Objective To investigate the effects of a highly selective dopamine D4 receptor (ABT-724)on behaviors in Spontaneously hypertensive rats (SHR),a rat model of attention-deficit hyperactivity disorder (ADHD).Methods After intervention of methylphenidate(5mg/kg) and three different doses of ABT-724(0.04mg/kg,0.16mg/kg,0.64mg/kg),behaviors of SHR were verified by open-field test,Morris water maze and Làt maze.Results Number of square crossing after intervention of methylphenidate and ABT-724 in SHR( 70.67 ± 8.59,76.50 ± 10.75,79.17 ± 10.44,65.67 ± 20.62) was less than the saline control group( 130.33 ± 1 1.40) (P＜0.05).During Morris water maze,SHRs(52.50 ± 4.04,52.17 ± 2.99,61 ± 8.15,53.83 ± 9.87 ) after intervention of both had a better spatial memory ability than control group(38.83 ±7.17) (P＜0.05).In Làt maze,number of rearing after intervention of both in SHRs(57.17 ± 5.67,60.83 ± 8.28,55.17 ± 9.45,65.33 ± 9.50 ) was less than saline control group(78.00 ± 13.84) (P＜0.05).Conclusion ABT-724 could improve behaviors of spontaneous locomotor activity,cognitive ability,non-selective attention in SHR.

Objective To study on the effect of DNA vaccine, p(Aβ3-10) 10-mIL-4, immunization on behavior and brain Aβ deposition in APP/PS1 transgenic mice.Methods DNA vaccine p(Aβ3-10) 10-mIL-4 was constructed which expressed fusion protein of ten tandem repeats of Aβ3-10 and mouse IL-4.APP/PS1 transgenic mice were vaccinated with p (Aβ3-10) 10-mIL-4.Aβ42 peptide and pcDNA3.1 (+) were injected to the control groups.Anti-Aβ antibody titers were detected before and after vaccination by enzyme-linked immunosorbent assay (ELISA) ,the spatial learning and memory ability of the mice were evaluated by Morris water maze.The senile plaques in mouse brains were detected by immunohistochemistry.Results Compare with pcDNA3.1 (+) group,immunization with the vaccine p (Aβ3-10) 10-mIL-4 in APP/PS1 mice induced high-titer Anti-Aβ antibodies ((27.49±4.51) μg/ml, P＜0.01) ,decreased Aβ deposition (reduced senile plaque number in the cortex and hippocampus by 52.86％ and 58.29％ respectively, P＜0.01) and improved their cognitive ability.Conclusions The immunization with p(Aβ3-10)10-mIL-4 in APP/PS1 mice achieve an ideal effect of vaccination, and p(Aβ3-10) 10-mIL-4 can be an alternative Alzheimer's disease vaccine to further study.

OBJECTIVE To compare the effect of four 5-hydroxytryptamine type 4 (5-HT4) receptor agonists:cisapride,zacopride,macopride and 2-[1-(4-piperonyl) piperazinyl]-benzothiazole (BZTZ),on rat cardiac inward rectifier potassium channel (IK1)and heart rhythm.METHODS The whole-cell configuration of patch-clamp technique was used to record effects of 5-HT4 receptor agonists onIk1 in enzymatic dissociated rat ventricular myocytes or Kir2.1 transfected HEK 293 cells.Western blotting was used to observe the expression of Kir2.1 channel exposed 24 h to agents in ventricular myocytes.Langendorff-perfused hearts were perfused with four agents respectively for 30 min.The electrocardiogram was recorded simultaneously.RESULTS BZTZ,cisapride and mosapride 0.1-10 μmol· L-1 decreasedIk1 in a concentrationdependent manner.At the same concentration (1 μmol· L-1),BZTZ showed the most potent inhibition onIκ1 (P＜0.01),followed by cisapride.Mosapride showed slight inhibition efficiency.However,zacopride enhanced Iκ1 (P＜0.01).In Kir2.1 heterologous expression systems,zacopride activated Kir2.1 current (P＜0.01) while mosapride had no effect.In ex vivo Langendorff-perfused hearts,BZTZ and cisapride 1μmol· L-1 elicited singnificant rhythm disturbances,and the total of premature ventricular beats (PVB) were 159±28 and 61±13.50％ (4/8) (P＜0.05) and 25％ (1/8) of the hearts exhibited ventricular tachycardia (VT),while 37.5％ (3/8) and 12.5％ (1/8) of the hearts exhibited ventricular fibrillation (VF),respectively.Mosapride and zacopride had no side effects on heart rhythm.Zacopride also suppressed BZTZ-or cisapride-induced arrhythmias.BZFZ had the strongest proarryhthmic potency among the 5-HT4 agonists,followed by cisapride,mosapride and zacopride.CONCLUSION Iκ1 might be an independent risk factor for arrhythmogenesis and a new target for screening safe 5-HT4 receptor agonists and gastrointestinal prokinetic agents.

