Objective To establish a fast and accurate technique of identifying the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation.Methods Using STA-PUT velocity sedimentation method to isolate the pachytene spermatocytes, round spermatids and elongating and condensing spermatids from mouse testes.To determine the cell populations` distribution,each tube of cell fraction was then partially transfered to the 96 plate well,and each well was added with acridine orange dye.Then each well was analyzed using fluorescence microscopy.Results Three types of spermatogenic cells can be identified quickly and accurately by it`s specific cytoplasm/nucleus character using the acridine orange dye staining under fluorescence detection.Conclusions A rubust method to quickly and accurately determine the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation is successfully developed.

ObjectiveTo study the effect of facilitation techniques combined with acupuncture on movement function and activity of daily living(ADL) of the stroke patients.Methods200 patients with stroke were divided randomly into 4 groups,group A for facilitation techniques with acupuncture,group B for facilitation techniques only,group C for acupuncture therapy only,and group D for control.The recovery grades of the Brunnstrom and Functional Independent Measure(FIM) were applied to assess movement function and ADL before and after treatment.ResultsThe number of patients whose limbs recoveried up to Brunnstrom 4 in group A、B、C after treatment was higher than that in group D as while as the scores of FIM(P<0.05).The FIM scores and the number of patients whose whose limbs recoveried grades(>4 grades) of the Brunnstrom in group B and C were no difference(P>0.05),The FIM scores and the number of patients whose recovery grades(>4 grades) of the Brunnstrom in group A were significant higher than that in group B and C (P<0. 05).ConclusionsFacilitation techniques combined with acupuncture can improve the movement function and ADL of the stroke patients.

BACKGROUND: Epidemiologic studies have shown an association between higher insulin levels and coronary artery disease, and metabolic studies have associated insulin resistance and compensatory hyperinsulinemia with non-insulin-dependent diabetes mellitus, hypertension, obesity,and lipid disorders.OBJECTIVE: To investigate the relationship between insulin resistance (IR) and erythrocyte insulin receptors (EIRs) in the patients with cerebral infarction (CI).DESIGN: Case-control trial.SETTING: Department of Neurology, China-Japan Union Hospital of Jilin University.PARTICIPANTS: From January 2004 to October 2004, 40 patients with CI, who were in-patients in China-Japan Union Hospital of Jilin University,were selected for the study. Meanwhile, 30 healthy doctors or nurses were recruited as normal controls.METHODS: The levels of blood glucose and serum insulin under fasting and 2-hour after oral glucose tolerance test (OGTT) were detected in the 40 patients with CI and 30 healthy doctors or nurses. Fasting blood glucose multiplied by fasting serum insulin was insulin resistance index (IRI). The number of insulin receptors and their binding affinity on every erythrocyte were determined using modified Gambhir's method. The correlation between the number of EIRs and IRI was analyzed.MAIN OUTCOME MEASURES: Comparison of CI group with controlRESULTS: Data of 40 patients with CI and 30 controls were analyzed, and under fasting and 2-hour after OGTT: The level of serum insulin under fasting and blood glucose, serum insulin at 2-hour after OGTT in CI group were higher than those in normal group [(13.30±5.15), (9.85±4.36) mU/L,(8.27±1.65), (6.32±1.37) mmol/L, (75.21±21.12), (28.26±6.31)mU/L,P ＜ 0.01,EIRs: The number of insulin receptors with high and low affinity and maximum specific binding rate in the patients with CI were significantly less than those in normal group [20.30±4.50, 23.80±4.10; 2 223.80±509.30,2 610.10±435.10; (10.62±3.55)%, (13.21±2.94)%, P ＜ 0.01]. Multiple linear regression analysis and correlation analysis showed the numbers of two types of EIRs in the patients with CI were negatively correlated with IRI (r=-0.458, -0.439, P ＜ 0.01, both).CONCLUSION: Insulin resistance can lead to cerebral infarction. Decrease in insulin receptors may play an importance role in cerebral infarction induced by insulin resistance.

