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SUMMARY: Despite comprehensive studies and reports about the properties of dental pulp stem cells (DPSCs) in vitro, we still need to confirm whether these in vitro characteristics coincide with the nature of DPSCs in situ. The anatomical location of DPSCs populations in the dental pulp has yet to be investigated. Moreover, the mesenchymal DPSCs have been much more studied than the neural crest-derived DPSCs. In this study, well-recognized neural/neural crest stem cell markers NCAM1, Nestin, SNAIL/SLUG, SOX9, and S100 are being investigated by immunohistochemistry to localize the precise location of these populations of DPSCs within the human adult dental pulp.All previously mentioned markers were expressed in the dental pulp, and their intensity and location of expression were reported.
A pesar de estudios e informes exhaustivos sobre las propiedades de las células madre de la pulpa dental (DPSC) in vitro, todavía necesitamos confirmar si estas características in vitro coinciden con la naturaleza de las DPSC in situ. La ubicación anatómica de las poblaciones de DPSC en la pulpa dental aún no se ha investigado. Además, las DPSC mesenquimales han sido mucho más estudiadas que las DPSC derivadas de la cresta neural. En este estudio, se están investigando mediante inmunohisto química marcadores de células madre de la cresta neural/ neural NCAM1, Nestin, SNAIL/SLUG, SOX9 y S100 para localizar la ubicación precisa de estas poblaciones de DPSC dentro de la pulpa dental humana adulta. Todos los marcadores mencionados anteriormente se expresaron en la pulpa dental y se informó su intensidad y ubicación de expresión.
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Humans , Adolescent , Young Adult , Stem Cells/metabolism , Dental Pulp/cytology , Neural Crest/cytology , Immunohistochemistry , S100 Proteins , CD56 Antigen , SOX9 Transcription Factor , NestinABSTRACT
Objective To investigate the effect of Mysm1 on the differentiation of neural stem cells(NSCs)into astrocytes and the possible mechanism.Methods NSCs were prepared from E12.5 cortices of wild-type C57BL/6 mice,cultured in vitro and induced to differentiate into astrocytes.Immunofluorescence staining,real-time quantitative PCR and Western blot assay were used to detect the expressions of Mysm1 during the differentiation of NSCs into astrocytes in vitro.Lentivirus was used to knock down Mysm1 expressions in NSCs before real-time quantitative PCR and Western blot assay were used to detect the knockdown efficiency.Immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after Mysm1 knockdown in vitro.Transcriptomics was adopted to detect the differential gene after knockdown of Mysm1 in NSCs in vitro.Western blot assay was used to verify the changes of proteins associated with the differential gene.Cut-Tag was used to detect the enrichment of Mysm1 in the promoter region of glial fibrillary acidic protein(GFAP)genes during the differentiation of NSCs into astrocytes in vitro.After overexpression of GFAP following knockdown of Mysm1,immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after overexpression in vitro.Results The expression of Mysm1 was gradually increased when NSCs were induced to differentiate into astrocytes in vitro.Mysm1 knockdown inhibited the differentiation of NSCs into astrocytes in vitro.Mysm1 affected the differentiation of NSCs into astrocytes by regulating the expression of GFAP.Overexpression of GFAP after Mysm1 knockdown partially rescued the ability of NSCs to differentiate into astrocytes.Conclusion Mysm1 regulates the differentiation of NSCs into astrocytes by epigenetically controlling GFAP transcription.
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Neonatal brain injury refers to the pathological damage of brain tissue caused by various factors in the perinatal period.The most common disorders are hypoxic-ischemic encephalopathy, periventricular-intraventricular hemorrhage, and periventricular leukomalacia.After a severe brain injury, the repair and reconstruction of the central nervous system (CNS) is crucial in restoring CNS architecture and function.The studies have shown that neural stem cells (NSC) have the potential for multidirectional differentiation and the ability to maintain self-renewal.Endogenous neural stem cells (eNSC) can proliferate, migrate to the lesion sites and finally differentiate into astrocytes, oligodendrocytes, and neurons, to provide new options for the treatment of neural regeneration.This paper aims to review the recent progress of eNSC in treating neonatal brain injury.
