1.Normalization of Red Cell Enolase Level Following Allogeneic Bone Marrow Transplantation in a Child with Diamond-Blackfan Anemia.
Jeong A PARK ; Yeon Jung LIM ; Hyeon Jin PARK ; Sun Young KONG ; Byung Kiu PARK ; Thad T GHIM
Journal of Korean Medical Science 2010;25(4):626-629
We describe a girl with Diamond-Blackfan anemia with accompanying red cell enolase deficiency. At the age of 9 yr old, the patient received allogeneic bone marrow transplantation from her HLA-identical sister who had normal red cell enolase activity. While the post transplant DNA analysis with short tandem repeat has continuously demonstrated a stable mixed chimerism on follow-up, the patient remains transfusion independent and continues to show a steady increase in red cell enolase activity for over two and a half years following bone marrow transplantation.
*Anemia, Diamond-Blackfan/blood/enzymology/surgery
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Bone Marrow Cells/cytology/metabolism
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*Bone Marrow Transplantation
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Child
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Child, Preschool
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Erythrocytes/*enzymology
;
Female
;
Humans
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Infant
;
Phosphopyruvate Hydratase/genetics/*metabolism
;
*Transplantation, Homologous
2.Inhibitory effects of Notch1 overexpression on proliferation and neuroendocrine marker expression in a small cell lung cancer cell line.
Jie-Xin WANG ; Xiu-ming ZHANG ; Ling-ling WANG ; Hui CHENG ; Gen-you YAO
Chinese Journal of Oncology 2009;31(5):335-339
OBJECTIVETo investigate the effects of Notch1 signal activation on proliferation and neuroendocrine marker expression in small cell lung cancer cells.
METHODSThe active form of Notch1 (NIC) was over-expressed in NCI-H446 cells by constitutive transfection and a stable transfected cell line was established. Proliferation of NCI-H446 cells was analysed by MTT assay on 6 successive days. Expression of neuroendocrine markers (CgA, NSE) was observed by immunohistochemistry and Western blot analysis. Statistical analysis was conducted to compare the results in cells with NIC transfected and those in control groups.
RESULTSMTT assay showed that absorbance (A) of cells overexpressing Notch1 was significantly depressed compared with that of the control cells (P<0.05). Immunohistochemistry of CgA showed that PUs in the NIC transfected group, sham group and negative control group were 8.81 +/- 0.77, 38.10 +/- 1.55, 38.97 +/- 0.80, respectively, the former one was significantly smaller than that of the latter two (P<0.01). Immunohistochemistry of NSE showed that PUs in the NIC transfected group, sham group and negative control group were 7.21 +/- 0.59, 28.25 +/- 1.46, 30.57 +/- 1.31, respectively, the former one was significantly smaller than that in the latter two (P<0.01). Western blot analysis showed that the gray scales of CgA in the NIC transfected group and sham group were 0.54 +/- 0.03 and 0.99 +/- 0.05, respectively, (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P<0.01). The gray scales of NSE in the NIC transfected group and sham group were 0.43 +/- 0.02 and 1.07 +/- 0.09, respectively (gray scale of the negative control set as 1.00), the former one was significantly smaller than that of the other two groups (P<0.01).
CONCLUSIONNotch1 may behave as a tumor suppressor in small cell lung cancer. Notch1 signal activation can inhibit the proliferation and neuroendocrine marker expression in small cell lung cancer cells, suggesting that Notch1 gene could be a new target for small cell lung cancer treatment and probable relief of paraneoplastic syndrome.
Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Chromogranin A ; metabolism ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; pathology ; Phosphopyruvate Hydratase ; metabolism ; Receptor, Notch1 ; genetics ; metabolism ; physiology ; Recombinant Proteins ; genetics ; metabolism ; Small Cell Lung Carcinoma ; metabolism ; pathology ; Transfection
3.Relationship between neuronal restricted silencing factor and induced differentiation from rat mesenchymal stem cells to neurons.
