Objective To investigate the efficacy of active immunization combined with dydrogesterone in the treatment of recurrent spontaneous abortion,and the expression of serum leptin(LP)and adiponectin(ADPN).Methods 51 cases of inpatient and outpatient treatment of repeated abortion of 51 pregnant women from January 2014 to December 2015 in Ningbo Fenghua People's Hospital of Zhejiang Province were selected,and 50 cases of normal pregnant women and normal nonpregnant women 50 cases as a control object.Depending on the treatment method,patients with recurrent spontaneous abortion were divided into combination group(n=26)and monotherapy group(n=25).The levels of LP,ADPN and β-HCG were measured before and after treatment,and the number of successful pregnancies.Results The levels of LP and ADPN in serum were measured in three groups of women,there were statistically significant differences between the two groups(P<0.05).The serum levels of β-HCG in the pregnant women with recurrent spontaneous abortion group were(3914.5±2548.2)mIU,and the normal pregnancy group was(7124.5±2847.6)mIU,the difference was statistically significant(P<0.05).After treatment,the levels of LP,ADPN and β-HCG in the combination group were significantly increased,and successful pregnancy as high as 88.46％,better than the level of monotherapy group,the difference was statistically significant(P<0.05).Conclusion Serum LP,ADPN lower levels of pregnant women prone to recurrent spontaneous abortion,through active immunization combined with dexamethasone treatment,can significantly improve the hormone level,improve the success rate of pregnancy.

Objective Based on the Chinese full version of the Temperament Evaluation of Memphis Pisa Paris and San Diego-Auto questionnaire ( TEMPS-A) , we aimed to validated a short version of TEMPS-A for Chinese adoles-cents.Methods Taking into account the item factor loading from our previously validated full version TEMPS-A, cultur-ally-determined items, and sexual activity related items, we derived a 60-item Chinese TEMPS-A for adolescents.The internal consistency and structural validity of the Chinese TEMPS-A for adolescents was evaluated in 822 participants aged 11~17 years old.Results The Cronbach’ s alphas coefficients for depressive, cyclothymic, hyperthymic, irritable and anxious subscales were 0.67, 0.78, 0.76, 0.77 and 0.83, respectively.The anxious, irritable, hyperthymic factors were effectively distinguished by exploratory factor analysis, while the depressive and cyclothymic factors tended to correlate.The males scored significantly higher on the hyperthymic subscale than the females [(5.407 ±2.842) vs.(4.852 ±2.963), P<0.01].The females scored significantly higher on the depressive [(3.521 ±2.221) vs.(3.144 ±2.295)], cyclothy-mic [(4.484 ±2.922) vs.(3.917 ±2.823)] and anxious [(5.236 ±3.719) vs.(4.366 ±3.658)] temperaments than the males ( P<0.05) .The scores of depressive subscale and cyclothymic temperaments subscale were significantly correla-ted (r=0.625, P<0.001), so did the scores of anxious subscale and irritable temperament subscale (r=0.628, P<0.001).Conclusions The Chinese Adolescent version of TEMPS-A is a reliable and valid instrument for investigating af-fective temperaments for adolescents.