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Objective:To study the role of SUMOylation in the process of therapeutic hypothermia on neural stem cells (NSCs) in neonatal hypoxic-ischemic encephalopathy.Methods:SUMOylation is an essential post-translational modification involving small ubiquitin-like modifiers (SUMOs). Primary-cultured NSCs from mice were assigned into four groups: control group, hypoxia group, hypothermia group and hypoxia+hypothermia group. Western Blot was used to detect the protein levels of SUMO2/3, hypoxia-inducible factor-1α (HIF-1α), peroxisome proliferator-activated receptor γ coactivator factor 1α (PGC-1α) and octamer binding transcription factor 4 (Oct4). The diameters of NSCs were compared. ELISA was used to detect lactate dehydrogenase (LDH) level. Apoptosis was examined using flow cytometry. Immunofluorescence method was used to measure the differentiation of NSCs into neuronal cells.Results:Compared with the control group, the levels of SUMO2/3, HIF-1αand PGC-1α in NSCs of the hypoxia group increased 33%, 126% and 140%, respectively ( P<0.05). Compared with the control group, the levels of SUMO2/3 and PGC-1α in NSCs of the hypothermia group increased 52% and 536%, respectively ( P<0.05). Compared with the hypoxia group, the levels of SUMO2/3, HIF-1α, PGC-1α and Oct4 in the hypoxia+hypothermia group increased 44%, 40%, 230% and 59%, respectively ( P<0.05). The diameters of NSCs in hypoxia group, hypothermia group and hypoxia+hypothermia group were smaller than control group, and hypoxia+hypothermia group smaller than hypoxia group ( P<0.05). No significant differences existed in LDH levels between hypothermia group and control group ( P>0.05). LDH level in hypoxia+hypothermia group were significantly lower than hypoxia group ( P<0.05). No significant differences existed in the cell death rates between hypothermia group and control group ( P>0.05). The cell death rate in hypoxia+hypothermia group was significantly lower than hypoxia group ( P<0.05). Compared with the control group, the expressions of Nestin in both hypoxia group and hypothermia group were increased, but neuron specific enolase (NSE) were decreased ( P<0.05). Compared with hypoxia group and hypothermia group, the level of Nestin in hypoxia+hypothermia group was further increased, while NSE was further decreased ( P<0.05). Conclusions:Therapeutic hypothermia may increase the tolerance of NSCs to hypoxia by enhancing SUMO modification of proteins, providing theoretical basis for the treatment of hypoxic-ischemic encephalopathy with therapeutic hypothermia.
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Objective:To investigate the effects of thermobaric charge explosion simulated gas on long-term neurobehavior and hippocampal neurogenesis in rats.Methods:A total of 48 male SPF grade SD rats aged 8-10 weeks were randomly divided into control group, 5 min exposure group, 10 min exposure group and 15 min exposure group, with 12 rats in each group. Twenty-eight days after inhalation of infection, the anxiety-like behavior of rats was evaluated by an elevated cross maze, and the learning and memory function of rats was evaluated by two-way active avoidance experiment. The number of positive cells of rat hippocampal dentate gyrus neural stem cells marker molecule neural epithelial cell protein (SOX2) and mature neuron marker molecular neuronal nuclei (NeuN) was detected by immunofluorescence staining. Western blot was used to detect SOX2 and NeuN protein expression in the hippocampal tissues of rats. GraphPad prism 8.0 software was used for data analysis.The comparison of repeated measurement design data was carried out by repeated measurement ANOVA.One-way ANOVA was used for inter group comparisons, and Tukey test was used for pairwise comparison. Hippocampal nerve cells were counted using the Image J software.Results:(1) The experimental results of the elevated cross maze showed that the percentage of arm opening and the percentage of open arm residence time in each group had significant group effects ( F=22.31, 5.43, all P<0.05). The percentage of open arm entry times of rats in the 5 min, 10 min and 15 min exposure group ((28.85±1.47)%, (15.04±4.69)%, (12.66±2.89)%) and the percentage of residence time in open arm ((12.12±2.64)%, (12.16±1.11)%, (8.73±3.52)%) were all lower than those of the control group ((65.40±1.86)%, (42.92±3.12)%) (all P<0.05). There were no statistically significant differences in pairwise comparison among the three exposure groups (all P>0.05). (2)During the memory acquisition period, the results of repeated-ANOVA showed that the time main effect ( F=56.46), the group main effect ( F=16.64) and the interaction effect had significant differences( F=4.21)(all P<0. 