Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mic...Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mice were used to examine the lineage differentiation of SOX2-positive cells during the development of cerebral cortex.Primary NSCs/astrocytes culture,ChIP-seq and Western Blot were adopted to analyze and verify the expression of candidate genes.Pharmacological manipulation,neurosphere formation,photochemical ischemia,immunofluorescence staining and behavior tests were adopted to evaluate the effects of activating DRD2 signaling on astrocytic dedifferentiation.Results:Immunofluorescence staining demonstrated the NSC-astrocyte switch of SOX2-expression in the normal development of cerebral cortex.ChIP-seq revealed enrichment of DRD2 signaling by SOX2-bound enhancers in NSCs and SOX2-bound promoters in astrocytes.Western Blot and immunofluorescence staining verified the expression of DRD2 in NSCs and reactive astrocytes.Application of quinagolide hydrocholoride(QH),an agonist of DRD2,significantly promoted astrocytic dedifferentiation both in vitro and in vivo following ischemia.In addition,quinagolide hydrocholoride treatment improved locomotion recovery.Conclusion:Activating DRD2 signaling facilitates astrocytic dedifferentiation and may be used to treat ischemic stroke.展开更多
[Objective] This study aimed to reduce the content of cell wall materials in tobacco stems and improve their internal quality. [Method] Pectinase was used to decompose cell wall materials in tobacco stems, followed by...[Objective] This study aimed to reduce the content of cell wall materials in tobacco stems and improve their internal quality. [Method] Pectinase was used to decompose cell wall materials in tobacco stems, followed by determination of the contents of four cell wall materials, six routine chemical components, as well as aroma constituents. [Result] Pectinase could effectively reduce the contents of cell wall materials in tobacco stems, with the largest decrease of 6.84%; after pectinase treatment,the content of reducing sugar in tobacco stems increased obviously, and the contents of total sugar, potassium ion, chloride ion and total nitrogen increased to varying degrees, of which the contents of potassium ion and reducing sugar displayed upward trends with the increase of pectinase concentration. Pectinase treatment significantly increased the contents of Maillard reaction products, with the most increase of 67.2%;the contents of carotenoid degradation products, phenylalanine degradation products and neophytadiene all increased to varying extents, and the contents of both Maillard reaction products and phenylalanine degradation products revealed ascending trends with the increase of pectinase concentration. [Conclusion] Pectinase treatment can effectively decompose cell wall materials in tobacco stems, improve routine chemical constituents, and increase the contents of aroma constituents.展开更多
HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infec...HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.展开更多
Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glio...Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.展开更多
OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrilla...OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrillary acidic protein （GFAP） was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α（RXRα）, p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity.RESULTS ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR （P〈0.05）. Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR （P〈0.05）. Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells （P〈0.05）.CONCLUSION ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRAinduced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.展开更多
Glial cell derived neurotrophic factor （GDNF） holds promises for treating neurodegenerative diseases such as Parkinson＇s dis- ease. Human neural stem cells （hNSCs） have proved to be a suitable cell delivery vehic...Glial cell derived neurotrophic factor （GDNF） holds promises for treating neurodegenerative diseases such as Parkinson＇s dis- ease. Human neural stem cells （hNSCs） have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin re- sistance gene as such vehicles. A modified tetracycline operator 7 （tetO7） was inserted into a region upstream of the EFI-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein （TTS）. TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline （Dox） the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-I positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases.展开更多
OBJECTIVE: To investigate the neural differentiation capacity of water extraction of velvet antler.METHODS: Velvet antler(Cervus Nippon Temminck) polypeptide(VAP) was used to differentiate neural stem cells(NSCs) towa...OBJECTIVE: To investigate the neural differentiation capacity of water extraction of velvet antler.METHODS: Velvet antler(Cervus Nippon Temminck) polypeptide(VAP) was used to differentiate neural stem cells(NSCs) towards neurons in the study. Firstly, we obtain the polypeptides of VAP by water extraction. Secondly, we observed the morphology, assayed the factors in the media by enzyme-linked immunosorbent assay, and detected the special neural molecules by immuno fl uorescence staining. NSCs were cultured on the cell climbing film. After neuronal differentiation, differentiated NSCs were mounted for immunocytochemistry with immunofluorescence technique.RESULTS: The differentiating cells look like neuron,some special factors, such as Glial cell line-derived neurotrophic factor, nerve growth factor, in the media can be detected while differentiated neuron-like cells can express the special neural molecules.CONCLUSION: Differentiation of NSCs towards neurons can be induced by velvet antler polypeptide.展开更多
文摘Objective:To explore the effects of dopamine receptor D2(DRD2)on astrocytic dedifferentiation based on SOX2-regulated genes in neural stem cells(NSCs)and astrocytes.Methods:Immunofluorescence staining and SOX2-GFP mice were used to examine the lineage differentiation of SOX2-positive cells during the development of cerebral cortex.Primary NSCs/astrocytes culture,ChIP-seq and Western Blot were adopted to analyze and verify the expression of candidate genes.Pharmacological manipulation,neurosphere formation,photochemical ischemia,immunofluorescence staining and behavior tests were adopted to evaluate the effects of activating DRD2 signaling on astrocytic dedifferentiation.Results:Immunofluorescence staining demonstrated the NSC-astrocyte switch of SOX2-expression in the normal development of cerebral cortex.ChIP-seq revealed enrichment of DRD2 signaling by SOX2-bound enhancers in NSCs and SOX2-bound promoters in astrocytes.Western Blot and immunofluorescence staining verified the expression of DRD2 in NSCs and reactive astrocytes.Application of quinagolide hydrocholoride(QH),an agonist of DRD2,significantly promoted astrocytic dedifferentiation both in vitro and in vivo following ischemia.In addition,quinagolide hydrocholoride treatment improved locomotion recovery.Conclusion:Activating DRD2 signaling facilitates astrocytic dedifferentiation and may be used to treat ischemic stroke.
