BACKGROUND: Cell labeling and nuclear magnetic resonance can non-invasively in vivo label the region, existing mode and some bionomics of transplanted neural stem cells (NSCs). OBJECTIVE: To observe the outcome of superparamagnetic iron oxide in vitro labeled human NSCs. DESIGN, TIME AND SETTING: The cytology, in vitro, observation study was performed at the Laboratory of Department of Pediatrics, Navy General Hospital of Chinese PLA from December 2006 to June 2007. MATERIALS: Aborted human embryo was provided by Navy General Hospital of Chinese PLA. Superparamagnetic iron oxide (Lot number 97060601) was produced by Advanced magnetics,inc., USA. METHODS: Monoplast suspension was isolated from human embryo hippocampus using the mechanical method, and in vitro incubated in NSC medium, supplemented with epidermal growth factor and basic fibroblast growth factor. 11.2 g/L superparamagnetic iron oxide was diluted into 28 mg/L using NSC medium, mixed with 1.0 mg/L polylysine (8 ?L), and then made into superparamagnetic iron oxide-polylysine composite labeled medium. NSC spheres with active proliferation were obtained, made into single cell suspension (1?109/L), and then treated with serum-free medium containing superparamagnetic iron oxide. One week later, various cytokines were removed, and 5% fetal bovine serum was used for 24 hours to induce the NSC differentiation. MAIN OUTCOME MEASURES: Prussian blue staining was utilized to determine marking positive rate. Immunofluorescence staining was applied to detect glial fibrillary acidic protein and neurofilament expression. RESULTS: Superparamagnetic iron oxide labeled NSCs were yellow, and the speed of clone formation was not stepped down compared with the non-labeled cells. 20 hours following superparamagnetic iron oxide labeling, blue iron particles in NSCs cytoplasm were found, with the positive rate of 90%. Following induction, superparamagnetic iron oxide labeled NSCs were positive for glial fibrillary acidic protein and neurofilament. CONCLUSION: Superparamagnetic iron oxide can highly effectively label NSCs in vitro, and not affect biological features following labeling. NSCs can normally amplify and orientedly differentiate into neurons and astrocytes.

BACKGROUND:Lingo-1 has been identified as a negative regulator of oligodendrocyte differentiation and myelination, which may be closely related to the white matter damage, but there is no systematic report on the dynamic changes of Lingo-1 after white matter damage.
OBJECTIVE:Toexplore the dynamic expression of Lingo-1 at different time points after white matter injury in newborn rats.
METHODS:Seventy-eight Sprague-Dawley rats aged 3 days old were equaly and randomly divided into sham operation group and model group. In the model group, models of white matter injury were established by unilateral ligation of the right common carotid artery combined with hypoxia. In the sham operation group, the right common carotid artery was isolated only, without ligation or hypoxia.
RESULTSAND CONCLUSION:At 7 days after model induction, hematoxylin-eosin staining and immunohistochemical staining for myelin basic protein showed that a selective white matter injury was seen at the injury site of a rat model, suggesting successful model establishment. Fluorescent quantitative PCR and western blot assay results demonstrated that the expression levels of Lingo-1 mRNA and protein were significantly up-regulated at 1 day and reached a peak at 7 days post-surgery. After 7 days, above expression wasgradualy decreased and the up-regulation of Lingo-1 protein lasted to the 28 days post-surgery compared to the sham operation group. These results show that Lingo-1 protein was closely related to the brain white matter injury.