05). The difference values of active avoidance number between the 4th day and 1st day among the four groups were significant different ( F=68.63, P<0.05). During the memory reproduction period, there were significant differences in active avoidance number and active avoidance time among the four groups ( F=8.17, 8.28, both P<0.05). The active avoidance numbers in 10 min and 15 min exposure groups((2.50±0.26) times, (2.33±0.06) times)were significantly lower than those in the control group ((8.33±3.72) times) (both P<0.05), and the active avoidance time ((6.25±0.40)s, (6.61±1.63)s) were significantly higher than those in the control group((3.69±1.41)s) (both P<0.05). The active avoidance numbers in 10 min and 15 min exposure groups were significantly lower than that in 5 min exposure group (both P<0.05). (3) The results of immunofluorescence staining showed that the numbers of SOX2-positive cells in the four groups were statistically significant ( F=5.33, P<0.05). The SOX2-positive cells in 15 min exposure group (4.33±1.12) was significantly lower than that in control group (7.67±1.52) ( P<0.05). The numbers of NeuN-positive cells in the four groups were significantly different ( F=11.06, P<0.05), and the NeuN-positive cells in the 10 min and 15 min exposure groups((105.67±8.50), (88.33±9.50)) were significantly lower than that in the control group (127.00±6.56) ( P<0.05). The NeuN-positive cells in 15 min exposure group were significantly lower than that in 5 min exposure group (110.67±8.32) ( P<0.05). (4) Western blot results showed that the relative expression of SOX2 and NeuN proteins in the four groups was statistically significant ( F=11.560, 7.035, both P<0.05). The relative expression of SOX2 and NeuN proteins in the 15 min exposure group were significantly lower than those in control group (both P<0.05). The relative expression of SOX2 protein in 15 min exposure group was significantly lower than that in 5 min exposure group ( P<0.05). Conclusion:Acute exposure to warm pressure charge explosion simulated gas can lead to anxiety-like behavior, learning and memory deficits in rats, and significantly reduce the protein expression levels of hippocampal dentate gyrus neural stem cells and mature neuronal marker molecules SOX2 and NeuN.
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Aim To study the ability of tetramethylpyrazine (TMP) on promoting neurogenesis in neural stem cell microenvironment after oxygen-glucose deprivation (OGD) injury in vitro. Methods Neural stem cells (NSCs), astrocytes (ACs) and cerebral microvascular endothelial cells (BMECs) were respectively extracted and separated to establish a co-culture system. The OGD modeling conditions were optimized by NSCs activity, and the concentration of TMP was optimized by Nissl staining. Then CCK-8 and Nestin
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[Abstract] Objective To investigate the effect of 6-gingerol treatment on cognitive behavior after hypoxic-ischemic brain injury (HIE) in neonatal mice, and to explore the protective mechanism of 6-gingerol on HIE brain injury in neonatal mice by observing the effects on neuronal survival and neural stem cell proliferation. Methods The right common carotid artery was ligated in Kunming mice (78) on the 7th day after birth and HIE model was established after 90 minutes of hypoxic treatment. 6-gingerol was injected intraperitoneally. The cognitive behavior was detected by Morris water maze test; 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used to observe the changes of brain injury; The changes of synaptic structure and number were obseved by transmission electron microscopy; HE staining, Nissl staining and dihydroethidium(DHE) staining were used to observe the pathomorphological changes of hippocampus in each group; The proliferation of neural stem cells and the expression of related transcription factors were detected by immunofluorescence and Real-time PCR; The changes of Akt signal pathway were detected by Western blotting. Results 6-gingerol treatment could improve the long-term learning and memory ability, reduce the brain injury and brain edema of neonatal mice after HIE, and improve synaptic plasticity of mice after HIE. In the 6-gingerol treatment group, the disorder of hippocampal cells in the diseased side of HIE was improved, the number of necrotic cells decreased, the proliferation ability of hippocampal neural stem cells and the expression levels of nestin and sex determining region box transcription factor 2 (Sox2) related transcription factors increased significantly, and the level of phosphorylated Akt (p-Akt) increased. Conclusion It is found that 6-gingerol can improve the learning and memory ability of HIE mice in adulthood and reduce brain tissue injury after HIE. 6-gingerol may play a role in inhibiting the production of reactive oxygen species(ROS), reducing neuronal injury and upregulating the expression of Akt signal pathway, promoting the proliferation of hippocampal neural stem cells, so as to provide potential drugs for the treatment of neonatal HIE.