基金Supported by the Special Fund of Hongyun Honghe Tobacco(Group)Co.,Ltd~~
文摘[Objective] This study aimed to reduce the content of cell wall materials in tobacco stems and improve their internal quality. [Method] Pectinase was used to decompose cell wall materials in tobacco stems, followed by determination of the contents of four cell wall materials, six routine chemical components, as well as aroma constituents. [Result] Pectinase could effectively reduce the contents of cell wall materials in tobacco stems, with the largest decrease of 6.84%; after pectinase treatment,the content of reducing sugar in tobacco stems increased obviously, and the contents of total sugar, potassium ion, chloride ion and total nitrogen increased to varying degrees, of which the contents of potassium ion and reducing sugar displayed upward trends with the increase of pectinase concentration. Pectinase treatment significantly increased the contents of Maillard reaction products, with the most increase of 67.2%;the contents of carotenoid degradation products, phenylalanine degradation products and neophytadiene all increased to varying extents, and the contents of both Maillard reaction products and phenylalanine degradation products revealed ascending trends with the increase of pectinase concentration. [Conclusion] Pectinase treatment can effectively decompose cell wall materials in tobacco stems, improve routine chemical constituents, and increase the contents of aroma constituents.
基金National Natural Science Foundation ofChina (30770105)Qingdao Technology Project (08-1-3-30-jch) Mt. Tai Scholar Construction Engineering Special Foundation of Shandong province, China.
文摘HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.
文摘Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.
基金the National Natural Science Foundation of China，Shandong Province Natural Science Foundation，Tianjin Natural Science Foundation
文摘OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrillary acidic protein （GFAP） was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α（RXRα）, p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity.RESULTS ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR （P〈0.05）. Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR （P〈0.05）. Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells （P〈0.05）.CONCLUSION ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRAinduced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.
基金supported by the National Basic Research Program of China (2007CB947704)the High-level Technical Personnel Training of Health Plan of Beijing
文摘Glial cell derived neurotrophic factor （GDNF） holds promises for treating neurodegenerative diseases such as Parkinson＇s dis- ease. Human neural stem cells （hNSCs） have proved to be a suitable cell delivery vehicle for the safe and efficient introduction of GDNF into the brain. In this study, we used hNSCs-infected with a lentivirus encoding GDNF and the hygromycin re- sistance gene as such vehicles. A modified tetracycline operator 7 （tetO7） was inserted into a region upstream of the EFI-α promoter to drive GDNF expression. After hygromycin selection, hNSCs were infected with a lentivirus encoding a KRAB-tetracycline repressor fusion protein （TTS）. TTS bound to tetO7 and suppressed the expression of GDNF in hNSCs. Upon administration of doxycycline （Dox） the TTS-tetO7 complex separated and the expression of GDNF resumed. The hNSCs infected with GDNF expressed the neural stem cell specific markers, nestin and sox2, and exhibited no significant change in proliferation rate. However, the rate of apoptosis in hNSCs expressing GDNF was lower compared with normal NSCs in response to actinomycin treatment. Furthermore, a higher percentage of Tuj-I positive cells were obtained from GDNF-producing NSCs under conditions that induced differentiation compared to control NSCs. The inducible expression of GDNF in hNSCs may provide a system for the controllable delivery of GDNF in patients with neurodegenerative diseases.
基金Supported by the National Natural Sciences Foundation of China(No.81470171)Basic Science Research Fund from Ministry of Finance of China(No.ZZ2012008 and ZZ2015014)
文摘OBJECTIVE: To investigate the neural differentiation capacity of water extraction of velvet antler.METHODS: Velvet antler(Cervus Nippon Temminck) polypeptide(VAP) was used to differentiate neural stem cells(NSCs) towards neurons in the study. Firstly, we obtain the polypeptides of VAP by water extraction. Secondly, we observed the morphology, assayed the factors in the media by enzyme-linked immunosorbent assay, and detected the special neural molecules by immuno fl uorescence staining. NSCs were cultured on the cell climbing film. After neuronal differentiation, differentiated NSCs were mounted for immunocytochemistry with immunofluorescence technique.RESULTS: The differentiating cells look like neuron,some special factors, such as Glial cell line-derived neurotrophic factor, nerve growth factor, in the media can be detected while differentiated neuron-like cells can express the special neural molecules.CONCLUSION: Differentiation of NSCs towards neurons can be induced by velvet antler polypeptide.