Bin LIU ; Hong-tu LI ; Tao ZHANG ; Fan-biao MENG ; Xiao-yu LIU ; Xi-ning PANG
Acta Academiae Medicinae Sinicae 2009;31(6):702-706
OBJECTIVETo analyze the change of the neuronal restricted silencing factor (NRSF) gene as well as the NRSF regulation genes in beta-mercaptoethanol induction of the marrow mesenchymal stem cells (MSCs) to neurons, and to discuss the function of NRSF in neural induction of the MSCs and the mechanism of the differentiation from MSCs to neurons.
METHODWe used beta-mercaptoethanol, serum-free DMEM, and dimethyl sulfoxide to induce rat MSCs to differentiate to neurons, and then analyzed the changes of the expressions of NRSF gene and NRSF-regulated genes through real-time PCR.
RESULTSThe rat MSCs were successfully induced to differentiate into neuron-like cells. The induced neuron marker, neuron-specific enolase, was positive. Real-time PCR showed that the expression of NRSF gene remarkably declined. The expressions of neurotrophic tyrosine kinase receptor, type 3, synaptosomal-associated protein 25, L1 cell adhesion molecular,neuronal pentraxin receptor in the NRSF-regulated genes also increased at varied extents.
CONCLUSIONSThe differentiation from MSCs to neurons is relevant with the decline of NRSF expression and the increase of the expressions of NRSF-regulated genes. The NRSF may be the key gene during the differentiation from MSCs to neurons.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Neurons ; cytology ; metabolism ; Phosphopyruvate Hydratase ; genetics ; metabolism ; Rats ; Rats, Wistar ; Repressor Proteins ; metabolism ; physiology ; Synaptosomal-Associated Protein 25 ; genetics ; metabolism
4.Neuron specific enolase gene silencing suppresses proliferation and promotes apoptosis of lung cancer cells in vitro.
Jian-jiang ZHOU ; Yuan XIE ; Yan ZHAO ; Zhen-xing LI
Journal of Southern Medical University 2011;31(8):1336-1340
OBJECTIVETo study the effects of neuron specific enolase (NSE) gene silencing on the cell proliferation and apoptosis of lung cancer cells in vitro.
METHODSNSE protein expression was detected in human small cell lung cancer cell line NCI-H446 and non-small cell lung cancer cell line A549 by immunocytochemistry, and a small interference RNA (siRNA) was transfected into the cells to inhibit NSE gene expression. The changes in the cell cycle, apoptosis, Ki67 protein and caspase-3 activity in the transfected cells were observed by flow cytometry, Western blotting and colorimetric assay, respectively.
RESULTSBoth A549 and NCI-H446 cells expressed NSE protein. Transfection of siRNA for NSE gene significantly inhibited the expression of NSE gene in the cells, resulting in an inhibition rate exceeding 90%. NSE gene silencing caused significantly decreased cell percentage in S phase and the expression of Ki67 protein, and increased the cell apoptotic rate and caspase-3 activity.
CONCLUSIONNSE gene expression promotes the cell proliferation and inhibits the cell apoptosis in lung cancer cells with neuroendocrine differentiation, which might be a causative factor contributing to increased malignancy of the cells.
Adenocarcinoma ; enzymology ; genetics ; pathology ; Apoptosis ; genetics ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Ki-67 Antigen ; metabolism ; Lung Neoplasms ; enzymology ; genetics ; pathology ; Phosphopyruvate Hydratase ; genetics ; RNA Interference ; Small Cell Lung Carcinoma ; enzymology ; genetics ; pathology
5.Granulosa cells model transfected by ENO1 overexpression adenovirus vector and the effect of ENO1 overexpression on progesterone secretion.
Wei-Hong ZHANG ; Hong JI ; Jiang-Lu WANG ; Rong-He HE ; Huan-Min YANG
Chinese Journal of Applied Physiology 2014;30(1):85-88
OBJECTIVETo construct primary cultured granulosa cells model of Zi Gooses tansfected by alpha-enolase (ENO1) overexpression adenovirus vector, and to detect the effect of ENO1 overexpression of granulose cells on progesterone secretion.
METHODSGranulosa cells were infected with Ad-CMV-ENO1 in gradient multiplicity of infection(MOI) levels:100, 250, 350 and 400 pfu/cell. Twenty four hours and 48 h after infection, green fluorescent protein (GDP) was respectively detected by fluorescence inverted microscopy. The effect of ENO1 overexpression of granulose cells on progesterone secretion was detected by the step double antibody sandwich enzyme-linked immunosorbent assay (ELISA).