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OBJECTIVE To study the improvement effects and mechanisms of Tujia medicine musk needle therapy on cognitive dysfunction in ischemic stroke model rats. METHODS Totally 44 rats were randomly divided into sham operation group, model group, musk needle treatment group and ordinary acupuncture group, with 11 rats in each group. Except for the sham operation group, ischemic stroke model was induced by modified suture method in other groups. After modeling, musk needle treatment group and the ordinary acupuncture group were treated with Tujia musk needle (containing 3 mg of artificial musk) and traditional filiform needle respectively to intervene in the muscle layer of the contralateral scalp motor area, with an intervention duration of 3 courses. The sham operation group and model group were not given any treatment. The neurological deficits score in rats were recorded and Morris water maze behavioral tests were conducted. The morphology of neurons in the cortical area of rats was observed, and the expression of DCX/BrdU and NeuN/BrdU co-labeled cells in the ischemic subependymal area was observed. The plasma levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor(VEGF) in rats were tested. RESULTS Compared with sham operation group, neurological deficit score of model group was increased significantly, escape latency prolonged significantly, and the times of crossing platform significantly reduced (P<0.05); the neuronal structure was significantly damaged, and the number of surrounding Nissl bodies decreased; the number of DCX/BrdU and NeuN/BrdU co- labeled cells in the ischemic subependymal area were significantly increased (P<0.05); the levels of HIF-1α and VEGF in plasma were significantly increased (P<0.05). Compared with model group, neurological deficits score, escape latency, the times of crossing platform were all reversed significantly in musk needle treatment group and ordinary acupuncture group (P<0.05); the neuronal structure was improved, and the number of Nissl bodies increased; the number of DCX/BrdU and NeuN/BrdU co-labeled cells in the ischemic subependymal area were significantly increased (P<0.05); the plasma levels of HIF-1α and VEGF were significantly increased (P<0.05). Compared with ordinary acupuncture group, the plasma level of HIF-1α was reduced (the difference was not statistically significant), while the level of VEGF was significantly increased (P<0.05). CONCLUSIONS Tujia medicine musk needle therapy can significantly improve the cognitive dysfunction in ischemic stroke model rats, and its mechanism of action may be associated with promoting migration and differentiation of neural stem cell in ischemic subependymal area, preventing the excessive release of HIF-1α and increasing the expression of VEGF.
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OBJECTIVE@#To investigate the effect of Zuogui Jiangtang Jieyu Decoction (ZJJ) on Shh signaling and self-renewal of neural stem cells in the hippocampal dentate gyrus of diabetic rats with depression.@*METHODS@#Diabetic rat models with depression were randomly divided into model group, positive drug (metformin + fluoxetine) group, and low-, medium-, and high-dose ZJJ groups (n=16), with normal SD rats as the control group. The positive drugs and ZJJ were administered by gavage, and the rats in the control and model groups were given distilled water. After the treatment, blood glucose level was detected using test strips, and behavioral changes of the rats were assessed by forced swimming test and water maze test. ELISA was used to examine the serum level of leptin; The expressions of nestin and Brdu proteins in the dentate gyrus of the rats were detected using immunofluorescence assay, and the expressions of self-renewal marker proteins and Shh signaling proteins were detected using Western blotting.@*RESULTS@#The diabetic rats with depression showed significantly increased levels of blood glucose and leptin (P < 0.01) and prolonged immobility time in forced swimming test (P < 0.01) and increased stage climbing time with reduced stage seeking time and stage crossings in water maze test (P < 0.01). The expressions of nestin and Brdu in the dentate gyrus, the expressions of cyclin D1, SOX2, Shh, Ptch1, Smo in the hippocampus and the nuclear expression of Gli-1 were decreased (P < 0.01) while hippocampal Gli-3 expression was increased significantly (P < 0.01) in the rat models. Treatment of rat models with high-dose ZJJ significantly reduced the blood glucose (P < 0.01) and leptin level (P < 0.05) and improved their performance in behavioral tests (P < 0.01). The treatment also obviously increased the expressions of nestin, Brdu, cyclin D1, SOX2, Shh, Ptch1, and Smo and the nuclear expression of Gli-1 in the dentate gyrus (P < 0.01) and reduced hippocampal expression of Gli-3 (P < 0.05) in the rat models.@*CONCLUSION@#ZJJ can significantly improve the self-renewal ability of neural stem cells and activate Shh signaling in dentate gyrus of diabetic rats with depression.
Subject(s)
Animals , Rats , Blood Glucose , Bromodeoxyuridine , Cell Self Renewal , Cyclin D1 , Dentate Gyrus , Depression , Diabetes Mellitus, Experimental , Hippocampus , Leptin , Nestin , Rats, Sprague-DawleyABSTRACT
Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.
