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.

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 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.

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

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

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

Objective To investigate the clinical characteristics,diagnosis and treatment of senile primary central nervous system lymphoma.Methods The patients were divided into two groups,one group of the patients underwent chemotherapy and radiotherapy treatment following surgical resection,the other received a combined treatment of chemotherapy and radiotherapy only.The survival were analyzed with log-rank test.Results All of the lesions showed the iso-to slightly high density in the CT scan,and the boundary was unclear,surrounded by edema.Contrast enhancement was found in all of the patients.MRI showed iso-to low T1 and iso-to slightly high T2 signal intensity to the adjacent gray matter.The lesions were surrounded by the edema belt.Contrast enhancement such as rim enhancement or homogeneous enhancement was found in all.The pathological diagnosis was B cell lymphoma(n=32)and T cell lymphoma(n=4).In addition,all of the patients were excluded out of HIV infection and their immune function was normal.The median survival time of the group receiving surgery combination treatment and the group receiving just chemotherapy and radiotherapy were 19 months and 14 months,respectively.There was no significant statistics between the survival time of the two groups.Conclusion It is necessary to differentiate primary central nervous system lymphoma from glioma,metastatic tumor,meningoma,and so on.The surgical resection seems no help for those senile central nervous system lymphoma patients with multi-focus.The diagnosis should be suspected after MRI and obtained by stereotactic biopsy in order to avoid an unnecessary extend of surgery in primary central nervous system lymphoma(PCNSL).

BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei appearances. ②The growth curve showed that the growing adaptive phase was the first day after AAV-293 cells were passaged, the actively growing phase was from the second day to the fifth day, and the growing platform phase was after the sixth day. ③ AAV-293 cells with green fluorescence observed by fluorescence inverted microscope, and cotransfection of the there AAV system plasmids was successful. AAV-293 cells gave out green fluorescence after infected with AAV supernatant, and AAV package succeeded.CONCLUSION: The culture method established by the authors in the experiment is simple and useful, and the cultured AAV-293 cells remain a good function of AAV package.

Objective To evaluate the curative effect of salvage liver transplantation and repeated hepatectomy for recurrent hepatocel-lular carcinoma.Methods The data of 72 patients with recurrent hepatocellular carcinoma fulfilling Child-Pugh A and the Milan criteria from September 2004 to August 2010 were retrospectively studied.According to different treatments,53 patients were divided into repeated hepatec-tomy group,and 19 patients were divided into salvage liver transplantation group.The overall survival rates and disease-free survival rates after operation were evaluated by Kaplan-Meier method.COX proportional hazard was used for univariate analysis and multivariate analysis to eval-uate the risk factors for prognosis.Results The 1-year,3-year and 5-year survival rates were 86.79%,62.26% and 45.28% in repeated hepatectomy group,and 89.47%,68.42%and 57.89% in the salvage liver transplantation group respectively.There was no significant differ-ence in the overall survival rates between the two groups (χ2 =2.530,P =0.112).The 1-year,3-year and 5-year disease-free survival rates were 67.92%,47.17% and 35.85%in the repeated hepatectomy group,94.74%,68.42% and 52.63% in the salvage liver transplantation group respectively.There was a significant difference in the disease-free survival rates between the two groups(χ2 =4.395,P =0.036).The univariate analysis and multivariate analysis indicated that microvascular invasion,satellite lesion and multiple tumors were the independent risk factors to influence the survival.Conclusion The salvage liver transplantation obtains a better effect for the patients fulfilling Child-Pugh A and the Milan criteria,which is an effective method in the treatment of hepatocellular carcinoma.