Research of the FLG mutation in various ethnic groups revealed non-overlapping mutation patterns. In addition, Japanese and Chinese atopic patients showed somewhat different mutations. These ethnic differences make the research on Korean patients mandatory; however, no systematic research on Korean atopic dermatitis (AD) patients has been performed. This study aims to investigate the genetic polymorphism of FLG in Korean atopic dermatitis patients. The study was made up of three groups including 9 Ichthyosis vulgaris (IV) patients, 50 AD patients and 55 normal controls: the ichthyosis group was incorporated due to the reported association between the FLG mutation and IV. In comparison to other sequencing methods, the overlapping long-range PCR was used. We revealed the genetic polymorphism of filaggrin in Koreans, and at the same time, we discovered nonsense mutations in p.Y1767X and p.K4022X in Korean AD patients. By using FLG sequencing techniques confirmed in this study, new mutations or genetic polymorphisms with ethnic characteristics would be detected and further larger studies of repeat number polymorphisms could be performed.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Codon, Nonsense ; DNA/blood/chemistry/metabolism ; DNA Mutational Analysis ; Dermatitis, Atopic/*genetics ; Female ; Genotype ; Heterozygote ; Humans ; Ichthyosis Vulgaris/genetics ; Intermediate Filament Proteins/*genetics ; Male ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

OBJECTIVETo investigate the target cells and molecules with sodium valproate induced differentiation of human glioma cells.

METHODSNude mice bearing human glioma xenogenic graft subcutaneously were treated with sodium valproate. The expressions of HDAC1 and Tob genes of xenografts were analyzed with semiquantitative RT-PCR. The CD133+ cells (BTSCs) were isolated from glioma specimens by immunomagnetic sorting, and cultured in the medium containing FCS or in the serum-free medium supplemented with growth factors, respectively, followed by treatment with sodium valproate in vitro for 21 days. The cell surface markers were detected with flow cytometry and confocal microscopy.

RESULTSSodium valproate inhibited the growth of subcutaneous xenografs bearing on nude mice (P<0.05), and up-regulated the HDAC1 expression (P<0.01), down-regulated the Tob expression (P<0.05). The cell surface markers of BTSCs were detected by flow cytometry after sodium valproate treatment for 21 days. In the FCS group, the GFAP or beta-tubulin III positive cells increased significantly (P<0.01), but in the growth factor group, no statistical differences were observed in the GFAP or beta-tubulin III expression (P>0.05). The results of confocal microscopy indicated that the GFAP+ or beta-tubulin III+ cells coexpressed with Nestin.

CONCLUSIONSHDAC1 and Tob genes were the potential target molecules in reversion of the differential inhibition of human glioma cells with sodium valproate. The BTSCs undergoing the processes of differentiation were the target cells for sodium valproate.

AC133 Antigen ; Actins ; analysis ; Animals ; Antigens, CD ; analysis ; Brain Neoplasms ; metabolism ; pathology ; prevention & control ; Cell Differentiation ; drug effects ; Flow Cytometry ; Gene Expression ; drug effects ; Glial Fibrillary Acidic Protein ; analysis ; Glioma ; metabolism ; pathology ; prevention & control ; Glycoproteins ; analysis ; Histone Deacetylases ; genetics ; Humans ; Intermediate Filament Proteins ; analysis ; Mice ; Mice, Nude ; Microscopy, Confocal ; Nerve Tissue Proteins ; analysis ; Nestin ; Peptides ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured ; Tumor Suppressor Proteins ; metabolism ; Valproic Acid ; pharmacology ; Xenograft Model Antitumor Assays ; methods

Animals ; Desmin ; analysis ; genetics ; Female ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; genetics ; Kidney Glomerulus ; chemistry ; Nephrosis ; metabolism ; Nerve Tissue Proteins ; analysis ; genetics ; Nestin ; Podocytes ; chemistry ; RNA, Messenger ; analysis ; Rats ; Rats, Inbred WKY ; Vimentin ; analysis ; genetics

OBJECTIVETo isolate, culture and identify glioma stem cells from human malignant glioma cell line U87, and investigate the changes of pro-angiogenic factors production by glioma stem cells followed by activation of CXCR4 and observe their tumorigenesis as well as the expression of vascular endothelial growth factor when implanted into nude mice.