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Animals , Mice , DNA-Binding Proteins/genetics , Cell Differentiation , Neural Stem Cells , Translocation, Genetic , Ubiquitins/genetics , Ligases/geneticsABSTRACT
Neurogenesis decline in hippocampal dentate gyrus (DG) participates in stress-induced depressive-like behaviors, but the underlying mechanism remains poorly understood. Here, we observed low-expression of NOD-like receptor family pyrin domain containing 6 (NLRP6) in hippocampus of stress-stimulated mice, being consistent with high corticosterone level. NLRP6 was found to be abundantly expressed in neural stem cells (NSCs) of DG. Both Nlrp6 knockout (Nlrp6-/-) and NSC-conditional Nlrp6 knockout (Nlrp6CKO) mice were susceptible to stress, being more likely to develop depressive-like behaviors. Interestingly, NLRP6 was required for NSC proliferation in sustaining hippocampal neurogenesis and reinforcing stress resilience during growing up. Nlrp6 deficiency promoted esophageal cancer-related gene 4 (ECRG4) expression and caused mitochondrial dysfunction. Corticosterone as a stress factor significantly down-regulated NLRP6 expression, damaged mitochondrial function and suppressed cell proliferation in NSCs, which were blocked by Nlrp6 overexpression. ECRG4 knockdown reversed corticosterone-induced NSC mitochondrial function and cell proliferation disorders. Pioglitazone, a well-known clinical drug, up-regulated NLRP6 expression to inhibit ECRG4 expression in its protection against corticosterone-induced NSC mitochondrial dysfunction and proliferation restriction. In conclusion, this study demonstrates that NLRP6 is essential to maintain mitochondrial homeostasis and proliferation in NSCs, and identifies NLRP6 as a promising therapeutic target for hippocampal neurogenesis decline linked to depression.
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Intense stimulation of most living cells triggers the activation of immediate early genes, such as Fos and Jun families. These genes are important in cellular and biochemical processes, such as mitosis and cell death. The present study focused on determining the temporal expression pattern of Fos and Jun families in fibroblasts and neural stem cells of cerebellum, hippocampus, and subventricular zone (SVZ) of rats of different ages at 0, 0.5, 1, 3, and 6 h after stimulation with fibroblast growth factor (FGF)-2. In neonates, a similar expression pattern was observed in all cells analyzed, with lower expression in basal condition, peak expression at 0.5 h after stimulation, returning to baseline values between 1 and 3 h after stimulation. On the other hand, cells from adult animals only showed Fra1 and JunD expression after stimulation. In fibroblasts and hippocampus, Fra1 reached peak expression at 0.5 h after stimulation, while in the SVZ, peak level was observed at 6 h after stimulation. JunD in fibroblasts presented two peak expressions, at 0.5 and 6 h after stimulation. Between these periods, the expression observed was at a basal level. Nevertheless, JunD expression in SVZ and hippocampus was low and without significant changes after stimulation. Differences in mRNA expression in neonate and adult animals characterize the significant differences in neurogenesis and cell response to stimulation at different stages of development. Characterizing these differences might be important for the development of cell cultures, replacement therapy, and the understanding of the physiological response profile of different cell types.
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Objectives: Investigating novel therapies for alcoholic encephalopathy (AE) would be part of the implementation of the concept of targeted pharmacological control of intracellular signalisation in regeneration-competent cells. This study aimed to explore the involvement of JNK and p53 in the implementation of the functions of different types of regeneration-competent cells of nervous tissue in alcoholic neurodegeneration (AN). Materials and Methods: The studies were conducted on C57B1/6 mice. AN was modelled in vitro and in vivo. The effects of the JNK and p53 inhibitors on the realisation of neural stem cell (NSC) and neuronal-committed progenitor (NCP) functions (their colony-forming ability, proliferative activity and intensity of specialisation), as well as on the secretion of neurotrophins by astrocytes, oligodendrocytes and microglial cells were studied. Individual cell fractions were prepared using an immunomagnetic separation method. Results: We showed that JNK and p53 stimulate the proliferation and specialisation of intact NSCs. An inversion of the role of these signalling molecules in the regulation of NSC proliferation in the conditions of modelling AN was revealed. It has been found that JNK and p53 are not involved in regulating the functions of NCP. The ambiguous role of JNK and p53 in the production of neurotrophic growth factors by different types of neuroglia cells was also found. Increased secretion of neurotrophins by oligodendrocytes and microglia during the blockade of JNK and p53 under conditions of exposure to ethanol cells was revealed. Conclusion: The results suggest the prospect of exploring the possibility of using JNK and/or p53 inhibitors as novel drugs to treat AE.
