 9 ( NEDD9) may be the target gene of miR-1249-5p. Compared with the negative control group, the NEDD9 gene expression in the miR-1249-5p group was significantly lower than that of the negative control group, the difference was statistically significant ( P<0.01). Conclusion:miR-1249-5p can inhibit the proliferation, metastasis and cell cycle of PC-3 cell in prostate cancer, which may be achieved by negatively regulating the expression of proto-oncogene NEDD9.
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Neural precursor cell Expressed‚Developmentally Down-regulated protein 4 (NEDD4-1‚ also known as NEDD4 in some papers) is a tumor-related protein that has attracted much attention in recent years. It belongs to the E3 HECT (homologous to E6 associated protein C terminus) ubiquitin ligase‚ which could ubiquitinate various proteins that are subsequently degraded in lysosomes or proteasomes‚ or mediate their nuclear-cytoplasmic translocation‚ or indirectly affect various signaling pathways of different malignant tumors. With the deepening of a large number of tumor-related experiments‚ it has been found that NEDD4-1 can affect the biological behavior of tumors by regulating cell cycle‚ invasion and metastasis of cancer cells‚ antagonize drug resistance and many other pathways. In digestive system tumors‚ NEDD4-1 mainly promotes the proliferation‚ invasion and migration of hepatocellular carcinoma through multiple pathways such as PTEN/ PI3K/ Akt‚ TGF-β‚ Hippo and LDLRAD4. In pancreatic cancer‚ NEDD4-1 acted as an oncogene in the PI3K/ Akt signaling pathway‚ but acted as a tumor suppressor gene in the Myc-Sirt2 signaling circuit. In gastric and colorectal cancer‚ the NEDD4-1-related signaling pathways are different from other digestive system tumors. NEDD4-1 promotes gastric cancer progression and metastasis (via the EGFR signaling pathway) and inhibits colorectal cancer tumor growth (via the Wnt signaling pathway) independently of the PTEN/ PI3K/ Akt pathway. NEDD4-1 has become a hot research direction for therapeutic purposes. In this paper‚ we summarize the functions‚ signaling pathways and potential inhibitors of NEDD4-1 in different digestive system tumors‚ and discuss the relationship between NEDD4-1 and different signaling pathways‚ aiming to provide important reference data for cancer therapy.
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BACKGROUND: Researchers believe that hydrogen sulfide (H2S), as an important cell protective molecule, may become a new treatment method to restore the physiological function of diseased cells or organ systems through the artificial regulation of endogenous H2S biosynthesis or in vitro administration of H2S donor. ADT-OH is a slow-release donor of H2S that can improve the survival rate of hippocampal nerve cells with glutamate-induced injury, but studies on the proliferation of cerebral cortical neural precursor cells are rare. OBJECTIVE: To investigate the effect of ADT-OH on the proliferation of neural precursor cells in embryonic cerebral cortex. METHODS: Neural precursor cells from cerebral cortical ventricular zone and subventricular zone of embryonic mice at embryonic 14.5 days were isolated. Neural precursor cells from one fetal mouse were inoculated into one well (24-well plate), and cultured with the medium containing 100 μmol/L ADT-OH. The size and number of neural spheres per well were measured at 3 days after culture. The proliferation rate of cultured neural precursor cells was detected by BrdU labeling. The proliferation of the cells was further verified by immunofluorescence staining with the specific antibody Ki67. The expression of cyclin D1 was finally detected by western blot assay. RESULTS AND CONCLUSION: Our experimental results showed that ADT-OH could promote the formation of neural spheres, and further detection by BrdU and Ki67 antibody showed that ADT-OH could promote the proliferation rate of neural precursor cells. Meanwhile, the expression of cyclin D1, a proliferation-related gene, was up-regulated in neural precursor cells after ADT-OH treatment. Overall, ADT-OH may promote the proliferation of neural precursor cells by regulating the expression of cyclin D1.
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Se presenta el caso de un paciente de 20 días de nacido, procedente de Cartagena (Bolívar), hospitalizado por presentar fiebre de 6 días de evolución asociado a sintomatología respiratoria con evaluación neurológica normal. La ecografía obstétrica evidenció una microcefalia con un percentil de perímetro cefálico <2, con hipoplasia del cuerpo calloso y tomografía axial computarizada de cráneo que reportó diámetros cefálicos disminuidos, finas calcificaciones residuales en región frontal-parietal y cambios atróficos cerebrales subcorticales. Se le inició terapia antibiótica por presentar sepsis neonatal, las pruebas serológicas y la PCR para Zika resultaron positivas. Se decidió dar el alta médica al 6 día por mejoría clínica y no presentar déficit neurológico aparente. Aunque no existe un tratamiento específico, el pilar del manejo de un recién nacido con microcefalia es el seguimiento y la vigilancia futura de las posibles comorbilidades, como epilepsia, parálisis cerebral o retraso cognitivo y motor.
