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 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.

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 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.

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 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 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 .

According to years of treatment experience on herpes zoster,combined with its current onset characteristics as well as modem medicine,the authors probed into the relation between liver depression and herpes zoster on the following two aspects:one was liver depression and healthy qi (immunity) ; the other was liver depression and the incidence of herpes zoster.We considered that long-term liver depression impairs healthy qi.Then herpes zoster appears because detriment of healthy qi leading to decreased immunity,reactivation and reproduction of virus.Liver depression is a significant factor for the onset of herpes zoster.

Aim To investigate the effect of resveratrol on proliferation and differentiation in K562 cells. Methods K562 cells were treated with different con-centrations of resveratrol for 6d. The colony number of K562 cells was detected with semi-solid culture assay. Expression of GATA-1 and PU. 1 in K562 cells was re-spectively measured with immunocytochemistry and Western blot. Expression of differentiation related anti-gen, CD11b, CD14 and CD42b, was measured with flowcytometry on K562 cells. Results Resveratrol
could significantly decrease the colony number of K562 cells in a dose-dependent manner, and enhance the ex-pression of GATA-1,PU. 1,CD11b, CD14 and CD42b in K562 cells. Conclusion Resveratrol could inhibit the proliferation and induce differentiation of K562 cells via up-regulating the expression of GATA-1 and PU. 1 protein.

Objective To analyze the clinical manifestations and the features of brain MRI and cerebrospinal fluid (CSF) findings in adult Chinese patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis.Methods We reviewed the clinical manifestations,brain MRI and CSF examinations of 29 patients who were diagnosed as anti-NMDAR encephalitis.Results The major clinical features of anti-NMDAR encephalitis patients included psychiatric symptoms (86％,25/29),seizures (83％,24/29),decreased consciousness (55％,16/29),involuntary movements (55％,16/29),central hypoventilation (34％,10/29),and hypersalivation (17％,5/29).Some patients also experienced autonomic instability,hemiplegia and aphasia.Underlying ovarian teratoma was identified in 14％ of affected patients(4/29).Brain MRI was found abnormal in up to 62％ patients (18/29),located in the temporal lobes,hippocampus,thalamus,brain stem,cingulate gyrus,frontal and parietal cortex,corpus callosum,internal capsule,basal ganglia and periventricular area.CSF findings were abnormal in 83％ of patients with anti-NMDAR encephalitis.Oligoclonal banding in CSF was positive in 95％ patients (19/20).The recurrence rate during 3 years was 31％ (9/29).Conclusions Anti-NMDAR encephalitis is a treatable disease,yet with high recurrence rate.Its predominant clinical features are psychiatric symptoms and seizures,while involuntary movements,central hypoventilation and hypersalivation are its characteristic manifestations.Lesions in MRI are widespread,not only restricted to limbic lobe.