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Chronic stress impairs radial neural stem cell (rNSC) differentiation and adult hippocampal neurogenesis (AHN), whereas promoting AHN can increase stress resilience against depression. Therefore, investigating the mechanism of neural differentiation and AHN is of great importance for developing antidepressant drugs. The nonpsychoactive phytocannabinoid cannabidiol (CBD) has been shown to be effective against depression. However, whether CBD can modulate rNSC differentiation and hippocampal neurogenesis is unknown. Here, by using the chronic restraint stress (CRS) mouse model, we showed that hippocampal rNSCs mostly differentiated into astrocytes under stress conditions. Moreover, transcriptome analysis revealed that the FoxO signaling pathway was involved in the regulation of this process. The administration of CBD rescued depressive-like symptoms in CRS mice and prevented rNSCs overactivation and differentiation into astrocyte, which was partly mediated by the modulation of the FoxO signaling pathway. These results revealed a previously unknown neural mechanism for neural differentiation and AHN in depression and provided mechanistic insights into the antidepressive effects of CBD.
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Animals , Humans , Mice , Cannabidiol/pharmacology , Cell Differentiation , Depression/prevention & control , Hippocampus/metabolism , Neural Stem Cells , Neurogenesis/physiologyABSTRACT
Aim To explore and optimize the different separation and purification methods of neural stem cells (NSCs) , and compare the cell viability and purity after separation and purification.Methods (X) Separation of NSCs: The brains of ICR mice born within 24 h were isolated ( the cerebellum and olfactory bulb removed) , chopped and digested by mechanical pipetting, trypsin digestion, and PDD enzyme digestion.After sieving and centrifugation, the brain tissues were inoculated at a concentration of 1 X 10 • L , and cultured in serum-free medium to observe and compare the activity, proliferation and number of miscellaneous cells.(2) Purification of NSCs: The brain tissues of mice born within 24 h were isolated, and NSCs were purified by differential centrifugation and sieving, and the purity was identi-fied by immunofluorescence.Results NSCs separated by PDD enzyme digestion had high survival rate and rapid proliferation rate; the Nestin positive rate of NSCs in both Sieving assay and Differential centrifugation assay groups was higher than that of primary NSCs (59.14% ± 0.16% , P <0.05) , and the former (93.54% ± 0.02%) was higher than the latter (86.12% ± 0.04% ) .Conclusions The three separation methods can obtain a large amount of NSCs.Among them, the PDD enzymatic digestion method has little damage to the NSCs, and the neuro-sphere proliferation speed is fast; the NSCs obtained by the sieving assay have higher purity.
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Objective:To investigate the role of subventricular zone (SVZ) irradiation in the prognosis of patients with glioblastoma (GBM), and to analyze the factors affecting the prognosis of patients with GBM.Methods:Clinical data of 52 patients with GBM treated in the Affiliated Cancer Hospital of Nanjing Medical University from 2017 to 2020 were analyzed retrospectively. According to the median dose of ipsilateral or contralateral SVZ, the patients were divided into the high-dose group and low-dose group. The prognostic differences between two groups were compared and the prognostic factors were analyzed.Results:The median progression-free survival (PFS) was 17.1 months (95% CI:12.4-30.7)and the median overall survival (OS) was 38.3 months (95% CI:20.4-44.5). Univariate analysis showed that whether the tumor invading SVZ ( P = 0.039), the degree of resection ( P = 0.009) and MGMT promoter methylation status ( P = 0.039) were the influencing factors of PFS. Age ( P = 0.018), Kanofsky performance score ( P = 0.043), whether the tumor invading SVZ ( P = 0.038), degree of resection ( P = 0.020) and MGMT promoter methylation status ( P = 0.019) were the influencing factors of OS. The analysis of SVZ dose as a continuous variable showed that SVZ dose was the influencing factor of PFS ( P < 0.05) rather than OS ( P ≥ 0.05). Whether the tumor invading SVZ or not, there was no significant difference in survival between the high-dose and low-dose groups. Multivariate analysis showed that whether the tumor invading SVZ and MGMT promoter methylation were the independent prognostic factor for PFS (both P < 0.05), and OS (both P < 0.05). The SVZ dose related variables were not statistically significant in multivariate analysis. Conclusions:Patients with tumors directly invading SVZ achieve worse survival. Increasing the ipsilateral or contralateral SVZ dose does not improve patient survival. Whether SVZ irradiation affects the survival of patients still needs to be further confirmed by prospective randomized clinical studies.
