Objective To explore the dissociation, purity and the culture method of spinal motoneurons in vitro and to find out an effective way to identify spinal motoneurons.Methods Spinal motoneurons were isolated from spinal cord of embryonic rat. Spinal tissues were digested and then the motoneurons through 6.8% metrizamide density gradient centrifugation, were obtained by collecting the top layer of metrizamide cushion. After the glia adhered to the plate wall, the enriched motoneurons were collected and plated in 24-well plates at a density of 4?10~5/ml. Cytosine arabinoside was added to the culture system after 24 hours in order to inhibit the outgrowth of glia. L-15 serum medium was used for the first 48 hours and then changed by serum-free medium. Later, neurons were cultured with 50% exchanged medium every two days. Spinal motoneurons were identified by immunostaining with polyclonal antibody to choline acetyltransferase (ChAT) according to ABC method. Laser scanning confocal microscope was used to observe its structure.Results Spinal motoneurons were immunoreactive to the antibody against ChAT. The present experiment revealed that ChAT-positive cells more than 85% might live about seven to nine days.Conclusions This study suggests a method of spinal motoneuron culture successful. Spinal motoneurons could be specially labeled with ChAT antibody.

Objective To study the immunoregulation effect of three kinds of polysaccharides extracted from different tissues of Octopus dollfusi on immunosuppression mice.Methods Two kinds of immunosuppression mice models were built by ip,cyclophosphamide(CY) and sc.hydrocortisone(HC),respectively.The spleen and thymus were weighed and the index of the immune organs was calculated.The white blood cells were counted by automated hematologic analyzer.Results In certain concentrations(20～80 mg?kg-1),the three kinds of polysaccharides could antagonize the atrophy of spleen and thymus caused by CY and HC,and increase the weight of spleen and thymus;The polysaccharides could protect against leukocytopenia induced by CY and HC,and especially in low and middle dose.Conclusion The results suggested that three kinds of polysaccharides extracted from Octopus dollfusi have potent enhancement effect on non-specific immunity for the immunosuppression mice.

Objective To Construct fusion gene DNA vaccine pcDNA3/STxB-VP1 and fusion gene DNA vaccine pcDNA3/MDC-VP1,then the two vaccines were inoculated to mice and stuy their immunological effects.Methods B subunit of Shiga Toxin(STxB)gene fragment were amplified by PCR from the DNA of Shigella dysenteriae and Macrophage-derived chemokine(MDC)gene fragment was amplified by RT-PCR from the total RNA of a mouse spleen cells.The DNA fragments of STxB and MDC waere respectively linked to coxsackievirus B3(CVB3)VP1 by a DNA sequence which encode a flexible polypeptide(15 amino acids)to construct eukaryotic expression plasmids pcDNA3/STxB-VP1 and pcDNA3/MDC-VP1.BALB/c mice were randomized to 6 groups which were respectively immunized pcDNA3,pcDNA3/STxB,pcDNA3/MDC,pcDNA3/VP1,pcDNA3/STxB-VP1 and pcDNA3/MDC-VP1 for 3 times at 3-week intervals,intramuscularly(i.m.)in tibialis anterior muscle.The levels of the serum neutralizing antibodies were detected 20d after each inoculation.The mice were challenged with 7 LD50 CVB3 3 weeks after the last immunization.Results The fusion gene vaccines pcDNA3/STxB-VP1 and pcDNA3/MDC-VP1 were constructed successfully.The survival rates of each group were 10%,10%,15%,40%,20% and 75%,respectively,and the levels of neutralizing antibody titers,virused titers,were all consistent with those survive rates in each group.Conclusion Comparing with pcDNA3/VP1,pcDNA3/MDC-VP1 vaccine can induce a high level of neutralizing antibody titers and result in a higher survival rate in mice,while pcDNA3/STxB-VP1 induce a low level of neutralizing antibody titers and result in a lower survival rate in mice.