METHODSThe ratio of CD133 positive cells was detected by flow cytometry. Magnetic separation of CD133 positive cells was carried out on the magnetic cell sorting system (MACS). Expression of nestin, glial fibrillary acidic protein (GFAP) and CXCR4 on tumorspheres was detected by indirect immunofluorescence under confocal laser scanning microscopy. The functional activation of CXCR4 was assessed by calcium mobilization experiments. ELISA was used to detect the production of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in conditioned medium. Glioma stem cells were implanted into nude mice to assess their tumorigenesis ability and the expression of VEGF.

RESULTSThe ratio of CD133 positive cells with stem cell property was 0.5% in U87 cells. Activation of CXCR4 on glioma stem cells induced calcium mobilization and increased VEGF and IL-8 protein secretion. CD133 positive cells secreted more VEGF and IL-8 than their negative counterparts in vitro. Tumors derived from CD133 positive cells grew more rapidly and expressed elevated level of VEGF than their negative counterparts.

CONCLUSIONSThere are a small fraction of glioma stem cells in human glioblastoma cell line U87. Expressing functional CXCR4 and secreting more pro-angiogenic factors may be involved in tumor angiogenesis mediated by glioma stem cells.

AC133 Antigen ; Animals ; Antigens, CD ; analysis ; Brain Neoplasms ; blood supply ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Glial Fibrillary Acidic Protein ; metabolism ; Glioblastoma ; blood supply ; metabolism ; pathology ; Glycoproteins ; analysis ; Humans ; Interleukin-8 ; metabolism ; Intermediate Filament Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; metabolism ; transplantation ; Neovascularization, Pathologic ; Nerve Tissue Proteins ; metabolism ; Nestin ; Peptides ; analysis ; Receptors, CXCR4 ; genetics ; metabolism ; physiology ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVEPrevious studies suggest that hyperbaric oxygen (HBO) treatment promotes the proliferation of neurocytes in neonatal rats following hypoxic-ischemic brain damage (HIBD). The Wnt signaling pathway is associated with neurogenesis. This study examined whether HBO promoted neural stem cells (NSCs) proliferation after HIBD, and whether that the proliferation correlated with Wnt-3 protein expression.

METHODSSeven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control, hypoxia-ischemia (HI), and HI-HBO. HI was induced by the ligation of left common carotid artery, followed by a 2-hr exposure to 8% O2 in the latter two groups. HBO was administered 3 hrs after HI in the HI-HBO group for continuous 7 days (2 atmospheres absolute, once daily). The proliferating NSCs in the subventricular zone (SVZ) was examined by BrdU/nestin immunofluorescence and the expression of Wnt-3 protein in NSCs was examined by nestin/Wnt-3 immunofluorescence at 6 and 24 hrs and at 3, 7 and 14 days of HI. The cellular expressions of nestin and Wnt-3 protein were analyzed by laser scanning confocal microscopy. The linear regression analysis was used to evaluate the correlation between cellular Wnt-3 and nestin protein. The expressions of nestin and Wnt-3 protein in the ischemic cerebral hemisphere were analyzed with Western blotting.

RESULTSThe number of BrdU/nestin positive cells in the SVZ increased 3 hrs after HBO therapy, peaked at 7 days and remained at a higher level until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of Wnt-3 protein in NSCs increased significantly 3 hrs after HBO therapy, peaked at 3 days and remained at high levels until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of cellular nestin protein was closely correlated with the level of cellular Wnt-3 protein (r = 0.893, P < 0.05). The Western blotting analysis demonstrated increased Wnt-3 and nestin protein expressions in the ischemic cerebral hemispheres.