Objective To investigate the effect of patent ductus arteriosus(PDA)on myocardial injury of premature infants.Methods From May 1,2010 to January 31,2011,110 preterm infants with gestational age from 28 to 36 weeks accepted echocardiography examination,and their blood samples were collected to determine cardiac troponin T(cTnT)and creatine kinase MB isoenzyme (CK-MB)levels 72 h and 3 d after deliveries.All subjects were divided into two groups according to the echocardiogram results:PDA group(n=44)and control group(n=66).The infants with PDA were treated with ibuprofen,and then echocardiography was taken again.cTnT and CK-MB were re-measured in both groups.Chi-square test,t-test,multi-variate linear regression and Spearman rank correlation test were perfomed for statistical analysis.Results Before treatment,cTnT[(0.259±0.134)μg/L]and CK-MB[(7.31± 2.69)μg/L]level of PDA group were significantly higher than those[(0.083±0.054)μg/L and(5.71±1.88)μg/L]of control group(t=9.557 and 2.588,P＜0.01 and ＜0.05,respectively).For 34 infants with successful treatment,cTnT and CK-MB levels decreased markedly to(0.062 ± 0.039)μg/L and(5.34 ± 1.50)μg/L,respectively(t =9.268 and 5.974,all P＜0.05),compared with those levels before treatment.For the ten infants failed to close ductus,the cTnT and CK-MB levels[(0.193±0.049)μg/L and(6.93±1.63)μg/L,respectively],were lower than those before treatment(t=1.525 and 0.766,all P＞0.05),while higher than those of the control group(t=9.068 and 4.055,P＜0.05).Level of cTnT positively related to the duration of ventilation,PDA and respiratory distress syndrome,while did not relate to gender,gestational age and birth weight.CK-MB level was associated to gender,gestational age,birth weight,duration of ventilatory support and PDA.In PDA group,the cTnT level was positively related to the diameter of the ductus,but not related to any indicators in echocardiography.Conclusions Symptomatic PDA could cause myocardial injury in preterm infants.The changes of blood cTnT and CK-MB were consistent with the severity of PDA.Serial measurements of blood cTnT and CK-MB might help to make early diagnosis and treatment for premature infants with PDA and myocardial injury.

Objective To investigate the effect of Calponin-1 suppression on human myometrium cells through adenovirus mediated siRNA. Methods Human uterine smooth muscle tissues were digested with enzymes, cultured and confirmed with immunocytochemistry. Aadenovirus siRNA-Calponin-1 plasmid was transfected into primary cultured uterine smooth muscle cells in vitro. The expressions of Calponin-1 mRNA and protein were analyzed by RT-PCR and Western blot, respectively.Results The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid was successfully constructed, and Calponin-1 siRNA mediated by recombinant adenovirus resulted in markedly reduced expression of Calponin-1 mRNA and protein in human myometrium cells. The gray values of Calponin-1 mRNA in the uterine smooth muscle cells in the experimental, blank control, and empty vector groups were 316.3±39.2, 1048.5±126.4 and 1027.2±127.5, respectively. The gray values of Calponin-1 protein were 323.3±43.2, 1021.5±143.4, and 1019.2±144.5,respectively. The difference between the experimental group and the blank control group as well as the empty vector group was significant (P< 0.05). There was no significant difference between the empty vector group and the blank control group (P>0.05).Conclusion The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid can inhibit the expression of Calponin-1 in human myometrium cells in vitro,which may be a useful approach to determine the role of Calponin-1 in delivery.

Objective To set up the quality control standard for Leaf of Chinese Holly; To provide basis for the utilization and development of Leaf of Chinese Holly.Methods Ten batches of Leaf of Chinese Holly samples from different habitats were collected, and the properties were described. The crosscutting and powder of leaf was under microscopic identification. Chlorogenic acid was set as reference substance to conduct thin-layer identification. Moisture, total ash, and acid-insoluble ash of the 10 batches were detected. HPLC was used to detect chlorogenic acid in Leaf of Chinese Holly.Results The properties and microscopic identification were described. Thin-layer chromatography method for chlorogenic acid in Leaf of Chinese Holly was formulated. Check items temporarily required that the moisture in Leaf of Chinese Holly should be less than 13%, total ash less than 13%, and acid-insoluble ash less than 5%. Content determination method for chlorogenic acid in Leaf of Chinese Holly was confirmed. It temporarily required that the content of chlorogenic acid should be more than 0.60%.Conclusion The established method can be used for the formulation of quality control of Leaf of Chinese Holly.