Objective Through the analysis of cases,to improve the cognition of clinicians on extracranial vas-cular embolism diseases(VED)in children.Methods The clinical information included incidence trend,thrombophi-lia,clinical features,treatment and prognosis of 29 children with VED,who were admitted from January 201 1 to Decem-ber 201 5 in Nanjing Children′s Hospital Affiliated to Nanjing Medical University,and retrospectively analyzed.Results The annual rate of VED increased from 0 to 1 .49 per 1 0 000 hospital admissions from January 201 1 to December 201 5.In 29 cases,there were 20 male and 9 female,with an average age of 6.03 years old (2 h -1 3.83 years old).In-fants accounted for >20%(6 /29 cases).The average age of the female,non infected group,rheumatic disease group and venous thrombosis group was higher than that of the male,the infection group,the non rheumatic disease group and the arterial embolization group,but there was no significant difference(all P >0.05).Thrombophilia were as follows:in-fection disease,heart disease,kidney disease syndrome,rheumatoid disease,tumor,and twins embolization syndrome. Risk factors included central venous line,immobility,dehydration,glucocorticoids use,etc.Heart disease was the leading cause of non thrombotic embolism,while other thrombophilic diseases resulted in thrombotic embolism.Pulmonary em-bolism was mainly seen in patients with severe pneumonia,especially mycoplasma pneumoniae pneumonia (MPP ).The older the age,the more typical clinical manifestations,and significantly elevated D dimer,fibrin/fibrinogen degradation products tips VED.Treatment depends on the degree of risk.Thirty -seven point five percent(3 /8 cases)of children with arterial embolism were less than 1 year old,and the mortality 25.00% (2 /8 cases)was significantly higher than that of venous thrombosis 1 7.65%(3 /1 7 cases)and 1 1 .76% (2 /1 7 cases).Conclusion Heart disease mainly cause arterial embolism and infection,while nephritic syndrome and rheumatic disease mainly cause venous embolism.Pulmona-ry embolism is mainly seen in children with severe pneumonia,especially MPP.The risk of arterial embolization is higher than that of venous embolism.

Objective This study is aimed to assess the accuracy of clinical measurement of remission and investigate the association between clinical remission and the ultrasound imaging assessment of synovitis and infla'mation and to explore the value of ultrasound imaging in the evaluation of disease activity in the tight control of rheumatoid arthritis (RA).Methods RA patients with a disease activity score in 28 joints (DAS28-ESR) ≤2.6 for at least 3 months were stratified-using a standard and more stringent DAS28 and simplified disease activity index (SDAI) remission thresholds and the corresponding clinical and ultrasound imaging measures of synovitis were recorded.In addition,a retrospective analysis of disease activity in patients was carried out.Mann-Whitney U test,x2 test and multi-factor analysis of variance of repeated measurements were used for statistical analysis.Results A total of 48 cases were recruited.Thirty-seven cases (77％) met the ACR remission criteria,29 cases (60％) fulfilled the SDAI remission criteria,and 32 (67％) cases reached 2010 ACR/EULAR Boolean-based definition of remission criteria.There was no significant differences on imaging disease activity between SDAI remission patients and those who were not in remission (GS score,P=0.38; PD score,P=0.32).Seventy-nine percent patients in SDAI remission had GS and/or PD activity,but the difference was not statistically different when compared with patients who did not reach SDAI remission (P=0.29).Sustainable remission obtained in patients who also had achieved image remission could have longer remission time (P＜0.01) Conclusion Using more stringent clinical remission criteria could help in reducing residual inflammatory activity of,but ultrasound PD activities may not be significantly reduced.Integrated clinical indicators such as physical examination and imaging tools such as ultrasound can provide more accurate assessment of disease activity and could be used to guide treatment to achieve trueremission.

OBJECTIVE To search a method for identifying Clostridium perfringens and genotyping their toxin for gene diagnosis by multiplex PCR.METHODS The mutiplex PCR was developed with three sets of primers(designed) based on the sequences of three C.perfringens toxin genes(CP?,CP? and CPE) published in GenBank to identify C.perfringens and genotype their three toxin genes.RESULTS Three expected(sequences) were (obtained) successfully by multiplex PCR and identified by electrophoresis.CONCLUSIONS The(specific) sequences of C.perfringens could be amplified and their three genes of toxins could be identified by this multiplex PCR(system).Such method should be helpful for developing gene diagnosis well.

0.05).Conclusion The increasing of IL-15 in peripheral blood after MP infection may play a role in bronchitic asthma pathogenesis.