Objective To realize the construction and expression of human dsFv genes to rabies virus. Methods The genes of dsFv VH and VL were amplified by PCR-based point mutagenesis strategy. Then the genes were cloned into plasmid pET-22b (+). E. coli BL21(DE3) was transformed with the recombinant expression plasmid and the protein was induced in Luris-Bertani medium by addition of IPTG. The inclusion body proteins of VH and VL were denaturalized by GuHCl, and then re-naturalized in refolding solution to form dsFv fragments. Afterwards, we evaluated the antigen-binding activity of the dsFv and its stability as compared with its originative scFv. Results The fragment genes of dsFv to rabies virus were constructed successfully by PCR-based point mutagenesis strategy. Sequence analysis proved that cysteines were introduced into the position 44 aa of VH and position 100 aa of VL. The dsFv VH and VL genes were expressed in PET22b(+)/BL21(DE3). The VH and VL protein folded into the active dsFv antibody fragments which showed specific binding capability to rabies virus. Conclusion We succeeded in achieving the stabilization of the human scFv to rabies virus and obtaining the active human dsFv antibody fragments. This established a solid basis for further study of the biological activaty and clinical application of dsFv to rabies virus.

0.05).However,There were significantly positive correlation between MMP-9 level and abnormal volume on mean transit time map(r=0.371,P=0.026),time to peak map(r=0.379,P=0.023),cerebral blood flow map(r=0.447,P=0.006) and great vessel occlution(r=0.416,P=0.004) on magnetic resonance.Conclusions The serum MMP-9 level of acute cerebral infarction are significantly positively correlated with the change of magnetic resonance,but there are no correlation between MMP-2 and MMP-13 levels and the change of magnetic resonance.It suggests that the MMP-9 may be involved in the pathology of acute cerebral ischemia.

Objective To investigate the changes of leukocyte derived cytokines,IL 1?,IL 6,IL 8,and TNF ?,in platelets concentrates (PCs) during storage,and to study the effect of leukodepletion on cytokine concentration in PCs and the incidence rate of febrile non hemolytic transfusion reaction (FNHTR).Methods 8 bags of apheresed PCs from 8 voluntary donors were each divided into two identical portions (A and B).Group A underwent leukocyte filtration,while group B did not.Then both groups were stored at 22℃ for 5 days.The levels of cytokines (pg/ml),white blood cell (WBC) count and platelet count in both groups were tested on the day 0,3 and 5 during storage.In clinic,240 patients were randomly divided into two groups with 120 in each group to receive non filtered or filtered PCs (1 bag/patient).The incidence of FNHTR was investigated.Paired t test and ? 2 test were used in data analysis. Results There was a significant positive correlation between the content of WBC in PCs and the level of cytokines.On the 5th day of storage,residual WBC in leukoreduced PCs were less than 1?10 6/PC,the levels of cytokines remained the same as those on 0;however,WBC in non filtered PCs was (351?81)?10 6/PC,and the levels of IL 1??IL 6?IL 8 and TNF ? were 105.0pg/ml,269.0pg/ml,1840pg/ml and 42.0pg/ml respectively and increased 18 ( P

Objective To explore the application of lentil lectin‐reactive alpha‐fetoprotein ratio (AFP‐L3% ) applied in the dif‐ferential diagnosis between hepatitis B infection of primary liver cancer and benign liver disease .Methods We included 108 cases of chronic HBV infection ,including 50 cases of primary liver cancer ,42 cases of cirrhosis ,16 cases of chronic hepatitis .Chemilumines‐cence detection was used to detect alpha‐fetoprotein (AFP) and AFP‐L3 content ,AFP‐L3 and the ratio of AFP (AFP‐L3% ) was calculated .Results AFP≥400 ng/mL as primary liver cancer diagnostic threshold ,the sensitivity and specificity were 36% ,84% , when used AFP‐L3% ≥ 10% as primary liver cancer diagnostic threshold ,the sensitivity and specificity were 62% ,83% . Conclusion AFP‐L3% is a better clinical indicator to distinguish between primary liver cancer and benign liver disease .AFP‐L3%can be used as a clinical indicator to differential diagnosis between HBV infection of primary liver cancer and benign liver disease .

OBJECTIVEA study was conducted to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of regulated upon activation normal T-cell expressed and secreted (RANTES) and fractalkine in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were incubated with different concentrations of Pg-LPS (200, 500, and 1000 ng x mL(-1)) for 1, 6, 12, and 24 h, respectively. Then real time quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent method (ELISA) were adopted to detect the protein levels and mRNA levels of RANTES and fractalkine.