RESULTSThe optimal infection rate (100%) was achieved when MOI was 800 pfu/cell,48h after infection. Real time RT-PCR and Western blot showed that the level of mRNA and protein expression ENO1 were increased significantly after infection (P < 0.01); The granulosa cells progesterone secretion of Ad-CMV-ENO1 group increased signigicantly (P < 0.01).
CONCLUSIONENO1 overexpression could make the primary culture follicle granulosa cells in vitro improve progesterone secretion.
Adenoviridae ; Biomarkers, Tumor ; genetics ; Cell Line ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; Female ; Genetic Vectors ; Granulosa Cells ; metabolism ; Green Fluorescent Proteins ; genetics ; Humans ; Ovarian Follicle ; cytology ; Phosphopyruvate Hydratase ; genetics ; Progesterone ; secretion ; RNA, Messenger ; genetics ; Transfection ; Tumor Suppressor Proteins ; genetics
6.A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression.
Jinsoo OH ; Youngsang YOU ; Yeomin YUN ; Hye Lan LEE ; Do Heum YOON ; Minhyung LEE ; Yoon HA
Yonsei Medical Journal 2015;56(4):1036-1043
PURPOSE: Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects. MATERIALS AND METHODS: In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo. RESULTS: NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons. CONCLUSION: We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.
Cell Differentiation/genetics/physiology
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*Gene Expression
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Gene Regulatory Networks
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*Genetic Therapy
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Humans
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Luciferases/genetics/*metabolism
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*Neural Stem Cells
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Neurons/metabolism
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Phosphopyruvate Hydratase/metabolism
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Promoter Regions, Genetic
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Spinal Cord Injuries/*therapy
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Stem Cells/*metabolism
7.Establishment of subcutaneously transplanted and metastatic neuroblastoma models in nude mice.
Hong-ting LU ; Qian DONG ; Qiang GAO ; Xi-wei HAO ; Hua SONG ; Nan ZHAO
Chinese Journal of Oncology 2010;32(4):258-261
OBJECTIVETo establish a tumor-bearing nude mouse model of human neuroblastoma in order to study the mechanisms of neuroblastoma invasion and metastasis, and to investigate potential therapeutic modalities in the experimental animal models.
METHODSA human neuroblastoma cell line was cultured in vitro. 1 x 10(7) cells undergoing exponential growth were collected in 0.1 ml of suspension and subcutaneously inoculated into the right flank next to the forelimb in nude mice. The biological characteristics of the developed tumors were observed, and histopathological and DNA microarray analyses were performed. The expressions of NSE in the subcutaneous tumor, metastatic tumor and the primary neuroblastoma tumor tissues from a pediatric patient were analyzed by immunohistochemistry.
RESULTSTumors successfully grew in 36 out of 48 injected mice, with a total tumor-formation rate of 75.0%. Metastasis occurred in 10 cases, and the metastatic rate was 20.8%. Tumors in five injected mice grew locally without metastasis. These tumors had large volume and the tumor weight reached up to half of the body weight of the host animal. Four mice exhibited systemic metastasis without tumor growth at the primary inoculation site. There were six mice with locally growing tumor accompanied by metastasis.
CONCLUSIONWe have successfully established a human neuroblastoma xenograft model in nude mice with high tumor growth and metastatic rates. This model depicting the natural cell growth, local infiltration and distant metastasis characteristics of human neuroblastoma, providing an ideal animal model for in vivo studies of neuroblastoma. In addition, the results of this study indicate the heterogeneous nature of neuroblastoma, it may play an important role in metastasis of this tumor.
Animals ; Cell Line, Tumor ; Child, Preschool ; Disease Models, Animal ; Female ; Gene Expression Profiling ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; secondary ; Male ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Transplantation ; Neuroblastoma ; genetics ; metabolism ; pathology ; secondary ; Phosphopyruvate Hydratase ; metabolism ; Tumor Burden
8.Human bone marrow mesenchymal stem cells differentiated into dopaminergenic neurons in vitro.