Objective To investigate the relationship between diabetes and bone metabolism and the potential epigenetic mechanisms. Methods BMSCs were cultured for 7 and 15 days in cell culture medium with different concentrations of glucose. The mRNA and protein expression of HDACs and osteogenesis-related genes were detected by RT-PCR and Western blot assay ,respectively. Moreover ,the combination of HDAC to the promoter region of Runx2 was tested by the chromatin immunoprecipitation (ChIP) assay. Results ThemRNA expression of osteogenesis-related genes ,incuding OCN(P < 0.05)and Col1(P < 0.05),in the bone marrow of diabetic mice was significantly reduced compared with the control mice. The mRNA and protein expression of ALP ,OCN ,Runx2 and OSX was gradually reduced with the increasing concentration of glucose ,while HDAC2 mRNA and protein expression was increased. The binding activity of HDAC2 to the upstream and downstream of Runx2 promoter region in 25mM glucose-treated BMSCs was higher than the control group(P < 0.05). Conclusoins Diabetes might repress osteogenesis of BMSCs via inhibiting the activity of Runx2 through upregu-lating the expression of HDAC2.

Objective To determine the effect of oxymatrine on the activity of HBV DNA polymerase in vitro. Methods Hepatitis B virus particles were purified from supernatant of cultured HepG2.2.15 cells by uhracentrifugation, and then were mixed with reaction buffer containing NP-40, β-mercaptoethanol, 32P-labelled nucleoside triphosphate (dCTP), MgCl2, and different concentrations of oxymatrine ( 1000 μg/ml, 800 μg/ml, 600 μg/ml, 400 μg/ml and 200 μg/ml) or adefovir dipivoxil ( 100 μg/ml, 80 μg/ml and 60 μg/ml, 40 μg/ml and 20 μg/ml). After incubation at 37 ℃ overnight, proteinase K was added to the reaction system for digestion and 35 μl of samples were spotted onto DE81 paper. Activities of endogenous polymerase in HBV particles were assessed by determining the radioactivity of 32P-labelled dCTP incorporated in the plus-strain of viral DNA. Results Compared with the blank control, the activity of endogenous polymerase in HBV particles treated with different doses of oxymatrine varied from 103% to 107%, and it varied from 91% to 101% when treated with different doses of adefovir dipivoxil. No significant difference was observed among treated groups and the control (P > 0. 05 ). Conclusion No direct inhibitory effect of oxymatrine on the activity of HBV polymerase was observed in vitro.

ObjectiveTo investigate the expression and significance of CD28- cells, CD4+ and CD8+T lymphocytes in the peripheral blood and synovial fluid in patients with rheumatoid arthritis ( RA ). Methods The expression of CD28, CD4+, CD8+ T lymphocytes and inducible co-stimulator(ICOS) in the peripheral blood and synovial fluid in 45 patients with RA were detected by three-color flow cytometry. Independent sample's t test was used for statistical analysis between the two groups. ResultsSynovial fluid CD4+CD28+ICOS+, CD4+CD28- ICOS+ , CD8 + CD28 + , CD8 + CD28 + 1COS+ T lymphocytes were significantly increased than the peripheral blood in RA patients[(36±19)％ vs (15±8)％, t=-4.234, P＜0.01; (2.1±2.2)％ vs (0.6±1.4)％, t=-3.143, P＜0.01; (62±15)％ vs (47±18)％, t=-2.885, P＜0.01; (9±9)％ vs (3±3)％,t=-2.131, P＜0.05], Synovial fluid CD8+CD28-T lymphoc-ytes were significantly reduced than the peripheral blood[(38±15)％ vs (54±18)％, t=2.975, P＜0.01], Synovial fluid CD8+ CD28-ICOS+, CD4+CD28+and CD4+ CD28- T lymphocytes had no significant difference than the peripheral blood (P＞0.05). Compared with peripheral blood in the same patients with RA, CD4+CD28+ ICOS+, CD8+ CD28+ T lymphocyteswere significantly increased[(38±18)％ vs (16±10)％, t=-4.065, P＜0.01 ; (61±16)％ vs (41±21)％, t=-4.065,P＜0.01], CD8+CD28-T was significantly reduced[(39±16)％ vs (59±21)％, t=2.949, P＜0.01]. The level of CD4+ CD28-, CD8+ CD28-, CD28-ICOS+ T lymphocytes in the active and remission patients with RA was not significantly different (P＞0.05). ConclusionSynovial fluid CD28T lymphocyte subsets disturbance and the abnormal expression of ICOS in patients with RA may play important roles in the mechanism of joint damage.