RESULTSThe RANTES protein levels and mRNA levels, as well as fractalkine mRNA levels, were significantly higher in all experimental groups of 1, 6, and 12 h than in the control group (P<0.05), except the expression of RANTES mRNA in 200 ng x mL(-1) group of 12 h and RANTES protein in 200 ng x mL(-1) group of 1 h. The expression levels of RANTES mRNA and fractalkine mRNA were highest in 1000 ng x mL(-1) group of 6 h and were 4.88- and 6.20-fold higher, respectively, than those in the control group. The expression levels of RANTES protein, mRNA, and fractalkine mRNA decreased 6 h after stimulation, and were significantly higher than those in the control group (P<0.05) in the RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis 500 ng x mL(-1) group of 24 h. There was a significant difference between the expression of fractalkine mRNA in 1000 ng x mL(-1) group of 6 and 12 h than in the control group (P<0.05).

CONCLUSIONPg-LPS infection might up-regulate the expression of RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis.

Atherosclerosis ; Cells, Cultured ; Chemokine CCL5 ; genetics ; metabolism ; Chemokine CX3CL1 ; analysis ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Porphyromonas gingivalis ; immunology ; isolation & purification ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

BACKGROUND: Skeletal muscle satellite cells are muscle-derived stem cells with proliferation and differentiation potential. Recently, foreign researches have reported that skeletal muscle satellite cells can be activated by some definite microenvironmental factors and differentiate into hematopoietic stem cells and thereby they will have the potential of hematopoietic reconstruction.OBJECTIVE: To initially validate the potential of adult muscle-derived stem cells- skeletal muscle satellite cells differentiating into hematopoietic stem cells.DESIGN: Validation animal experiment.SETTING: Department of Histology and Embryology, College of Basic Medical Science, Tianjin University of Traditional Chinese Medicine.MATERIALS: Sixty-five male Kunming adult mice, weighing 25-28 g, were involved in this study. Five Kunming neonate rats, aged 5 days, were provided by the Laboratory Animal Center, Department of Medicine, Peking University.METHODS: This experiment was carried out in the Laboratory for Cell Culture, Department of Human Anatomy and Histo-embryology, School of Basic Medical Sciences, Peking University Health Science Center between August 2001 and August 2003. Skeletal muscle satellite cells of 5 neonate rats were isolated by collagenase and trypsin digestion. Bone marrow mononuclear cells of 5 adult Kunming mice were isolated. Sixty adult female mice were used as recipients, irradiated with 60Coγ 8.0 Gy and then randomized into 4 groups: control group, in which, the mice were untouched; culture fluid infusion group, in which, the mice were injected with DMEM/F-12 medium through caudal vein; satellite cell infusion group, in which, the mice were injected with 0.3 mL satellite cell suspension through caudal vein (cell concentration 1×109 L-1); bone marrow-derived cell infusion group, in which, the mice were injected with 0.3 mL bone marrow-derived cell suspension (cell concentration 1×109 L-1) through caudal vein.MAIN OUTCOME MEASURES:①The survival rate of 14-day-old mice in each group. ②The surviving recipient mice were euthanized 14 days after irradiation, and tubercles on the surface of spleen were counted by naked observation; Bone marrow mononuclear cell smear was stained by Wright-Gimesa.RESULTS:① Determination of colony forming unit-spleen (CFU-S): No significant difference in the number of spleen tubercles of mice existed between satellite cell infusion group and bone marrow-derived cell infusion group 14 days after irradiation (P＞0.05). ②Histological identification of bone marrow-derived mononuclear cells: Many hematopoietic cells appeared at the early stage in the bone marrow-derived mononuclear cell smears between satellite cell infusion group and bone marrow-derived cell infusion group. Their morphology meets the biological characteristics of hematopoietic cells at the early stage. ③ The survival condition of irradiated mice: All the mice in the control group and culture fluid infusion group died 9 to 13 days after irradiation. In contrast, 8 mice from the satellite cell infusion group and 13 the bone marrow-derived cell infusion group survived 14 days after irradiation.CONCLUSION: Skeletal muscle satellite cells have the function of differentiating into hematopoietic stem cells.