We present the case of a 20-day-old patient from Cartagena (Bolívar), hospitalized for presenting a 6-day fever associated with respiratory symptoms with normal neurological evaluation. The obstetric ultrasound showed a microcephaly with a percentile of cephalic perimeter <2, with hypoplasia of the corpus callosum and computed tomography of the skull that reported decreased cephalic diameters, fine residual calcifications in the frontal-parietal region and atrophic subcortical cerebral changes. Antibiotic therapy was initiated due to neonatal sepsis, the serological tests and the PCR for Zika were positive. It was decided to discharge the hospital after 6 days due to clinical improvement and for not presenting apparent neurological deficit. Although there is no specific treatment, the pillar of the management of a newborn with microcephaly is the monitoring and future surveillance of possible comorbidities, such as epilepsy, cerebral palsy or cognitive and motor retardation.
Subject(s)
Humans , Male , Infant, Newborn , Zika Virus , Microcephaly , Stem Cells , Pregnancy , Diagnostic Imaging , Fever , Anti-Bacterial AgentsABSTRACT
The concept of cellular reprogramming was developed to generate induced neural precursor cells (iNPCs)/dopaminergic (iDA) neurons using diverse approaches. Here, we investigated the effects of various nanoscale scaffolds (fiber, dot, and line) on iNPC/iDA differentiation by direct reprogramming. The generation and maturation of iDA neurons (microtubule-associated protein 2-positive and tyrosine hydroxylase-positive) and iNPCs (NESTIN-positive and SOX2-positive) increased on fiber and dot scaffolds as compared to that of the flat (control) scaffold. This study demonstrates that nanotopographical environments are suitable for direct differentiation methods and may improve the differentiation efficiency.
Subject(s)
Cellular Reprogramming , Nanofibers , Neurons , TyrosineABSTRACT
The direct lineage conversion of fibroblasts into neuronal or neural precursor cells (NPCs) has become a hot issue in recent years as an attractive approach in the field of stem cell regenerative medicine. In this study, we adopted the stromal feeder co-culture method during the early conversion period to enhance conversion efficiency. Stromal cells are often used in directed differentiation of dopaminergic (DA) neurons from pluripotent stem cells. We co-cultured rat embryonic fibroblasts (REFs) on γ-irradiated sonic hedgehog-overexpressing MS5 stromal (MS5-SHH) cells after transduction with Brn2, Ascl1, Myt1L, and BclxL-GFP (BAMXGFP) transcription factors to REFs. One week after co-culture, transduced cells (GFP+ cells) that proliferated on MS5-SHH cells were separated from MS5-SHH cells through a 40 µm cell strainer. Subsequently, the converted cells (GFP+ cells) were expanded on fibronectin-coated culture plates in NPC expansion medium. The induced NPCs (iNPCs) expressed NPC potential (NESTIN+/SOX2+) earlier than seen with non-co-culture methods and were efficiently differentiated into DA neurons by overexpression of Nurr1 and Foxa2 genes, which are specific transcription factors for midbrain DA neuron development. These observations indicated that direct conversion to NPCs using an MS5 stromal cells co-culture method is a suitable technique for efficient generation of iNPC/DA neurons from fibroblasts.