Objective Cell therapy is a possible effective way to treat myelination disorder diseases.Cell therapy needs to apply a method for culturing oligodendrocyte precursor cells.Therefore,this study was to develop a stable,efficient and economical method for obtaining human oligodendrocyte precursor cells (OPCs) in order to provide cell required for clinical treatment.And this will provide a new option for clinical applications.Methods Human OPCs were obtained through magnetic bead sorting and cultured in OPCs proliferation medium.For a short-time (2,4,6,8days) and long-time culture,morphology of OPCs was observed.The fourth generation of OPCs was analyzed for expression of OPCs specific markers O4,Soxl0 and platelet derived growth factor alpla receptors(PDGFαR) and the capacity to differentiate into oligodendrocytes by immunofluorescence staining.At the same time,the effects of B27 and N1 on OPCs growth state were inspected as well.Results For a short-time culture,OPCs had typical bipolar or tripolar morphology and proliferated in good condition.For a long-time culture,all 4 generations OPCs had typical bipolar or tripolar morphology;the fourth generation OPCs highly(＞ 90％) expressed 04,Sox10 and PDGFαR,after induction,OPCs could be differentiated into oligodendrocytes.After 4 generations of long-time culture,OPCs already maintained the original sharp,high purity and had the capacity to differentiate into oligodendrocytes.It was indicated that this culture system was suitable for human OPCs for a long-time culture.Conclusions Overall,using this culture system,isolated human OPCs not only can be stably cultured and proliferated in vitro,but also have the capacity to differentiate into oligodendrocytes.From this reproducible method,a large number of human OPCs can be stably obtained in vitro as convenient as possible.And this will provide a new option for clinical applications.This method uses fewer cytokines.Therefore,this method will provide stable,efficient and economical OPCs for cell therapy of myelination disorders or myelin damage diseases.

Objective To explore the protective effect of miconazole on white matter damage (WMD) in neonatal rats. Methods Three-day-old neonatal SD rats were randomly divided into sham group, WMD model group, 10 mg/(kg·d) miconazole group and 40 mg/(kg·d) miconazole group with 15 rats each. Rats in WMD model group were subjected to the ligation of right carotid artery, and then kept in a chamber with 6% oxygen and 94% nitrogen for 80 min to establish the white matter damage model. The rats in miconazole group were intraperitoneally injected with different doses (10 and 40mg/kg) of miconazole, dissolved in dimethyl sulfoxide (DMSO), for five consecutive days, and rats in WMD model group were injected with the same volume of DMSO. Myelin basic protein (MBP) of white matter was detected by immunofluorescence staining and western blot. Myelin sheaths of corpus callosum were observed by transmission electron microscopy. Weight changes of rats were compared among groups. Results Immunofluorescence staining and western blot showed that, after treatment with miconazole, the MBP expression level of corpus callosum was higher than in WMD model group (P<0.05). In WMD model group, the myelin sheath of corpus callosum had loose structure and a large number of small vacuoles with decreased thickness of myelin sheath. After treatment with miconazole, myelinolysis induced by anoxia and ischemia could be improved significantly. The increase in weight of rats in WMD model group was significantly less than that in sham group. And after miconazole treatment, the rate of weight gain of rats was increased. Conclusion Miconazole can significantly reduce the brain white matter damage induced by anoxia and ischemia through promoting myelination, and then improves the growth and development in rats.

ObjectiveTo explore the effect of different hypoxic duration on the brain white matter injury.Methods ewborn rats were randomly divided into two groups, normal group (n=24) and model group (n=45). The model group was di-vided into 3 subgroups (n=15) according to the time of hypoxia (50 min, 70 min, and 90 min). The animal model of white matter injury was established by unilateral carotid artery ligation in model group. After different duration of hypoxia, the mortality rate was recorded, the morphological changes of brain pathology was observed by hematoxylin eosin (HE) staining, myelin basic pro-tein (MPB) of white matter was detected by immunolfuorescence staining and motor function was evaluated by climbing slope test.ResultsThe mortality rates signiifcantly increased with prolonged hypoxia. The mortality rate was as high as 60% in 90 min subgroup. The HE staining showed that there were no obvious injury in 50 min subgroup, selective white matter injury on the operative side appeared in 70 min subgroup, and a wide range of infarction of white matter, hippocampus, and cortex appeared in 90 min subgroup. MBP semi-quantitative scores of white matter injury were higher in 70 min subgroup (3.89 ± 0.47) and 90 min subgroup (4.72 ± 0.57) than that in the normal group (0.06 ± 0.24), the difference was statistically signiifcant (P <0.05). In climb-ing slope test, the subgroups had different degrees of motor dysfunction on affected side with 90 min subgroup being the most serious.ConclusionsWhite matter injury model could be established by unilateral carotid artery ligation, and different hypoxic duration signiifcantly affects the range and degree of injury.