Li-Hui CHAI ; Su-Xia WU ; Wen-Hai YAN ; Yuan-Fang MA
Chinese Journal of Biotechnology 2007;23(2):252-256
Midbrain dopamine (DA) neurons play an essential role in modulating motor control. Defects in central DA neurons affect a wide range of neurological disorders including Parkinson's disease (PD). The greatest motivation in the field has been the potential use of DA neurons for cell transplantation therapy in Parkinsonian patients. Recent studies indicated that BMSCs could differentiate into DA neurons in vitro as neural stem cells (NSC) and embryonic stem cells (ESC) could. However, there are no direct evidences about functional DA neurons derived from BMSCs. According to the protocols which had been applicated in inducing neuronal stem cells and embryonic stem cells differentiate into DA neurons in vitro, the present study provides a protocol by using 50 micromol/L brain derived neurotrophy factor (BDNF), 10 micromol/L forskolin (FSK) and 10 micromol/L dopamine (DA) to induce BMSCs differentiate into DA neurons. After 2 weeks of differentiation, the cells expressed the character of neurons in ultrastructure. RT-PCR discovered mRNA of NSE (neuron specific enolase), Nurr1, Ptx3, Lmx1b and Tyrosine hydroxylase (TH) were positive. Immunocytochemistry staining indicated the ratio of TH-positive neural cells was significantly increased after induced 2 weeks (24.80 +/- 3.36) % compared to that of induction of 3 days (3.77 +/- 1.77) %. And the DA release was also different between differentiated and undifferentiated cells detected by high performance liquid chromatography (HPLC). That is to say BDNF and FSK and DA can induce BMSCs differentiate into DA neurons in vitro, and the transdifferentiated cells express mature neurons characters. BMSCs might be a suitable and available source for the in vitro derivation of DA neurons and cell transplantation therapy in some central neural system diseases such as PD.
Adult
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Aged
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Bone Marrow Cells
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cytology
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metabolism
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ultrastructure
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Brain-Derived Neurotrophic Factor
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pharmacology
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Cell Transdifferentiation
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drug effects
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genetics
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physiology
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Cells, Cultured
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Chromatography, High Pressure Liquid
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Colforsin
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pharmacology
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Dopamine
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metabolism
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pharmacology
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Female
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Humans
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Immunohistochemistry
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Male
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Mesenchymal Stromal Cells
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cytology
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metabolism
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ultrastructure
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Microscopy, Electron, Transmission
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Middle Aged
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Neurons
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cytology
;
metabolism
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ultrastructure
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Phosphopyruvate Hydratase
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genetics
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Reverse Transcriptase Polymerase Chain Reaction
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Tyrosine 3-Monooxygenase
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genetics
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metabolism
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Young Adult
9.Laminar Distribution of Neurochemically-Identified Interneurons and Cellular Co-expression of Molecular Markers in Epileptic Human Cortex.
Qiyu ZHU ; Wei KE ; Quansheng HE ; Xiongfei WANG ; Rui ZHENG ; Tianfu LI ; Guoming LUAN ; Yue-Sheng LONG ; Wei-Ping LIAO ; Yousheng SHU
Neuroscience Bulletin 2018;34(6):992-1006
Inhibitory GABAergic interneurons are fundamental elements of cortical circuits and play critical roles in shaping network activity. Dysfunction of interneurons can lead to various brain disorders, including epilepsy, schizophrenia, and anxiety. Based on the electrophysiological properties, cell morphology, and molecular identity, interneurons could be classified into various subgroups. In this study, we investigated the density and laminar distribution of different interneuron types and the co-expression of molecular markers in epileptic human cortex. We found that parvalbumin (PV) and somatostatin (SST) neurons were distributed in all cortical layers except layer I, while tyrosine hydroxylase (TH) and neuropeptide Y (NPY) were abundant in the deep layers and white matter. Cholecystokinin (CCK) neurons showed a high density in layers IV and VI. Neurons with these markers constituted ~7.2% (PV), 2.6% (SST), 0.5% (TH), 0.5% (NPY), and 4.4% (CCK) of the gray-matter neuron population. Double- and triple-labeling revealed that NPY neurons were also SST-immunoreactive (97.7%), and TH neurons were more likely to express SST (34.2%) than PV (14.6%). A subpopulation of CCK neurons (28.0%) also expressed PV, but none contained SST. Together, these results revealed the density and distribution patterns of different interneuron populations and the overlap between molecular markers in epileptic human cortex.