Subject(s)
Animals , Rats , Coculture Techniques , Dopaminergic Neurons , Fibroblasts , Mesencephalon , Methods , Neurons , Pluripotent Stem Cells , Regenerative Medicine , Stem Cells , Stromal Cells , Transcription FactorsABSTRACT
OBJECTIVE: Recently, regenerative therapies have been used in clinical trials (heart, cartilage, skeletal). We don't make use of these treatments to spinal cord injury (SCI) patients yet, but regenerative therapies are rising interest in recent study about SCI. Neural precursor/stem cell (NPSC) proliferation is a significant event in functional recovery of the central nervous system (CNS). However, brain NPSCs and spinal cord NPSCs (SC-NPSCs) have many differences including gene expression and proliferation. The purpose of this study was to investigate the influence of neural growth factor (NGF) on the proliferation of SC-NPSCs. METHODS: NPSCs (2×10⁴) were suspended in 100 µL of neurobasal medium containing NGF-7S (Sigma-Aldrich) and cultured in a 96-well plate for 12 days. NPSC proliferation was analyzed five times for either concentration of NGF (0.02 and 2 ng/mL). Sixteen rats after SCI were randomly allocated into two groups. In group 1 (SCI-vehicle group, n=8), animals received 1.0 mL of the saline vehicle solution. In group 2 (SCI-NGF group, n=8), the animals received single doses of NGF (Sigma-Aldrich). A dose of 0.02 ng/mL of NGF or normal saline as a vehicle control was intra-thecally injected daily at 24 hour intervals for 7 days. For Immunohistochemistry analysis, rats were sacrificed after one week and the spinal cords were obtained. RESULTS: The elevation of cell proliferation with 0.02 ng/mL NGF was significant (p<0.05) but was not significant for 2 ng/mL NGF. The optical density was increased in the NGF 0.02 ng/mL group compared to the control group and NGF 2 ng/mL groups. The density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group (p<0.05). High power microscopy revealed that the density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group. CONCLUSION: SC-NPSC proliferation is an important pathway in the functional recovery of SCI. NGF enhances SC-NPSC proliferation in vitro and in vivo. NGF may be a useful option for treatment of SCI patients pending further studies to verify the clinical applicability.
Subject(s)
Animals , Humans , Rats , Brain , Cartilage , Cell Proliferation , Central Nervous System , Gene Expression , Immunohistochemistry , In Vitro Techniques , Microscopy , Nerve Growth Factor , Nestin , Spinal Cord Injuries , Spinal CordABSTRACT
@#Objective To observe dynamic variation of neural precursor cells in dentate gyrus of hippocampus and effect of Yangxue Qingnao Granule on it in vascular dementia rats. Methods 72 Sprague-Dawley rats were randomly divided into sham group (n=24), vascular dementia group (model group, n=24) and Yangxue Qingnao Granule group (treatment group, n=24). The vascular dementia model was established with modified Pulsineli's four-vessel occlusion. The expression of Nestin was detected with Western blotting, the expression of 5-bromodeoxyuridine (BrdU) and BrdU/Nestin were detected with immunofluorescence in dentate gyrus of hippocampus 1, 2, 4 and 8 weeks after modeling. Results The expression of Nestin, BrdU and BrdU/Nestin increased in the model and treatment groups with time, peaked at 4 weeks after modeling, and it was more than that of the sham group on all the time points (P<0.01). However, it was more in the treatment group than in the model group on all the time points (P<0.01). Conclusion Yangxue Qingnao Granule promotes the proliferation of neural precursor cells in dentate gyrus of hippocampus in vascular dementia rats.
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Objective To observe dynamic variation of neural precursor cells in dentate gyrus of hippocampus and effect of Yangxue Qingnao Granule on it in vascular dementia rats. Methods 72 Sprague-Dawley rats were randomly divided into sham group (n=24), vascular dementia group (model group, n=24) and Yangxue Qingnao Granule group (treatment group, n=24). The vascular dementia model was estab-lished with modified Pulsineli's four-vessel occlusion. The expression of Nestin was detected with Western blotting, the expression of 5-bro-modeoxyuridine (BrdU) and BrdU/Nestin were detected with immunofluorescence in dentate gyrus of hippocampus 1, 2, 4 and 8 weeks af-ter modeling. Results The expression of Nestin, BrdU and BrdU/Nestin increased in the model and treatment groups with time, peaked at 4 weeks after modeling, and it was more than that of the sham group on all the time points (P<0.01). However, it was more in the treatment group than in the model group on all the time points (P<0.01). Conclusion Yangxue Qingnao Granule promotes the proliferation of neural precursor cells in dentate gyrus of hippocampus in vascular dementia rats.