Objective To assess the clinical efficacy of human neural stem cell (hNSC) transplantation in the treatment of severe cerebral palsy (CP) in children.Methods hNSCs were obtained from the forebrain of 10 to 12-week-fetus.Forty children with CP were voluntarily received hNSC transplantation that were injected into cerebral ventricle.The development of motor and fine motor functions were evaluated by GMFM and PDMS-FM 1 month before hNSC transplantation.as well as 3 and 6 months after hNSC transplantation.Results Twenty six (65％) cases displayed improvement from day 5 to month 6 after hNSC transplantation.GMFM assessment showed that the percentage was (4.52±2.50) ％ 1 month before hNSC transplantation,(7.74±2.94) ％ 3 months after hNSC transplantation and (13.01±6.71)％ 6 months after hNSC transplantation,indicating a significant improvement by the treatment of hNSC transplantation(P＜0.05).The percentage in PDMS-FM evaluation was (15.01± 12.00)％,(20.34± 11.91) ％ and (30.02± 12.50) ％ one month before hNSC transplantation,3 and 6 months after hNSC transplantation,respectively,also suggesting a significant improvement induced by hNSC transplantation treatment (P＜0.05).Moreover,the developmental improvement was the most prominent among 1-3 months post hNSC transplantation.Then the development slowed down.Significantly,patients received no hNSC transplantation experienced serious adverse events or complications.Conclusions hNSC transplantation is an effective and safer therapy for severe CP.Future observations are needed to evaluate long-term clinical efficacy of the therapy.

Objective To identify the best technique of postmastectomy radiation therapy (PMRT).Methods Twenty-eight patients with stage Ⅱ or Ⅲ invasive breast cancer were treated with modified radical mastectomy and radiotherapy sequaciously involving the supraclavicular region and the chest wall.Three different techniques were developed for each patient:two tangential conformal fields ( half field) in the chest wall plus supraclavicular intensity modulated radiotherapy (3D-CRT + IMRT),integrated chest wall and supraclavicular IMRT(IMRT),and two tangential conformal fields (half field) in the chest wall plus single field electron beam radiotherapy in the supraclavicular region( 3D-CRT + E).The dose distributions of the target areas and the irradiated volumes of the ipsilateral lung ( V5,V10,V20,and V45)were estimated with the dosage volume histogram (DVH).The dosage prescription was 50.4 Gy (1.8 Gy × 28 f).Results The conformity index (CI) of the 3D-CRT + IMRT group was (0.61 ± 0.03),not different from that of the IMRT [ (0.62 ±0.03),q =2.16,P ＞0.05],and the CI levels of these 2 groups were both higher than that of the 3D-CRT + E group [ (0.44 ± 0.02 ),q =20.50,22.66,P ＜0.01 ].The heterogeneity index (HI) of the 3D-CRT + IMRT group was ( 1.17 ±0.02),not different from that of the IMRT [ (1.15 ±0.02),q =1.66,P ＞0.05],and the HI levels of these 2 groups were both lower than that of the 3D-CRT + E group[ ( 1.24 ±0.04),q =3.91,5.58,P ＜0.01 ].The levels of V5 and V10 of the ipsilateral lungs of the 3D-CRT + E group(48.70％ ±3.24％,38％.56％ ±3.70％ ) and 3D-CRT + IMRT group (49.12％ ±3.03％,38.38％ ± 3.56％ ) were all significantly lower than those of the IMRTgroup [(77.18％ ±8.01％,53.07％ ±6.85％),V5,q =20.35,20.05,P＜0.01; V10,q=12.10,12.24,P ＜0.01 ] and there were not significant differences in the V5 and V10 levels between the 3D-CRT + E and 3D-CRT + IMRT groups ( q =0.30,0.14,P ＞ 0.05 ).The levels of V20 of the ipsilateral lungs of the 3D-CRT + IMRT group (26.57％ ±2.51％ )and IMRT group (25.22％ ±2.77％) were all significantly lower that those of the 3D-CRT + E group [ (31.79％ ± 3.00％ ),q =5.27,8.21,P ＜ 0.01 ]and there were not significant differences in the V20 level between the 3D-CRT + IMRT and IMRT groups (q=2.76,P ＞ 0.05 ).There were not significant differences in the V45 levels among these 3 groups (F =0.69,P ＞ 0.05).Conclusions The 3D-CRT + IMRT technique in PMRT effectively reduces the radiated dose on the ipsilateral lung.

