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Objective To explore the difference of DNA methylation levels between normal Schwann cells (NSCs) and activated Schwann cells (ASCs) in rats. Methods The adult Wistar rats were received sciatic nerve ligation and fed for 7 days. The ASCs and NSCs were separated from ligated sciatic nerves and brachial plexus respectively. Immunocytochemical staining of S-100 antibody was used to identify the cells. The growth condition of cells was detected by CCK-8 method. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) was applied to filter the differentially methylated regions in ASCs and NSCs. The distribution of differentially methylated genes related with axonal regeneration in chromosome was analyzed, and Gene ontology(GO)and PATHWAY analysis were also conducted. Results High purity of ASCs and NSCs were obtained successfully, which were both positive for S-100 antibody. In the same culture condition, ASCs showed a faster proliferation than that of NSCs. A total of 177 176 differentially methylated regions were found by MeDIP-Seq. Among them, 1 097 were located in the promoter (≤1 kb), 1 136 in the promoter (1-2 kb) and 567 on the CpG. After functional annotation of differentially methylated genes, 214 differentially methylated genes related with axonal regeneration were found in ASCs and NSCs. Compared with NSCs, 191 genes were up-regulated and 23 genes were down-regulated in ASCs. These genes were located on different chromosomes, most of which on chromosome 12 (22 genes) and the least on chromosomes M (2 genes). GO analysis indicated that the differential methylated genes were involved in axon growth, axon formation, axon elongation and axon guidance. The MAPK, cell adhesion molecules, Ras signaling pathway may be related with the differential methylated genes. Conclusion The methylation levels between ASCs and NSCs are significantly different, which are probably related with axon regeneration.
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Objective To observe the role of baicalin on the expression of phosphorylated protein of signal transduc-ers and activators of transcription signaling proteins (STATs) during the process that neural stem cells (NSCs) differentiating into neurons. Methods NSCs were isolated from the embryonic cerebral cortex of the 14-15-day pregnant SD rats, which were cultured and passaged in vitro. The 3rd generation of NSCs was used in the experiment. NSCs were randomized into nat-ural differentiation control group, three different doses of baicalin groups (7.5μmol/L, 15μmol/L and 30μmol/L), leukemia inhibitory factor (LIF)+basic fibroblast growth factor (bFGF) group and baicalin+LIF+bFGF group. After 6 d culture in vi-tro, the immunohistochemical method was used to observe the expressions of microtubule-associated protein 2(MAP-2) and glial fibrillary acidic protein (GFAP) in different groups. The expression levels of phosphorylation protein of STAT 3 in NSCs were detected by Western blotting method after 2 h and 6 d of culture. Results The expression of MAP-2 in NSCs was in-creased by baicalin, but the expression of GFAP in NSCs decreased. The expression of GFAP in NSCs was enhanced in LIF+bFGF group, which was inhibited by baicalin+LIF+bFGF. The phosphorylation level of STAT3 in NSCs was downregulat-ed by baicalin, but the phosphorylation level of STAT3 was upregulated in LIF+bFGF group. The upregulated phosphoryla-tion level of STAT3 was inhibited in baicalin+LIF+bFGF group(P＜0.05). Conclusion Baicalin can induce NSCs to dif-ferentiate into neurons, which may be caused by the downregulation of the phosphorylation level of STAT3 in NSCs.