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Objective To study the effect of neuronal Nogo-66 receptor (NgR1) antagonist,soluble Nogo-66 receptor (sNgR1-Fc),on promoting the endogenous neural precursor cells (NPCs) differentiating into neurons in order to clarify the mechanism.Methods The cortical infarction was induced by photochemistry,named photothrombotic cortical injury (PCI).Twelve Sprague Dawley rats were randomly divided (random number) into three groups:Sham-operated group,PBS group,and sNgR1-Fc group.PBS (PBS group) or sNgR1-Fc (sNgR1-Fc group) was injected into the lateral ventricle of brain with a minipump.BrdU (Bromodeoxyuridine) was injected into the peritoneal cavity 4-6 days after PCI.The subdentate gyrus zone (SGZ) of brain from sacrificed rat was harvested for Immunohistochemistry to observe the ratio of NeuN +/BrdU + cells 35 days after PCI.Proteins including Nestin、Notch1 and Mash1 were detected by Western Blot.Results The cortical infarction in rat was successfully induced by photochemistry.Thirty-five days after PCI,the BrdU + cells number and theratio of NeuN +/BrdU + in the SGZ of the ipsilateral cerebrum hemisphere with PCI were significantly higher in sNgR1-Fc group than those in PBS group (P ＜ 0.05).The levels of Notch1,Mash1 and Neuro D in the sNgR1-Fc group were significantly higher than those in the PBS group (P ＜ 0.05),which were significantly higher than those in the Sham-operated group.Conclusions sNgR1-Fc could promote the endogenous NPCs differentiating into neurons in a cortical infarction model.The mechanisms may be attributed to the Notch/bHLH (proneural basic helix-loop-helix genes) signaling way.
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BACKGROUND:Whether embryonic stem cel transplantation can be an effective method for treatment of cerebrovascular disease has become a hot spot. OBJECTIVE:To investigate the effect and feasibility of embryonic stem cel-differentiated neural precursor cel transplantation for the treatment of cerebrovascular disease. METHODS:The rat models of Parkinson’s disease, ischemic brain injury and vascular dementia were established and the embryonic stem cells were in vitro cultured and induced to differentiate into the neural precursor cells. The embryonic stem cel-differentiated neural precursor cells were transplanted into the rat brains with corresponding cerebrovascular disease, and then the rotational behavior changes, pathological changes in the brain tissue as wel as changes in the hippocampal structure and the number of nerve cells in the rats with cerebrovascular disease were observed. RESUTLS AND CONCLUSION:After the embryonic stem cel-differentiated neural precursor cells transplanted into the rat brains with Parkinson’s disease, the frequency of apomorphine-induced rotation was decreased and in the downward trend, while the striatal dopamine levels were significantly increased. After transplantation of embryonic stem cel-differentiated neural precursor cells into the rat brains with ischemic brain injury, the cells could survive for a long time, and then migrated and distributed in the injured hippocampus to form the hippocampal structure;the cells could differentiate into neurons, and the number of nerve cells in the injured hippocampus was significantly increased. The results indicate that the transplanted glial cel line-derived neurotrophic factor gene-modified embryonic stem cells can improve the learning and memory function of vascular dementia rats, enhance neural plasticity, and induce self-directed migration and differentiation into mature neurons.
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The transplantation of neural precursor cells (NPCs) is known to be a promising approach to ameliorating behavioral deficits after stroke in a rodent model of middle cerebral artery occlusion (MCAo). Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and differentiate into mature neurons to some extent. However, the spatiotemporal dynamics of NPC migration following transplantation into stroke animals have yet to be elucidated. In this study, we investigated the fates of human embryonic stem cell (hESC)-derived NPCs (ENStem-A) for 8 weeks following transplantation into the side contralateral to the infarct region using 7.0T animal magnetic resonance imaging (MRI). T2- and T2*-weighted MRI analyses indicated that the migrating cells were clearly detectable at the infarct boundary zone by 1 week, and the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the signals were slightly increased or unchanged. At 8 weeks, we performed Prussian blue staining and immunohistochemical staining using human-specific markers, and found that high percentages of transplanted cells migrated to the infarct boundary. Most of these cells were CXCR4-positive. We also observed that the migrating cells expressed markers for various stages of neural differentiation, including Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, indicating that the transplanted cells may partially contribute to the reconstruction of the damaged neural tissues after stroke. Interestingly, we found that the extent of gliosis (glial fibrillary acidic protein-positive cells) and apoptosis (TUNEL-positive cells) were significantly decreased in the cell-transplanted group, suggesting that hESC-NPCs have a positive role in reducing glia scar formation and cell death after stroke. No tumors formed in our study. We also performed various behavioral tests, including rotarod, stepping and modified neurological severity score tests, and found that the transplanted animals exhibited significant improvements in sensorimotor functions during the 8 weeks after transplantation. Taken together, these results strongly suggest that hESC-NPCs have the capacity to migrate to the infarct region, form neural tissues efficiently and contribute to behavioral recovery in a rodent model of ischemic stroke.