CONCLUSIONSHBO treatment promotes the proliferation of NSCs in HIBD neonatal rats, which is correlated with the activation of Wnt signaling.

Animals ; Animals, Newborn ; Blotting, Western ; Bromodeoxyuridine ; metabolism ; Cell Proliferation ; Female ; Fluorescent Antibody Technique ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Intermediate Filament Proteins ; Male ; Nerve Tissue Proteins ; Nestin ; Neurons ; cytology ; Rats ; Stem Cells ; cytology ; Wnt Proteins ; analysis ; Wnt3 Protein

OBJECTIVETo explore the expression change of stem cell-derived neural stem/progenitor cell supporting factor (SDNSF) gene in the injuried spinal cord tissues of rats, and the relation between the expressions of SDNSF and nestin.

METHODSThe spinal cord contusion model of rat was established according to Allen's falling strike method. The expression of SDNSF was studied by RT-PCR and in situ hybridization (ISH), and the expression of nestin was detected by immunochemistry.

RESULTSRT-PCR revealed that SDNSF mRNA was upregulated on day 4 after injury, peaked on day 8-12, and decreased to the sham operation level on day 16. ISH revealed that SDNSF mRNA was mainly expressed in the gray matter cells, probably neurons, of spinal cord. The immunohistochemistry showed that accompanied with SDNSF mRNA upregulation, the nestin-positive cells showed erupted roots, migrated peripherad and proliferation on the 8-day slice. However, the distribution pattern of these new cells was different from that of SDNSF-positive cells.

CONCLUSION(1) SDNSF is expressed in the gray matter of spinal cord. The expression of SDNSF mRNA in the spinal cord varies with injured time. (2) The nestin-positive cells proliferate accompanied with spinal cord injury repair, but do not secrete SDNSF.

Analysis of Variance ; Animals ; Disease Models, Animal ; Female ; Gene Expression Regulation ; physiology ; In Situ Hybridization ; methods ; Intermediate Filament Proteins ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Spinal Cord Injuries ; metabolism ; Time Factors ; Vesicular Transport Proteins ; genetics ; metabolism

OBJECTIVEBasic fibral growth factor (bFGF) might have a role in the restoration and regeneration of injured neurons following hypoxic-ischemic brain damage (HIBD), but its mechanism has not been fully elucidated. Nestin is an intermediate filament protein expressed in dividing cells during the early stages of CNS development, but it can be reinduced in adults during regeneration of injured neurons after CNS injury. This study investigated the effect of exogenous bFGF on nestin expression in neonatal rats following hypoxia-ischemia (HI) and to explore the possible mechanism.

METHODSEighty-four 7-day-old SD rats were randomly assigned into a Sham-operation group, a HIBD group and a bFGF intervention group (n=28 each). HIBD was induced by ligation of the left carotid artery along with 8% oxygen exposure in neonatal rats from the latter two groups. The Sham-operation group was not subjected to HI. The bFGF intervention group received an intraperitoneal injection of bFGF daily (4000 U/kg). Each group was randomly subdivided into groups sacrificed immediately, at 3, 12 and 24 hrs and 3, 7 and 14 days after HI (n=4). The expression of nestin in the cerebral cortex, hippocampus and extraventricular zone was examined with immunohistochemical staining and image quantitative analysis.

RESULTSNestin was weakly expressed in the hippocampus and extraventricular zone and not expressed in the cortex in the Sham-operation group. The nestin in the cortex, hippocampus and extraventricular zone was significantly increased after HIBD, peaking at 7 days. bFGF treatment increased the nestin expression in the cortex, hippocampus and extraventricular zone and statistical differences were observed from 1 to 14 days after HI when compared with the untreated HIBD group.

CONCLUSIONSExogenous bFGF can up-regulate the nestin expression in neonatal rats following HIBD. The effects of restoration and regeneration of bFGF on injured neurons may be associated with increased nestin expression in neonatal rats.