Objective:To investigate the potential mechanism of electroacupuncture(EA)at bilateral Fengchi(GB20)in treating cerebral ischemia-reperfusion injury and to provide a scientific basis for future experimental research and clinical applications. Methods:Forty male specific-pathogen-free Sprague-Dawley rats were randomly divided into four groups:a normal group,a normal with EA group,a model group,and a model with EA group,with 10 rats in each group.The normal group received no intervention.The normal with EA group received EA at bilateral Fengchi(GB20).The model group underwent middle cerebral artery occlusion(MCAO)using the suture.The model with EA group underwent MCAO and received EA at bilateral Fengchi(GB20).Cerebral blood flow was monitored using a laser Doppler cerebral blood flow meter.Neurologic damage was assessed using the neurologic deficit score,and motor ability was observed using the CatWalk gait system.The expression of glial fibrillary acidic protein(GFAP)and neuronal nuclei(NeuN)protein,the neuron markers,was detected by Western blotting.The protein expression levels of GFAP and NeuN,as well as the number of positive cells in the motor cortex,were detected using immunofluorescence. Results:Compared to the normal group,the cerebral blood flow values in the model group and the model with EA group decreased by more than 50%during the modeling process(P<0.01)and returned to pre-modeling levels after reperfusion(P>0.05).The neurologic deficit score increased(P<0.05),the average motor velocity decreased(P<0.05),GFAP protein expression and the number of positive cells in the motor cortex increased(P<0.05),and the NeuN protein expression and the number of positive cells decreased(P<0.05)in the model group.Compared to the model group,the neurologic deficit score decreased(P<0.05),the average motor velocity accelerated(P<0.05),GFAP and NeuN protein expression and the number of positive cells in the motor cortex increased(P<0.01)in the model with EA group. Conclusion:EA at bilateral Fengchi(GB20)can reduce neuronal loss and increase GFAP and NeuN protein expression in the motor cortex of rats after ischemia-reperfusion,improve the motor function after ischemic stroke,and accelerate the recovery of balance and stability of the affected limbs.

Objective To explore the influence of the lentiviral vectors mediated mouse genetic engineering regulatory T cells(Tr) infused after allogeneic bone marrow transplantation(allo-BMT) on graft-versushost disease(GVHD) in mice. Methods Lentivirus-mediated expression of forkhead box P3 (Foxp3) converted CD4 + CD25 - T cells from BALB/c mice into engineered Tr in vitro. An allo-BMT model of BALB/c→C57BL/6 mice was established. After irradiation, the recipients were injected with donor cells along with genetic engineering Tr. Survival time, histopathological analysis, serum levels of inflammatory cytokines were observed after allo-BMT. Results The mean survival times in radiation group, transplantation control group, engineering Tr group and empty vector control group were ( 8.8 ± 0.6 ) d, ( 36.7 ± 2.5 ) d, ( 51.6 ± 4.0 ) d and ( 34.1 ± 2. 3 ) d. The survival time in engineering Tr group was significantly increased as compared to other groups as judged by the log-rank test ( P ＜0.05 ). Histopathological analysis in several target organs( skin, liver and small intestine) confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD were observed in recipients in engineering Tr group. The serum levels of IFN-γ, IL-2 and TNF-α were all increased after transplantation in above groups. The peaks of concentrations of IFN-γ, IL-2 and TNF-α in engineering Tr group were significantly decreased compared to transplantation control group and empty vector control group at day 21 ( P ＜ 0. 05 ). Conclusion Co-injection of genetic engineering Tr can efficiently prevent recipients from lethal GVHD during allo-BMT in mice by reducing the serum levels of inflammatory cytokines.