Subject(s)
Animals , Humans , Male , Rats , Apoptosis , Cell Differentiation , Cell Movement , Embryonic Stem Cells/cytology , Glial Fibrillary Acidic Protein/genetics , Infarction, Middle Cerebral Artery/metabolism , Neural Stem Cells/cytology , Psychomotor Performance , Rats, Sprague-Dawley , Receptors, CXCR4/geneticsABSTRACT
Objective To reseach the time point of the highest percentage of neural precursor cells derived from adipose stromal cells (ADSCs) in vitro, and to observe the ultrastructure features of neural precursor cells. Methods Used the β-mercaptoethanol to induce ADSCs to differentiate into neural precursor cells and neuron-like cells. The morphology of the uninductedcells and inducted cells were observed with inverted phase contrast microscope. The expression of nestin which was the marker of neural precursor cell in each group was detected using immunofluorescence staining method. The ultrastructural feature of cells which was induced for 3 hours were observed. Results The highest ratio of positive expression of nestin was 3 hours following induction,with the ratio ( 86.25 ± 4.82) %. There were many protuberance on the cell membrane under transmission electron microscopy.There were plenty of organelles in the neural precursor cells. The neural precursor cells had a large size nucleus,large nucleoplasmic index, much extended chromatin,and less condensed chromatin. The nucleus had double-layer nuclear envelope, more nuclear pore on the nuclear envelope. Conclusion The time point of the highest percentage of neural precursor cells derived from ADSCs is 3 hours,and the ultrastructral feature of induced neural precursor cells confirm that cells at this time point are in a state of split active period.
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Cell therapy for neurological disorders has advanced, and neural precursor cells (NPC) may become the ideal candidates for neural transplantation in a wide range of diseases. However, additional work has to be done to determine either the ideal culture environment for NPC expansion in vitro, without altering their plasticity, or the FGF-2 and EGF mechanisms of cell signaling in neurospheres growth, survival and differentiation. In this work we evaluated mouse neurospheres cultured with and without FGF-2 and EGF containing medium and showed that those growth factors are responsible for NPC proliferation. It is also demonstrated that endogenous production of growth factors shifts from FGF-2 to IGF-1/PDGFb upon EGF and FGF-2 withdrawal. Mouse NPC cultured in suspension showed different patterns of neuronal localization (core versus shell) for both EGF and FGF-2 withdrawal and control groups. Taken together, these results show that EGF and FGF-2 removal play an important role in NPC differentiation and may contribute to a better understanding of mechanisms of NPC differentiation. Our findings suggest that depriving NPC of growth factors prior to grafting might enhance their chance to effectively integrate into the host.
As terapias celulares para doenças neurológicas têm avançado e células precursoras neurais (NPC) surgem como candidatas ideais para o transplante de células neurais em muitas doenças. No entanto, trabalhos adicionais devem ser feitos para determinar o ambiente de cultivo ideal para a expansão in vitro das NPC, sem alterar sua plasticidade, e os mecanismos de sinalização celular do fator de crescimento epidérmico (EGF) e fator de crescimento de fibroblasto 2 (FGF-2) no crescimento, sobrevivência e diferenciação da neuroesfera. Nesse trabalho avaliamosNPCcultivadas na presença e na ausência de FGF-2 e EGF e mostramos que esses fatores de crescimento são responsáveis pela proliferação das NPC. Também foi demonstrado que a produção endógena de fatores de crescimento alterna de FGF-2 a fator de crescimento de insulina 1 (IGF-1) e fator de crescimento derivado de plaquetas b (PDGFb) após remoção de EGF e FGF-2. NPC de camundongo cultivadas em suspensão mostraram padrões de localização neuronal distintos (centro versus borda) tanto no grupo controle como no grupo sem EGF e FGF-2. Juntos, esses resultados mostram que a remoção de EGF e FGF-2 exerce importante ação na diferenciação de NPC e possivelmente contribui para melhor compreensão dos mecanismos envolvidos na diferenciação. Nossos achados sugerem que, privando as NPC de fatores de crescimento antes do transplante, talvez aumente as chances de que as células efetivamente se integrem ao hospedeiro.