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Objective:To explore the effects of Chaihu-Shugan San (CSS) on the behavior and neurogenesis function of depression model mice induced by chronic unpredictable mild stress (CUMS).Methods:Thirty clean grade healthy male C57BL/6 adult mice were randomly divided into control group (Con group), model group (CUMS group) and Chaihu-Shugan San treatment group (CSS group), with 10 mice in each group.The mice in CUMS group and CSS group were given CUMS intervention to establish depression model. At the same time of modeling, the mice in CUMS group and CSS group were given distilled water and CSS(2.7 g/kg) by gavage respectively.While the mice in Con group were only given equal volume distilled water by gavage without CUMS stimulation.After the intervention, the depressive-like behavior of mice was evaluated by increased body weight, sugar water preference test (SPT), forced swimming test (FST) and tail suspension test (TST). The number of newborn neurons was detected by immunofluorescence staining. The mRNA expression levels of brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2) and spindle and kinetochore-associated protein 2(SKA2) in mice hippocampus were detected by qRT-PCR.Statistical analysis was performed by SPSS 22.0 software. One-way ANOVA was used for multi group comparison, and Tukey test was used for pairwise comparison.Results:(1) After modeling, there was significant difference in body weight increment among the three groups ( F=8.859, P <0.05). The body weight increment of CUMS group was lower than those of Con group and CSS group (both P< 0.05). There were significant differences in sugar water preference rate, tail suspension immobility time and swimming immobility time among the three groups ( F=10.544, 12.957, 8.095, all P<0.05). The sugar water preference rate in CUMS group was lower than that in Con group ((87.46±2.78)%, (93.90±3.31)%, P<0.05), and that in CSS group was higher than that in CUMS group ((91.65±2.61)%)( P<0.05). The tail suspension immobility time ((198.00±27.57) s) and swimming immobility time ((322.20±46.98) s) in CUMS group were higher than those in Con group ((138.80±38.50) s, (238.50±50.51) s, both P<0.05). The tail suspension immobility time ((139.00±21.29) s) and swimming immobility time ((265.20±44.90) s) in CSS group were lower than those in CUMS group (both P<0.05). (2) Immunofluorescence showed that there was significant difference in the number of newborn neurons labeled by BrdU and NeuN in the dentate gyrus of hippocampus among the three groups ( F=9.486, P<0.05). The number of double labeled cells (31.66±3.21) in CUMS group was lower than that in Con group(63.66±15.17) and CSS group (58.00±6.00) (both P<0.05). (3) RT-PCR results showed that the mRNA levels of BDNF, FGF2, SKA2 in hippocampal dentate gyrus of the three group were significantly different( F=14.522, 9.337, 8.701, all P<0.05). The levels of BDNF mRNA (0.79±0.06), FGF2 mRNA (0.74±0.18) and SKA2 mRNA (0.52±0.32) in the dentate gyrus of hippocampus in CUMS group were lower than those in Con group (BDNF mRNA (1.03±0.10), FGF2 mRNA (1.04±0.11), SKA2 mRNA (1.05±0.37), all P<0.05). Compared with CUMS group, the mRNA levels of BDNF (1.07±0.80), FGF2 (1.30±0.29) and SKA2 (1.40±0.55) in CSS group were higher (all P<0.05). Conclusion:CSS can alleviate the depressive like behavior of depression model mice, which may be related with increasing the mRNA expression levels of BDNF, FGF2, SKA2 and promoting the proliferation of neural stem cells in hippocampus.
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Objective:To evaluate the effect of propofol on proliferation of neural stem cells (NSCs) in mice and the role of specificity protein-1 (Sp-1)-epidermal growth factor receptor (EGFR)-protein kinase B (Akt) signaling pathway.Methods:Primary NSCs harvested from both the cortices and hippocampus of C57BL/6 mouse embryos were identified by immunofluorescent staining of Nestin.NSCs at passages 3-6 were divided into 3 groups ( n=21 each) using a random number table method: normal saline control group (C group), propofol group (P group) and propofol plus Sp1 inhibitor plicamycin group (PP group). Propofol at a final concentration of 10 μmol/L was added in group P. Propofol at a final concentration of 10 μmol/L and plicamycin at a final concentration of 100 nmol/L were added in group PP.The equal volume of normal saline was added in group C. The medium was replaced after 6 h of incubation and the cells were continuously incubated.The proliferation of NSCs was assessed by direct cell counting at 24, 36, 48, 60 and 72 h after the end of treatment with drugs.At 6 h after the end of treatment with drugs, the expression of Sp1 and EGFR mRNA was detected by real-time fluorescent quantitative polymerase chain reaction, and the expression of Sp1, Akt and phosphorylated Akt (p-Akt) by Western blot. Results:Compared with group C, the count of NSCs was significantly increased at 48, 60 and 72 h after treatment with drugs, and the expression of EGFR mRNA, Sp1 protein and mRNA and p-Akt was up-regulated in group P ( P<0.05 or 0.01), and no significant change was found in each parameter in group PP ( P>0.05). Compared with group P, the count of NSCs was significantly decreased at 48 and 60 h after treatment with drugs, and the expression of EGFR protein and mRNA and p-Akt was down-regulated in group PP ( P<0.05 or 0.01). Conclusions:Propofol can promote the proliferation of NSCs, and the mechanism may be related to activation of Sp1-EGFR-Akt signaling pathway in mice.
