OBJECTIVETo identify potential mutations of the FLG gene in two Chinese families affected with ichthyosis vulgaris.

METHODSAll coding exons and exon-intron boundary of the FLG gene were amplified by polymerase chain reaction (PCR) and analyzed by direct sequencing. The results were compared with those of 100 unrelated healthy controls.

RESULTSTwo novel missense mutations, c.1360A>G (p.T454A) and c.10363G>T (p.D3455Y), were detected in all affected individuals from family 1 and family 2 respectively but none of the controls.

CONCLUSIONThe c.1360A>G (p.T454A) and c.10363G>T (p.D3455Y) of the FLG gene may lead to alteration of the structure and function of the FLG protein and cause ichthyosis vulgaris in the two families.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; DNA Mutational Analysis ; Exons ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; ethnology ; genetics ; Humans ; Ichthyosis Vulgaris ; ethnology ; genetics ; Intermediate Filament Proteins ; genetics ; Introns ; genetics ; Male ; Mutation, Missense ; Pedigree

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

OBJECTIVEThe purpose of this study was to culture and identify neural stem cells from mouse embryos in vitro using a modified method and provide a basis for further study of the biology of neural stem cells under hypoxia.

METHODSThe cells were isolated mechanically from the front cortex of fetal Institute of Cancer Research (ICR) mice on embryonic day 14. They were passaged by mechanical dissociation and enzymatic digestion. The neurospheres were identified by immunofluorescent staining of nestin. Cell differentiation was induced by 1% fetal bovine serum and then the cells were identified by immunohistochemistry of β-tubulin III and GFAP.

RESULTSThe cells obtained from the front cortex of fetal ICR mice had the capacity of forming neurospheres which showed nestin immunoreactive positivity. After being induced by 1% fetal bovine serum, the cells were differentiated into β-tubulin III-positive cells and GFAP-positive cells.

CONCLUSIONSUsing mechanical dissociation of primary cells and mechanical dissociation with enzymatic digestion of primary cells, the NSCs from the front cortex of mouse embryos can be obtained.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Intermediate Filament Proteins ; analysis ; Mice ; Mice, Inbred ICR ; Nerve Tissue Proteins ; analysis ; Nestin ; Neural Stem Cells ; chemistry ; cytology ; Tubulin ; analysis

OBJECTIVETo study the effects of ganglioside 1 (GM1) and nerve growth factor (NGF) on neural stem cells (NSCs) proliferation in vitro.

METHODSNSCs were isolated and cultured in vitro. NSCs were cultured in the medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) or without the two agents. Different concentrations of GM1 and NGF were added into the two different medium. MTT and cell ball counting methods were used to ascertain the proliferation of NSCs. Immunohistochemical technology was used to observe the effect of GM1 and NGF on the proliferation of NSCs.

RESULTSHigh concentrations of GM1 (100 ng/L and 200 ng/L) promoted significantly the proliferation of NSCs in the medium containing EGF and bFGF (p<0.05). In the differentiation medium containing serum but no EGF and bFGF, NSCs proliferation increased with increasing concentration of GM1; the proportion of neurons and gliacytes increased with increasing concentration of NGF.

CONCLUSIONSHigh concentration of GM1 can promote NSCs proliferation and NGF can promote NSCs differentiation.

Animals ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Female ; G(M1) Ganglioside ; pharmacology ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Growth Factors ; pharmacology ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; drug effects ; physiology

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