BACKGROUND: Human embryonic stem cells (hESCs) are pluripotent cells which may differentiate into tissues of all three germ layers. Such research as the feeder-free growth of hESCs is few in China. Fibroblast growth factor (FGF) is a major factor to maintain the undifferentiated state of hESCs.OBJECTIVE: To evaluate the ability of FGF at different concentrations in maintaining the undifferentiated state and pluripotency of hESC lines in the long-term culture.METHODS: Two cell lines of hES-8 and hES-18 were cultured with mouse embryonic fibroblast condition medium for 3 passages and then transferred into mouse embryonic fibroblast condition medium containing different concentrations of FGF: 100, 160, 250 μg/L for 8 passages. The hESCs were removed from the petri dish, cell clusters were digested with collagenase IV and gathered. Cell differentiation and pluripotency were observed. The eighth generation of the hESCs were collected and incubated into severe combined immunodeficiency mice, so as to observe teratoma formation. Morphologies of the cells were evaluated. Alkaline phosphatase staining, surface labeling immunocytochemical analysis and RT-PCR assay method were utilized to determine the OCT-4 expression and tumorigenesis in vivo.RESULTS AND CONCLUSION: Cultured in mouse embryonic fibroblast condition medium containing 160 and 250 μg/L FGF, two cell lines of hESC could maintain undifferentiated state: Clones were round with a high ratio of nucleus to cytoplasm. Large areas in the center of clones were undifferentiated cells, while surrounding the clones were differentiated cells; Strong positive expression for alkaline phosphatase staining was observed; Two cell lines showed high levels of OCT-4 transcription factor protein; The surface markers SSEA-4, TRA-1-60, TRA-1-81 were all positive on both two lines; The hESC clusters could form embryoid body in vivo 10 days later; 3 germ layers of teratomas were also obtained after implanted into severe combined immunodeficiency mice. Mouse embryonic fibroblast condition medium containing 100 μg/L FGF was not sufficient to maintain the long-term proliferation of hESCs, and most of the cells differentiated and died after 4 passages. Alone with concentration 160 μg/LbFGF or more could maintain two hESC lines undifferentiated stably in vitro, has no influence on the differentiation and totipotency of two cell lines.

The synapsins are a family of neuron specific phosphoproteins associated with the membranes of synaptic vesicles. The synapsins play important roles in the neurotransmitter release and during the early neuronal development. In this review, we focus on the family members, gene location, distribution, structure and function of the synapsins.

AIM: Integrins act as receptors for molecules of the extracellular matrix (ECM) and play a major role in mediating cell morphology change,metabolism,function,migration,proliferation and differentiation. It is still unclear that whether ECM is associated with regulation of microglial activation and integrin expression. The study investigated the effects of three different ECM (fibronectin, vitronectin and laminin) on the expression of rat microglia integrin. METHODS:Experiments were performed in Research Center for Clinical Neurology, Tongji Medical College of Huazhong University of Science and Technology from May 2006 to August 2007. ①Neonatal Wistar rats were bought from Experimental Animal Center of Tongji Medical College. Animal intervention met the animal ethical standard. ②Rat microglia was cultured and purified. Culture plate was coated with fibronectin, vitronectin and laminin, and non-ECM served as control. ③The influence of individual ECM on microglia integrin expression and microglia surface expression of MHC Ⅰ by flow cytometry. RESULTS:①MHC classⅠexpression on rat microglia was significantly increased by fibronectin and vitronectin(P

Objective:To study if embryonic stem cell derived dendritic cells(esDCs) could induce transplant tolerance to syngeneic neural progenitor cells ( NPCs) in ischemic rat brain.Methods:Neural progenitor cells ( NPCs) were differentiated from 129/svj pCX-eGFP ES-D3 embryonic stem cells and dendritic cells were directly differentiated from 129/svj ES-D3 respectively.All of SD rats were accepted MCAo surgery and subdivided in two groups based on pretreatment with or without esDCs through tail vein injection 1 week after MCAo.pCX-eGFP NPCs were then injected into the lateral ventricle of animals 2 weeks after MCAo.A proliferation assay of lymphocytes dissociated from cervical lymph nodes by MTT method,counting of the survival of the grafted cells, histological evaluation of CD4,CD8 and ED1 positive cells in brain and detection of mRNA level of IL-10 and IFN-γin ischemic lesions by reverse transcriptase-polymerase chain reaction(RT-PCR) were performed 2 weeks after graft (4 weeks after MCAo).Results:Pre-treatment with esDCs decreased CD4 positive cells infiltration (134.7 ±36.2 vs.198.8 ±59.6,P<0.05) and enhance the survival of NPCs in ischemic striatum,but had no effect on ED1 (298.8 ±75.9 vs.302.2±88.5),CD8 positive cells (145.8 ±45.4 vs.134.3 ± 39.0) distribution (P>0.05).There was also no difference in lymphocytes proliferation (PI,1.245 ±0.211 vs.1.331 ±0.235) or mRNA expression level of IL-10 ( 1.147 ±0.260 vs.1.264 ±0.119 ) and IFN-γ( 1.697 ±0.273 vs.1.829 ±0.250 ) between two groups ( P>0.05).Conclusion:The results indicate that pretreatment with esDCs may prolong syngeneic NPCs survival though reducing CD4 positive cells reaction in ischemic striatum,which provides some evidence for the tolerogenic function of esDCs.However,there was lack of evidence for cytokine-dependent routine involving in this mode and further investigation was needed to make certain the cardinal principle.