ABSTRACT
Objective:To observe the adhesion, growth and differentiation of rat neural stem cells (NSCs) on spinal cord acellular scaffold (SCAS) to evaluate its feasibility for spinal cord tissue engineering. Methods:NSCs derived from neonatal Sprague-Dawley rat cerebral cortex were cultured and identified. SCAS were prepared from female Sprague-Dawley rat spinal cord tissues using modified chemical extraction and physical oscillation, and evaluated. The third generation NSCs were planted on SCAS and co-cultured, the morphology of the cells on the scaffold was observed with immunofluorescence, immunohistochemistry and scanning electron microscope. Results:The cultured cells were NSCs, which could proliferate and differentiate. The porosity, water content and enzymatic hydrolysis rates of the prepared SCAS were significantly higher than that of normal spinal cord (|t| > 4.679, P < 0.01). The matrix structure of SCAS was loosely network-like, with few residual nuclei. NSCs adhered and grew well, and differentiated into neurons and glial cells on SCAS. Conclusion:This kind of SCAS shapes multi-channel spatial structure and is suitable for NSCs adhesion, growth and differentiation, which can be used for spinal cord tissue engineering.
ABSTRACT
Objective:To investigate the effect of Buyang Huanwutang (BHT) on proliferation and differentiation in neural stem cells (NSCs) after oxygen-glucose deprivation/reoxygenation (OGD/R) injury. Method:NSCs isolated from the hippocampus of SD rats were cultured and randomly divided into a normoxia group, a model group, a BHT group, a rapamycin (Rapa) group, and a combination group [autophagy inhibitor 3-methyladenine (3-MA) combined with BHT]. The 20% blank serum was used in the normoxia group, and 20% BHT-medicated serum in the BHT group. The doses of Rapa and 3-MA were 1 μmol·L<sup>-1</sup> and 5 mmol·L<sup>-1</sup>, respectively. The cells were subjected to OGD/R except those in the normoxia group. The cell morphology was observed under a light microscope. NSCs were confirmed by immunofluorescence detection of nestin expression. The viability and proliferation of NSCs were assessed by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2-deoxyuridine (EdU) labeling, respectively. Furthermore, Ad-mCherry-GFP-LC3B fluorescence assay was performed to investigate autophagy. The effect of BHT on autophagy-related protein expression was detected by western blot assay. Brain derived neurotrophic factor (BDNF), <italic>β</italic>-tubulin Ⅲ, and glial fibrillary acidic protein (GFAP) were evaluated by immunofluorescence assay. Result:OGD/R significantly reduced the cell viability of rat NSCs as compared with the normoxia group. Compared with the model group, the BHT group exhibited significantly improved viability of rat NSCs (<italic>P</italic><0.01). BHT induced the production of autophagosomes in NSCs after OGD. The BHT group showed increased expression of microtuble-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) and Beclin-1 (<italic>P</italic><0.05,<italic>P</italic><0.01) and slightly changed p62 compared with the normoxia group, and significantly up-regulated LC3Ⅱ and Beclin-1 (<italic>P</italic><0.05,<italic>P</italic><0.01) and down-regulated expression of p62 (<italic>P</italic><0.01) compared with the model group. The Rapa group had similar effect as the BHT group (<italic>P</italic><0.05,<italic>P</italic><0.01), while the combination group inhibited the activity of autophagy (<italic>P</italic><0.01). As indicated by the results of ad-mCherry-GFP-LC3B, compared with the normoxia group, the model group showed increased fluorescence intensity (<italic>P</italic><0.01), and the BHT and Rapa groups could further increased the fluorescence intensity of autophagy (<italic>P</italic><0.01), while the combination group inhibited autophagy activity (<italic>P</italic><0.01). Immunofluorescence results revealed that compared with the normoxia group, the model group displayed significantly reduced positive cells of EdU, <italic>β</italic>-tubulin Ⅲ, GFAP, and BDNF (<italic>P</italic><0.01), and the BHT and Rapa groups exerted similar protective and promoting effects (<italic>P</italic><0.05,<italic>P</italic><0.01), while the combination group partially blocked the neuroprotection and differentiation ability of BHT (<italic>P</italic><0.05). Conclusion:BHT pretreatment can effectively protect rat NSCs against OGD-induced injury and promoted proliferation and differentiation by up-regulating autophagy.