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Animals , Mice , Cell Differentiation/drug effects , Epidermal Growth Factor/pharmacology , /pharmacology , Neuronal Plasticity/physiology , Neurons/drug effects , Stem Cells/drug effects , Cell Culture Techniques/methods , Cell Differentiation/physiology , Neurons/cytology , Neurons/physiology , Stem Cells/cytology , Stem Cells/physiologyABSTRACT
Objective To investigate the cellular localization of the neural precursor cell-expressed, developmentally downregulated isoforms(Nedd4), Nedd4- 1/2 and Nedd4- 2, and the serum glucocorticoid- inducible kinasel(SGK1) in various subregions of the rat cochlea. Methods The expression patterns of Nedd4-1/2, Nedd42 and SGK1 in the cochlea of rat were studied by immunohistochemistry with the specific polyclonal rabbit antibodies against the rat Nedd4-1/2, Nedd4-2 and SGK1. Results All three proteins were extensively expressed in various regions of the rat cochlea. They were found in the stria vascularis, spiral ligament, organ of Corti, spiral limbus, spiral ganglion and Reissner's membrane. Conclusion Our findings suggest that there exists a Na+ transport system in the cochlea consisting of SGK1, Nedd4 isoforms and ENaC, which may work in concert to transport Na+ and to maintain homeostasis in the inner ear as they do in other tight epithelia.
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The differentiation of neural precursor cells (NPCs) into neurons and astrocytes is a process that is tightly controlled by complicated and ill-defined gene networks. To extend our knowledge to gene networks, we performed a temporal analysis of gene expression during the differentiation (2, 4, and 8 days) of spinal cord-derived NPCs using oligonucleotide microarray technology. Out of 32,996 genes analyzed, 1878 exhibited significant changes in expression level (fold change>2, p<0.05) at least once throughout the differentiation process. These 1878 genes were classified into 12 groups by k-means clustering, based on their expression patterns. K-means clustering analysis revealed that the genes involved in astrogenesis were categorized into the clusters containing constantly upregulated genes, whereas the genes involved in neurogenesis were grouped to the cluster showing a sudden decrease in gene expression on Day 8. Functional analysis of the differentially expressedgenes indicated the enrichment of genes for Pax6- NeuroD signaling-TGFb-SMAD and BMP-SMAD-which suggest the implication of these genes in the differentiation of NPCs and, in particular, key roles for Nova1 and TGFBR1 in the neurogenesis/astrogenesis of mouse spinal cord.
Subject(s)
Animals , Mice , Astrocytes , Gene Expression , Gene Regulatory Networks , Neurogenesis , Neurons , Oligonucleotide Array Sequence Analysis , Spinal CordABSTRACT
Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.
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Objective To observe the morphology of embryonic stem cell-derived neurons after transplanted into A?-injured rat hippocampus. Methods Neural precursor cells (NPCs) were generated from mouse embryonic stem cells (ESCs) expressing EGFP with modified serum-free methods and then transplanted into the hippocampus of A?1-40-injuried rats. The morphology, neurotransmitter phenotypes and receptors of EGFP-positive donor cells were observed with immunofluorescene methods. Results The engrafted NPCs survived and differentiated into glutamatergic and GABAergic neurons and expressed both glutamatergic and GABAergic neurotransmitter receptors. Synaptophysin-positive dots were found surrounding somata and dendrites of transplanted neurons, suggesting the presence of presynaptic terminals adjacent to their membranes. Conclusion NPCs derived from ESCs can differentiate into excitatory and inhibitory neurons after grafted into Alzheimer's disease model rats, and maybe form synapse with the host neurons.
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Aim To study the effects of agmatine on the proliferation ability of neural progenitor cells from neonatal rat hippocampus.Methods Hippocampus of neonatal rat was isolated and made into single-cell suspension,which was cultured in serum-free medium and then using CCK-8 kit assay,the survival rate of neurospheres incubated with various concentrations of agmatine and efaroxan.Furthermore,the effect of these drugs was measured with()~3H-thymidine incorporation assay.Results Cells we cultured could continuously proliferate and cultured as floating neurospheres.CCK-8 kit assay revealed that agmatine 1 ?mol?L~(-1) and 10 ?mol?L~(-1) enhanced the survival rate of neural stem cell,and the result alike with()~3H-thymidine incorporation assay.Efaroxan 10 ?mol?L~(-1) inhibit proliferation effect of agmatine on neural stem cell.Conclusion Agmatine was found to increase the proliferation of neural stem cell and efaroxan can block the proliferation effect.It suggested that imidazolineⅠ receptor may be related to the proliferation effect.