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Objective To seek an optimal method for the separation,culture of mouse embryonic germ cells (EGCs) in vitro,and to observe the influence of Activated Schwann cells (ASCs)-derived neurotrophins on the differentiation capability of mouse EGCs into neurogenic cells.Methods The gonadal ridges and a few abdominal tissues of the 11-day postcoitum (dpc) mouse embryos were isolated and disaggregated by 0.125％ trypsin-0.02％ EDTA,followed by culture of the mouse EGCs on mouse embryonic fibroblast (MEF) feeders.Monoclonal formation of the mouse EGCs was observed,and the staining of stage specificity embryo antigen-1 (SSEA-1),alkaline phosphatase (AKP),periodic acid-Schiff staining (PAS) were applied to identify the mouse EGCs.Two groups were divided as followed:mouse EGCs+basic medium (control group) and mouse EGCs+ASCs (experimental group).Immunofluorescence (NeuN,MBP,GFAP)analysis was used to evaluate the neurogenic differentiation of mouse EGCs and then to calculate the statistical positive rates of cell staining.All experimental results were analyzed statistically.Results (1) Identification ofmouse EGCs:Mouse EGCs were characterized by a dome-shaped colony containing a large nucleus and a relatively small amount of cytoplasm.All mouse EGCs were positive staining of SSEA-1,AKP,and PAS;(2)The neural induction of mouse EGCs:After one week induction,there were few round or oval cells with long axon-like processes migrating from the edge of the EGCs clones.3 weeks later,the neurogenic-like cells increased quickly.The results of immunofluorescence (NeuN,MBP,GFAP)staining demonstrated that mouse EGCs could differentiate into neurogenic cells under the influence of ASCs.The positive rate of cell staining was significant.Conclusion In this study,a simple,economical method was applied to successfully separate the mouse EGCs in vitro; mouse EGCs can differentiate into neurogenic cells under the influence of ASCs-derived neurotrophins.
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Objective To observe whether immature Brain Derived Neurotrophic Factor (proBDNF)can affect the activities of OPCs in the fields of cell proliferation and migration after SCI,and to investigate the relationship between proBDNF and p75NTR signal pathway on OPCs.Methods OLN-93 cell line was cultured and maintained for in vitro experiments.Immunofluorescence were used to check the expression of endogenous proBDNF,p75NTR and sortilin on OPCs.MTT assay was used to illustrate the inhibitory effect of proBDNF.The effects of anti-proBDNF was also observed by BrdU staining to find a probably signal pathway for proBDNF on OPCs.The Sprague-Dawley rats were administered for T9 spinal cord injury animal model.BBB score was applied to observe the situation of functional recovery after treated by anti-proBDNF.BrdU staining was managed to observe the situation of OPCs proliferation and migration after SCI.Results Endogenous proBDNF inhibited proliferation and migration of OPCs after SCI.BrdU staining showed that population of proliferative OPCs in lesion site of spinal cord was less in proBDNF in treated group than that in control group and anti-proBDNF group.While anti-proBDNF could inhibit proBDNF specifically and might induced a better functional recovery which was illustrated by BBB scores.The in vitro experiments found the inhibitory effect of proBDNF is dose-dependent and can be neutralized by anti-proBDNF properly.Moreover,the expression levels of p75NTR and sortilin are down regulated by proBDNF antibody treated group.This indicated that proBDNF may inhibit OPCs via p75NTR pathway.Conclusion Endogenous proBDNF can inhibit cell proliferation of OPCs after SCI and can be neutralized by specific antibodies of proBDNF.This kind of detrimental effect may be induced by p75NTR-sortilin pathway.Furthermore,proBDNF antibody treatment is effective to block proBDNF and promote the functional recovery.