Objective To determine the conditioning regimen suitable for mice allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Twelve BALB/c mice were randomly divided into 2 equal groups to undergo X-ray irradiation by linear accelerator at the dose of 7.0 Gy (pure X-ray group) or 60Co source irradiation at the dose of 7.0 Gy (pure γ-ray group).Thirty mice were randomly divided into 2 equal groups to undergo X-ray irradiation and then infusion of bone marrow from donor mice via caudal vein (X-ray + transplantation group) or γ-ray and then infusion of bone marrow via caudal vein (γ-ray + transplahtation group).3,5,7,10,15,20,and 30 d later peripheral blood samples were collected to calculate the number of white blood cells (WBCs) and detect the chimeric rates of lymphocytes by flow cytometry.5,10,and 20 d after irradiation 15 mice were killed with their lung,liver,small intestine,spleen,and femurs taken out to undergo pathological examination.Results The survival rates during the period 5-15 days of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group.The pathological changes of organs of the X-ray +transplantation group were all more severe than those of the γ-ray + transplantation group.Since the fifth day after transplantation cells originating from the donor began to appear in the peripheral blood.The chimeric rate of the γ-ray + transplantation group 10 days after transplantation was (95.53± 2.57) %.The chimeric rates 5,10,and 20 days after transplantation of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group (t = 15.263,3.256,P < 0.05).The WBC count of both irradiation groups decreased to the lowest level 5 d later and began to increase 10 days after transplantation and the WBC counts of the γ-ray + transplantation group 10 and 20 days aftertransplantation were both significantly higher than those of the X-ray + transplantation group (t = 3.624,6.695 ,P < 0.05).The chimeric rats of the peripheral lymphocytes 10 and 20 days after transplantation of the γ-ray + transplantation group were both significantly higher than those of the X-ray + transplantation group (t = 12.317,8.295,P < 0.05).The homogeneity rate of transplantation of the γ-ray +transplantation group was better than that of the X-ray + transplantation group.Conclusions As a conditioning regimen in allogeneic hematopoietic stem cell transplantation γ-ray irradiation causes milder injury and accelerated reconstitution of hematopoiesis and immunity,in comparison with X-ray irradiation.

Objective To explore prevention of cyclosporine A (CsA) combined with Cobalt protoporphyrin (CoPP) against murine graft versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods C57BL/6 (H-2Kb) mice were used as donors and BALB/c (H-2Kd) mice as recipients,which were randomly divided into 4 groups.The mice in total body irradiation group (TBI group) were lethally irradiated and injected intravenously with PBS; The mice in Allo-HSCT group (BS group) were lethally irradiated and injected intravenously with bone marrow cells and spleen cells; The mice in CsA intervention group (CsA group) were injected with CsA intraperitoneally after allo-HSCT; The mice in CsA combine with CoPP intervention group (combination group) received both CsA and CoPP intraperitoneally after alloHSCT.Recipients were monitored for condition,survival rate and weight.The liver,small intestine and skin in the recipients were gained and pathological changes of GVHD were assessed.The kidney was stained with Masson staining dye to observe the tissue fibrosis.The expression levels of renal HO-1 mRNA in the recipients were detected.Results In contrast to BS and CsA groups,GVHD degree in combination group was mild,with less reduction and quick recovery of weight.On the day 30 after HSCT,survival rate in BS group was 36.8％,and that in combination group and CsA group was 69.6％ and 53.5％ respectively (P＜0.05).In comparison with BS and CsA groups,pathological changes in combination group were mild,cellular edema and degeneration degree of the liver,small intestine and skin were slight,and few necrosis and infiltrated inflammatory cells were observed.Tubulointerstitial fibrosis hardly occurred in combination group,but it occurred in CsA group abundantly.As compared with BS group,the expression levels of HO-1 mRNA was increased in combination group,while decreased in CsA group (P＜0.05).Conclusion CsA combined with CoPP enhanced the protective effect of CsA against GVHD,moreover,CoPP could alleviate the side effects of CsA,which might be related with up-regulation of the expression levels of HO-1.

Objective This study aimed to establish the leukemia mouse model by using EL4/DsRed cell line expressing red fluorescent protein (DsRed) and to evaluate the model. Methods After total body irradiation with X-ray of 7.0 Gy, C57BL/6 mice were inoculated 5×106 bone marrow cells mixed different numbers of EL4/DsRed cells via tail vein. The model was evaluated by flow cytometry (FCM), reverse transcriptase-polymerase chain reaction (RT-PCR), and histopathology. Results The incidence of leukemia was 100 %. The presence of EL4/DsRed cells was found in liver, spleen, bone marrow and peripheral blood of recipients by FCM two weeks after transplantation. Pathological section revealed that all recipients had several organs infiltration apparently. With the increase in the number of inoculated tumor cells, the survival time of recipients was reduced and the infiltration of leukemia cells in organs was more serious. Conclusion Mouse leukemia model was successfully established when C57BL/6 mouse was intravenously transplanted with ≥5×102 EL4/DsRed cells. The model could be employed usefully in the future research such as the pathogenesis of leukemia and minimal residual disease (MRD).