Animals ; Animals, Newborn ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; therapeutic use ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Tissue Proteins ; analysis ; Nestin ; Rats ; Rats, Sprague-Dawley

OBJECTIVEThis study investigated the effect of hyperbaric oxygenation (HBO) on neural stem cells (NSCs) and myelin in neonatal rats following hypoxic-ischemic brain damage (HIBD) and aimed to explore the possible mechanism of the protective effect of HBO on HIBD.

METHODSSeven-day-old Sprague-Dawley rat pups were randomly assigned into 4 groups: Normal control, HIBD, hyperbaric air (HBA), and HBO groups (n=30 each). The HIBD model was produced by permanent occlusion of the left common carotid artery and 2 hrs hypoxemia exposure (8% O2 at 37 degrees C). HBA and HBO treatment was administered (2 ATA, once daily for 7 days) in the HBA and HBO groups respectively 1 hr after HIBD. BrdU immunohistochemistry was used to detect the NSCs in the sub-ventricle zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. The myelin damage was assessed by myelin basic protein (MBP) immunostaining.

RESULTSThe BrdU-positive cells in the SVZ and the DG of the ischemic hemisphere in the HIBD group were dramatically decreased compared with those of the Normal control group at 3 weeks post-HIBD (P < 0.01). The HBO treatment resulted in an increase of BrdU-positive cells in the DG from 153.7 +/- 37.0 to 193.7 +/- 38.8 (P < 0.05). The nestin expression in the HIBD and HBA groups was reduced compared with that in the Normal control group. There was no difference in the nestin expression between the HBO and the Normal control groups. Hypoxia-ischemia (HI) led to marked myelin damage at 1 week post-HIBD. HBO or HBA treatment alleviated the damage.

CONCLUSIONSThe HBO treatment can result in the proliferation of BrdU-positive cells and alleviate the myelin damage following HIBD in neonatal rats, thereby offering neuroprotectivity against HI insults.

Animals ; Animals, Newborn ; Bromodeoxyuridine ; metabolism ; Female ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Male ; Mice ; Myelin Basic Protein ; analysis ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

To investigate the effect of forced running in motor-driven wheel on neurogenesis in the hippocampal dentate gyrus (DG) of adult rats, 5-bromo-2-deoxyuridine (BrdU), a thymidine analog was applied to mark cell proliferation. Neuroepthelial stem cell protein (nestin) expression was used to identify neural stem/precursor cells. The BrdU- and nestin-positive cells were examined by immunohistochemical technique. The ability of learning was evaluated by Y-maze test to explore the functional role of the newborn cells in the DG after forced running. It was found that the number of BrdU- and nestin-positive cells in the DG in running groups was significantly increased compared to that in the control group (P<0.05). The effect of forced running on neurogenesis was intensity-dependent. In addition, an improvement of learning ability in Y-maze test was observed after forced running. These findings suggest that forced running in motor-driven wheel could enhance neurogenesis in the hippocampal DG of adult rats and improve learning ability.
Animals ; Bromodeoxyuridine ; metabolism ; Cell Survival ; Dentate Gyrus ; cytology ; physiology ; Intermediate Filament Proteins ; analysis ; Learning ; Male ; Maze Learning ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; physiology ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Running

OBJECTIVETo explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro.

METHODSMSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1beta, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days.

RESULTSMSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032 +/- 2.489%, 41.580 +/- 5.101% and 34.958 +/- 5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1beta, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days.

CONCLUSIONMSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application.

Adipocytes ; cytology ; Animals ; Blotting, Western ; Bone Marrow Cells ; Carboxylesterase ; analysis ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; cytology ; Culture Media, Conditioned ; Dopamine ; analysis ; Intermediate Filament Proteins ; analysis ; Mesencephalon ; cytology ; Mesenchymal Stromal Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; metabolism ; Phosphoprotein Phosphatases ; analysis ; Rats ; Rats, Wistar