Adolescent
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Adult
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Brain Chemistry
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genetics
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physiology
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Cerebral Cortex
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metabolism
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pathology
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Child
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Cholecystokinin
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metabolism
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Epilepsy
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etiology
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pathology
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Female
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Gene Expression Regulation
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physiology
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Humans
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Interneurons
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metabolism
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Male
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Middle Aged
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Neuropeptide Y
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metabolism
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Parvalbumins
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metabolism
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Phosphopyruvate Hydratase
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metabolism
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Somatostatin
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metabolism
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Tyrosine 3-Monooxygenase
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metabolism
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Young Adult
10.Salidroside via ERK1/2 and PI3K/AKT/mTOR signal pathway induces mouse bone marrow mesenchymal stem cells differentiation into neural cells.
Ya-Nan CHEN ; Hui LIU ; Hong-Bin ZHAO ; Yang LIU ; Jie BAI ; Xiao-Juan ZHU ; Yu WANG
Acta Pharmaceutica Sinica 2013;48(8):1247-1252
To investigate the role of the extracellular signal-regulated kinase (ERK1/2) and PI3K/AKT/ mTOR signal pathway inducing bone marrow mesenchymal stem cells (BMSCs) differentiation into neural cells, mouse bone marrow-derived mesenchymal stem cell lines D1 cells were used as research object. And they were divided into control groups and salidroside (SD) groups. Different concentrations (5, 25, 50, 100 and 200 microg x mL(-1) of SD were used and SD (100 microg x mL(-1)) was used to induce at different time (0.5, 1, 3, 6, 9, 12, 24, 48 and 72 h). The immunofluorescence staining chemical technology, real-time PCR and Western blotting were used to detect the positive rates of NSE, MAP2, beta-Tubulin III, NES, GFAP and the expression levels of beta-Tubulin III, NSE, ERK1/2, AKT. The expression of ERK1/2 and NSE was detected when the ERK1/2 and PI3K/AKT/ mTOR signal pathway was blocked by PD98059 and LY294002. It indicated that the positive rates of NSE, MAP2, beta-Tubulin III, NES and GFAP were gradually enhanced with time increased. The expression level of NSE and beta-Tubulin III protein were significantly higher than those in control groups (P < 0.01). The expression of ERK1/2, AKT mRNA and protein were higher with concentration and time increased. When the ERK1/2 and PI3K/AKT/mTOR signal pathway were blocked, the expression levels of NSE, NES and beta-Tubulin III mRNA and NSE protein were inhibited significantly. It points out that SD can stimulate the ERK1/2 and PI3K/AKT/mTOR signal pathway to promote BMSCs differentiation into neural cells.
Animals
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Bone Marrow Cells
;
cytology
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Cell Differentiation
;
drug effects
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Cells, Cultured
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Chromones
;
pharmacology
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Enzyme Inhibitors
;
pharmacology
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Flavonoids
;
pharmacology
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Glial Fibrillary Acidic Protein
;
metabolism
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Glucosides
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antagonists & inhibitors
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isolation & purification
;
pharmacology
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MAP Kinase Signaling System
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drug effects
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Mesenchymal Stromal Cells
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cytology
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Mice
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Microtubule-Associated Proteins
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metabolism
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Mitogen-Activated Protein Kinase 1
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genetics
;
metabolism
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Mitogen-Activated Protein Kinase 3
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genetics
;
metabolism
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Morpholines
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pharmacology
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Nestin
;
metabolism
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Neurons
;
cytology
;
metabolism
;
Phenols
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antagonists & inhibitors
;
isolation & purification
;
pharmacology
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Phosphatidylinositol 3-Kinases
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metabolism
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Phosphopyruvate Hydratase
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genetics
;
metabolism
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Plants, Medicinal
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chemistry
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Protein Kinase Inhibitors
;
pharmacology
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Proto-Oncogene Proteins c-akt
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genetics
;
metabolism
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RNA, Messenger
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metabolism
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Rhodiola
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chemistry
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Signal Transduction
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drug effects
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TOR Serine-Threonine Kinases
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metabolism
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Tubulin
;
metabolism