AIM: To investigate the kinetics of interleukin-6(IL-6) protein and mRNA expressions in the co-culture of rat brain vascular endothelial cells(VECs) with fibrin.METHODS: The IL-6 concentration were measured by rat IL-6 ELISA kit and the IL-6 mRNA was measured by real-time PCR after development an in vitro model of in situ fibrin polymerization on rat brain vascular endothelial cells.RESULTS: Fibrin induced IL-6 expression.The IL-6 antigen concentration increased significantly in 1.0 g/L media group compared to the low dose fibrin group or control media group(P

Objective To investigate the expressions of hypoxia-inducible factor-1 alpha (HIF-1?) and its relation with secondary brain damage in perihematomal issue in human intracerebral hemorrhage. Methods Perihematomal brain issue were collected in the course of evacuation of hematoma in 32 patients with hypertensive intracerebral hemorrhage. Expressions of HIF-1? were observed by immunohistochemistry and the neuronal apoptosis were observed by terminal deoxynucleotidyl transterrase mediate dUIP nick end labeling (TUNEL) staining and HE staining. Results HIF-1? protein immunohistochemical staining positive cells ((2.8?0.8)/HP) were identified dispersedly from 4 h after acute hemorrhagic stroke in perihematomal brain issue, and reached the peak at 24—48 h ((12.5?3.9)/HP). The expressions of HIF-1? kept high at 49—72 h ((12.2?1.8)/HP) after acute hemorrhagic stroke. Nervous cells and vascular endothelium cells had been swelled at 4 h after acute hemorrhagic stroke. TUNEL positive cells appeared from 12 h ((11.2?4.1)/HP), increased markedly at 24—48 h ((29.7?8.4)/HP), and reached the peak ((33.2?4.3)/HP) at 49—72 h after acute hemorrhagic stroke. There was a statistically significant correlation between HIF-1? expressions and TUNEL positive cells (r=0.788, t=7.02, P

Using different models of focal cerebral ischemia, the temporal and spatial rules of metabolism and energy changes in the post-ischemia brain tissue were measured by proton magnetic resonance spectroscopy (1HMRS) to provide valuable information for judging the prognosis of acute focal cerebral ischemia and carrying out effective therapy. Nine healthy Sprague-Dawly rats (both sexes) were randomly divided into two groups: The rats in the group A (n = 4) were occluded with self-thrombus for 1 h; The rats in the group B (n = 5) were occluded with thread-emboli for 1 h. The 1H MRS at 30, 40, 50, 60 min respectively was examined and the metabolic changes of NAA, Cho and Lac in the regions of interest were semiquantitatively analyzed. The spectrum integral calculus area ratio of NAA, Cho, Lac to Pcr + Cr was set as the criterion. The values of NAA.Cho in the regions of interest were declined gradually within 1 h after ischemia, especially, the ratio of Cho/(Pcr + Cr), NAA/(Pcr + Cr) at 60 min had significant difference with that at 50 min (P < 0.05). The ratio of Lac/(Pcr + Cr) began to decrease at 40 min from initial increase of Lac in both A and B groups. MR proton spectrum analysis was a non-invasive, direct and comprehensive tool for the study of cellular metabolism and the status of the biochemical energy in acute ischemia stroke.
Brain Ischemia/*diagnosis ; Energy Metabolism ; Infarction, Middle Cerebral Artery/diagnosis ; *Magnetic Resonance Spectroscopy ; Phosphorylcholine/metabolism ; Random Allocation ; Rats, Sprague-Dawley

The article introduces the pathogenesis of genetic and acquired thrombophilia and elucidates the relationships among thrombophilia, pediatric stroke, adult ischemic stroke and cerebral venous thrombosis. Meanwhile, it briefly introduces the relationships between acquired thrombophilia and cerebrovascular diseases.

Objective To investigate the dynamic changes of ATP content in rat cerebral cortex after transient ischemia followed by reperfusion and the relationship between the change of energy and the recovery of neural function.Methods The rats were subjected to 10 min of middle cerebral artery occlusion (MCAO). At the time point of 0 h, 1 h, 3 h, 6 h, 12 h, 24 h and 72 h after reperfusion, ATP contents of frontal and parietal cortex were measured by capillary zone electrophoresis.Results At the end of 10 min ischemia, ATP content fell dramatically to less than 20% of the control level. After reperfusion, ATP content recovered gradually. After 1 h, 3 h, 6 h and 12 h of reperfusion, ATP content returned to 70.5%, 65.7%, 84.8% and 86.9% of the control level ( P=0.052, 0.030, 0.332 and 0.491). From 24 h on until 72 h after reperfusion, ATP content decreased again, reaching half of the control level ( P=0.003 and P=0.023). After 10 min ischemia, limb function recovered gradually and completely at last. From 24 h on until 72 h after reperfusion, unwillingness of action and eating was found.Conclusions The recovery of cellular energy system function is delayed even though the reperfusion is in time after transient cerebral ischemia. Furthermore, secondary failure of cellular energy system function occurrs with the reperfusion proceeding. These phenomena are probably responsible for the delayed recovery of neural function after cerebral ischemia in spite of reperfusion.

Objective To find a new source to produce hESC lines.Methods D3 embryos with low morphological scores were cultured to blastocyst stage.Trophectoderm cells were separated from the ICMs by immunosurgery and isolated ICMs were cultured for 5-8 days on mitomycin-treated mouse embryonic fibroblasts(MEFs).Colonies derived from the ICMs were passed every 4-7 days and evaluated for cell surface markers including AKP,OCT-4,SSEA-4,SSEA-1,TRA-1-60,TRA-1-81,differentiation potentials and karyotypes.Results A total of 19 blastocysts were obtained from 130 embryos(quality score