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ObjectiveTo observe and analyze the synapses developing process of newly generated connections of autologus activated Schwann cells (AASCs) in combination with fetal spinal cord cell suspension(FSCS) in the surrounding area of the spinal cord injury site.MethodsA total of 42 Wistar rats underwent unilateral ligation of the saphenous nerve.The portion of nerve tissues distal to the ligation site were harvested 1 week after operation.AASCs were isolated,cultured and purified.Spinal cord injury model produced in 42 Wistar rats on T7 by modified Allen impact method.Three days after injury,20 μl FSCS with a density of 1×105/μl prepared from pregnant rats (El4) in combination with AASCs were injected into the epicenter of the traumatized cavity.Animals were sacrificed at 2,4,6,8,10,12 weeks post transplantation.Light and electronmicroscopic studies as well as immunohistochemical assay were carried out to evaluate the graft survival,its differentation and integration with the host.ResultsIn the transplantation area,AASCs showed good growth and differentiation,and glial scarring surrounding the lesions was less.The neuroblast stretched out the terminal endings 4 weeks after implantation,followed by the presenting of the pre- and post-synaptic membrane.Eight weeks post transplantation,the dense or developed projections were observed in the pre- and post-synaptic membrane,the high electron dense substance full filled the synaptic cleft.All the spherical cleat vesicles,granular vesicles,elliptical vesicles and flattened-f type vesicles were discovered under the electron microscope.Ten weeks after injury,the axosomatic,dendrosomatic,dendro-dendritic,axoaxonic,and dendro-axonic synapses coexisted.Light microscopy showed that the graft cell grew gradually.Immunohistochemical assay showed that NF,5-HT,CGRP and GFAP positive fibers were in the graft.Synapses,glia fibers and blood brain barrier integrated each other.Conclusion1) The transplanted FSCS combined with AASCs can develop mature synapses with miscellaneous synaptic vesicles in the acute injured spinal cord.2) Co-existing indicate the possibility of synaptic connection between FSCS and host.
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Objective To explore the clinical characteristics and risk factors of re-fracture in patients suffering from osteoporosis-related fractures as well as effective interventions.Methods From January 2006 to January 2008,a total of 273 patients with osteoporosis-related fracture were entered in the study,including out-patients and in-patients who were over 50 years old.The patients were divided into fracture group(n=225)and re-fracture group(n=48).The re-fracture rate was followed up for 2 years,during which 11 patients developed re-fracture.General data including age and sex,fracture types,femoral neck bone mineral density(BMD)T-scores tested by dual-energy X-rays absorptiometry(DEXA),Charlson index,timeinterval between two fractures as well as mobility skill assessment were collected and analyzed.Results The average age at the first fracture was 67.7±8.5 years vs.72.7±9.5 years for the re-fracture cases.Female accounted for 70.2% of the fracture group and 77.1% of the re-fracture group.The most common re-fracture type was vertebral fracture for the first time and femoral neck fracture for the second time during the followup.Risk factors for a second fracture in osteoporotic fractures patients include age(＞75 years,HR=1.23; ＞85years,HR=1.68),female sex(HR=1.36),prior vertebral fractures(HR=1.62),prior hip fractures(HR=1.27),BMD T-score＜-3.5(HR=1.38)and weakened motor skills(HR=1.27).The refracture rate in osteoporosis-related fractures was 4.9% followed up for 2 years.The second fracture happened 3.7 years after the first one on average.Conclusion The risks of second fracture among patients with initial brittle fracture are substantial.Mobility skill assessment is an important risk factor for osteoporosis fractures recurrence.There is adequate time between fracture and re-fracture for effective interventions to prevent or reduce the risks of refracture,especially for the old women with a vertebral or hip fracture.Medication,motor function rehabilitation and fall-down prevention training would be helpful.
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Objective To observe the effect of treating acute spinal cord injury in rats by transplantation of autologous activated Schwann cells(AASCs). Methods Unilateral saphenous nerves were ligated directly, free it and culture Schwann cells 1 week later using the tissue clot method. Nerve growth factor(NGF) and brain-derived neurotrophic factor(BDNF) in medium were detected in different periods. 90 female Wistar rats[(200?30) g] were randomly assigned to 3 different study groups as follows: control group A(n=30): 20% DMEM injection; research group B(n=30): autologous Schwann cells(ASCs) transplantation; research group C (n=30): AASCs transplantation. The cells were purified before transplantion to the injuried T10 spinal cord site of rats (New York University type weight drop apparatus, NYU). The recoveries of the lower extremity were observed using Basso-Beattie-Bresnahan(BBB) locomotor scoring system and somatosensory evoked potential and motor evoked potentials(SEP & MEP). And then observe the coticospinal tract(CST) using the biotinylated dextran amine(BDA) tracing. Results BBB score was higher in research group than the control group 4 weeks after injury, the statistical difference was significant(P