Objective To explore the effect of paracrine extracts derived from human adipose stem cells on white matter injury of neonatal rats and to compare the difference of therapeutive effect between the cerebellum medulla oblongata pool injection and the jugular vein injection.Method A total of 73 three-day-old SD rats were chosen to establish the model of white mater injury.After 24 hours,the 73 rats were randomized into the experimental group (n =46) and the control group (n =27).Then the experimental group was reclassified into ventricular group (n =23) and intravenous group (n =23).In the ventricular group,the paracrine extracts of human adipose stem cells was injected locally into the cerebellum medulla oblongata pool injection,while the extracts was injected into the jugular vein in the intravenous group.The control group was reclassified ventricular control group (n =15) and intravenous control group (n =12),and equivoluminal saline was injected the same way as the experimental group.Frozen sections of the brain tissue from 3 rats of each experimental group one day after injection were stained with fluorescein-conjugated streptavidin to study the distribution of the extracts.The brain tissue of 3 rats from each subgroup 3 days after injection were stained with hematoxylin eosin (HE) to observe the pathomorphological changes.While 7 days later,myelin basic protein (MBP) of white matter which was obtained from 7 rats of each group was detected by immunofluorescence staining.28 days after injection,the remaining rats were assessed by neurobehavior tests.For the rats that died during the experiment,the same number of the rats would be substituted in this study.Result The paracrine extracts were found to transfer to the brain lesion area,and the amount of the extracts was more in the ventricular group.The results of the HE staining showed that the white matter injury was more severe in the ventricular control group,and extensive area of infarction were found in this group.White matter injury was mild in the experimental group,and the structure of the corpus callosum was more complete in the ventricular group.MBP semi-quantitative scores of the ventricular group (0.7 ± 0.3) and intravenous group (1.7 ± 0.3) were lower than those of ventricular control group (3.4 ± 0.4)and intravenous control group(3.3 ±0.3).And the MBP scores of ventricular group was significantly lower than that of intravenous group (P ＜ 0.05).The scores of the neurobehavioral tests of the experimental group were significantly higher than those of the control group,while the scores of the ventricular group were significantly higher than those of the intravenous group (P ＜ 0.05).Conclusion The paracrine extracts derived from adipose stem cells could improve the prognosis of white matter injury,and cerebellum medulla oblongata pool injection showed better curative effect than the jugular vein injection.

Lingo-1 as a dark horse is a potent negative regulator of neuron and oligodendrocyte survival,neurite extension,axon regeneration,oligodendrocyte differentiation,axonal myelination and functional recovery in central nervous system injury.The structure,distribution and biological characteristics of Lingo-1 and its role in the central nervous system injury are collected and analyzed;the role and status of Lingo-1 in the field of nerve regeneration are further clarified.The further research of Lingo-1 will broaden the treatment method and target of new drugs in the field of nerve regeneration

The transcriptional repressor RE1 silencer transcription factor(NRSF/REST) is an important factor that restricts some neuronal traits in neurons.Since these traits are also present in pancreatic islet cells,NRSF-regulated genes involved in islet function are searched.A NRSE-like motif was analysed in human insulin promoter.The role of NRSE was evaluated by generating a model of insulin-secreting cells that firmly express NRSF.The presence of NRSF led to a decrease in activity of human insulin promoter by stable or transient transfection with human insulin-promoter luciferase.The predicted NRSE-like motif also confers NRSF-dependent transcriptional repression in the context of a surrogate gene promoter.Specific binding activity of NRSF/REST to the NRSE-like motif was confirmed by EMSA.Moreover,the binding activity is competed by consensus NRSE sequence.These data showed that human insulin promoter is regulated by the transcriptional repressor NRSF/REST via the NRSE-like motif.