BACKGROUND:For pediatric patients with aplastic anemia in China, it is difficult to find human leucocyte antigen-matched sibling donors that are mostly replaced by parental donors.
OBJECTIVE:To retrospectively analyze the clinical efficacy and safety of parental haploidentical peripheral blood hematopoietic stem cel transplantation in children with relapsed and refractory severe aplastic anemia.
METHODS:Seventeen children with relapsed and refractory severe aplastic anemia who had no matched sibling or unrelated donor and failed to respond to immunosuppressive therapy were subjected to parental haploidentical peripheral blood hematopoietic stem cel transplantation. A conditioning regimen of fludarabine+cyclophosphamide+rabbit anti-human thymocyte immunoglobulin antibody and the triple therapy of methotrexate, cyclosporine A and mycophenolate mofetil were applied to prevent graft-versus-host disease.
RESULTS AND CONCLUSION: (1) Of the 17 children, 16 cases (94%) reached hematopoietic reconstitution, and the median time of neutrophils≥ 0.5×109/L and platelets≥ 20×109/L was 13 (11-15) days and 17 (12-28) days, respectively. (2) Incidence of acute graft-versus-host disease was 47% (8 of 17 cases), including 29% (5/17) of grades I-II and 18% (3/17) of grades III-IV. Incidence of chronic graft-versus-host disease was 41% (7/17). (3) With a median folow-up duration of 268 (43-753) days, the overal survival rate was 70.6% (12/17). Five dead cases (29%) belonged to transplantation-related death, including one case of fungal skin infections, one case of graft-versus-host disease, three cases of severe lung infection. No relapse case was reported. These findings indicate that if there are no matched sibling or unrelated donors and the immunosuppression effect is poor, parental haploidentical peripheral blood hematopoietic stem cel transplantation is a safe and effective salvage treatment for children with relapsed and refractory severe aplastic anemia.

Objective:To construct pGEX-3X/hTSHa Escherichia coli(E.coli)expression system and prepare purified recombinant GST-recombinant human thyroid stimulating hormone(rhTSH)α protein.Methods:The complete coding sequence of hTSHα was obtained by RT-PCR with total RNA extracted from fresh chorial tissue as the template,and thereafter cloned into expression vector pGEX-3X by EcoRl and BamHI digestion.The recombinant plasmid was transformed into E.coli Mach1-T1 and then induced expression by WrG.The GST-rhTSHα fusion protein was identified by SDS-PAGE and its antigenicity was verified by a modified competitive ELISA.Results:A specific protein band of 36 ku,in accordance with predicted molecular weight,could be visualized in SDS-PAGE.As the result of ELISA,the recombinant GST-hTSHα protein can inhibit the intact TSH molecular binding with anti-TSHα antibody in a dose dependent manner.Conclusion:The cDNA of hTSHα was cloned and the recombinant expression vector pGEX-3X/hTSHα was constructed successfully.The recombinant GST-rhTSHα protein could be highly expressed in E.coli Machl-T1 and was approved of possessing antigenicity.

Aim To examine the influence of tetramethylpyrazine on learning and memory function of hypoxic hypoxia rats, and the expression of gamma aminobutyric acid(GABA) receptor and forkhead box P2(FOXP2) in hippocampus of rats.Methods A total of 120 Sprague Dawley rats were randomly divided into low hypoxic hypoxia and high hypoxic hypoxia groups, then according to different time points every group was divided into 1 d, 3 d, 7 d 15 d, 30 d group, with 12 rats per each group.Experiment group and the control group were treated with tetramethylpyrazine and 0.9% normal saline, respectively.The hypoxic hypoxia environment was achieved by putting the rats in a hypobaric chamber at a simulated altitude of 5 500 meters for different days.The capabilities of learning and memory of rats were detected by Morris water maze test.The expression of GABA receptor and FOXP2 protein in hippocampus of rat was determined by Western blot.Results ① Morris water maze test showed that the total distance of rats in the simulated hypobaric hypoxia control group was longer than that in the tetramethylpyrazine group(P<0.01) and the number of crossing the bestride platform increased compared with that in the hypobaric hypoxia control group(P<0.01) from the third day;② Western blot results showed that the expression of GABAAα1 receptor have no statistical significance(P>0.05);however,GABAB1 receptor and FOXP2 protein rose from the third day(P<0.05).The expression of GABAAα1 receptor and FOXP2 protein expression were correlated to total distance of Morris water maze in the control group(r=-0.738, P<0.05;r=-0.693, P<0.05), and the expression of GABAB1 receptor was correlated with FOXP2 protein level(r=0.834, P<0.05).Conclusion The simulated high-altitude hypobaric hypoxia can decrease the learning and memory abilities of rats, which may be ameliorated by tetramethylpyrazine intervention, and this effect might be related to the increase of GABAB1R receptor and FOXP2 expression in hippocampus of rats.

Objective:To construct suitable vectors for the secretory expression of hirudin in Pichia pastoris.Methods:The α-facor-hirudin gene was amplified from pPIC9-hirudin by PCR and sub-cloned into PA0815.The multi-copy recombinant plasmid pA0815-(α-Hirudin)n was constructed.The recombinant was transformed into P.pastoris strain GS115 for induction expression and then the activity of secreted products was identified.Results:A new multi-copy vector pA0815-(αHirudin)n was successfully constructed and was capable of secreting recombinant hirudin efficiently,which was confirmed respectively by PCR and SDS-PAGE.The products possessed the activity of thrombin inhibitor.Conclusion:This result offers efficient P.pastoris stains for mass production of biological active hirudin.

Objective To study the validity of RAND-UCLA (Rand Corporation and University of California at Los Angeles) consensus panel method in developing guidelines of referral indications for low back pain (LBP).Methods Evidence-based clinical guidelines for LBP management at community level and its referral guidelines published since 2001 and other tools were retrieved with varied tools.All clinical guidelines met inclusion criteria were evaluated with clinical studies and evaluation tools (AGREE).An pool of indication items was established based on evidence for developing referral indications for LBP, which were added by RAND-UCLA consensus panel method, and alternative referral indications were selected and clinical guidelines for LBP referral were established.Results A total of 15 copies of clinical guidelines from nine countries or regions were included in it after critical appraisal.Four copies of referral guidelines from two countries were included.Referral indications for LBP were derived directly from the RAND-UCLA consensus panel process, consisting of 44 referral indications for three groups (immediate, urgent and routine referral).Conclusions The RAND-UCLA consensus panel method is a more useful and practical tool in developing clinical guidelines, referral guidelines, which is worthwhile being recommended and spread.

Objective: To evaluate the reliability and validity of warning signs checklist developed by the National Health and Family Planning Commission of the People′s Republic of China (NHFPC), so as to determine the screening effectiveness of warning signs on developmental problems of early childhood. Method: Stratified random sampling method was used to assess the reliability and validity of checklist of warning sign and 2 110 children 0 to 6 years of age(1 513 low-risk subjects and 597 high-risk subjects) were recruited from 11 provinces of China. The reliability evaluation for the warning signs included the test-retest reliability and interrater reliability. With the use of Age and Stage Questionnaire (ASQ) and Gesell Development Diagnosis Scale (GESELL) as the criterion scales, criterion validity was assessed by determining the correlation and consistency between the screening results of warning signs and the criterion scales. Result: In terms of the warning signs, the screening positive rates at different ages ranged from 10.8%(21/141) to 26.2%(51/137). The median (interquartile) testing time for each subject was 1(0.6) minute. Both the test-retest reliability and interrater reliability of warning signs reached 0.7 or above, indicating that the stability was good. In terms of validity assessment, there was remarkable consistency between ASQ and warning signs, with the Kappa value of 0.63. With the use of GESELL as criterion, it was determined that the sensitivity of warning signs in children with suspected developmental delay was 82.2%, and the specificity was 77.7%. The overall Youden index was 0.6. Conclusion The reliability and validity of warning signs checklist for screening early childhood developmental problems have met the basic requirements of psychological screening scales, with the characteristics of short testing time and easy operation. Thus, this warning signs checklist can be used for screening psychological and behavioral problems of early childhood, especially in community settings.

Objective To study the effect of recombinant human platelet activating factor acetylhydrolase (rPAF-AH) on candidate plasticity-related gene 15 (CPG15) expression in focal cerebral ischemia rats.Methods Forty-five SD rats were randomly divided into sham operation group,saline group and rPAF-AH group (15 in each group).A middle cerebral artery occlusion model of rats was established for the latter two groups.Each group was divided into 2 d group,7 d group,14 d group.Five rats in each of 2 d group,7 d group,14 d group were used in experiment.The total protein was extracted from coronary sections using homogenate.CPG15 protein expression was detected by Western blot.Results The CPG15 protein expression level was significantly higher in saline group and rPAF-AH group than in sham operation group on days 2,7,14 and reached its peak on day 7,and in rPAF-AH group than in saline group on day 7 (1.48±0.04 vs 1.12±0.05,P＜0.01).Conclusion PAF-AH plays a positive role in neuroplasticity of cerebral ischemia by upregulating the CPG-15 expression in focal cerebral ischemia rats.