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 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 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 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 study the repair function of united endothelial progenitor cells (EPC)transplantation on injured liver endothelium by bone marrow transplantation (BMT) conditioning.Methods C57BL/6 mice were divided into four groups randomly: normal control group, without any treatment; irradiation alone group, administered a total body irradiation(TBI) pretreatment, without BMT; (3) BMT alone group: C57BL/6 mice were infused with bone marrow mononuclearcells (MNC) 5 × 106/only through caudal vein not more than 4 h after the same TBI pretreatment as the irradiation alone group; united transplantation group: receiving the same way as the BMT alone group, but C57BL/6 mice were infused with EPC 5 × 105/only at the same time. Two, 4, 7, 14, and 21 days after the TBI, the changes of the liver weight were observed regularly. The histopathological examination of liver was done at the 4th, 7th, 14th, and 21st day after the TBI. Results In irradiation alone group, BMT alone group and united transplantation group the liver weight began to increase significantly on the day 2 and peaked at 14th day after the TBI, and the peaks were respectively (1.65±0. 15) times (P＜0. 05), (1.61 ±0.06) times (P＜0.05), and (1.11 ±0.40)times (P＜0. 05) of those in normal control group. At the day 14, the liver weight in irradiation alone group, BMT alone group and united transplantation group began to decrease, and on the day 21 the liver weight in united transplantation group had been completely restored to normal level, however the liver weight in irradiation alone group and BMT alone group were still significantly heavier than that in normal control group (P＜0. 05). Liver histopathological examination revealed that there were obvious sinusoidal endothelial cells (SEC) injury, hepatocyte edema and severe inflammatory cell infiltration in irradiation alone group, and on the day 7 the hepatocyte edema and necrosis were significantly worse than before, and almost no alive SEC were found. On the day 14 the injury of SEC in BMT alone group was lighter than before, but on the day 21 the injury had not returned to normal. On the day 7 the injury of SEC, hepatocyte edema and necrosis were alleviated in united transplantation group as compared with irradiation alone group and BMT alone group, and on the day 14 the injury had returned to normal basically. Conclusion The transplantation conditioning could damage recipient liver endothelium and the injury would persist, and united EPC infusion could repair the injured SEC following BMT.

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 study the relationship between graft-versus-host disease (GVHD) and endothelium injury following hematopoietic stem cells transplantation in mice. Methods C57BL/6 mice as donors and Balb/c mice as recipients were randomly divided into 4 groups: control group, bone marrow transplantation group, GVHD group, GVHD mitigation group. The clinical manifestations,circulating endothelial cells and tissue pathological changes were observed at different time points after transplantation. Results No manifestations of GVHD were found in each group at the day 5, while those were found in GVHD group at the day 9 and all died within 15 days. The counts of endothelial cells in peripheral blood showed no significant difference at the day 5 between GVHD group (7. 34 ±1.26 cells/μl) and bone marrow transplantation group (11.51 ± 7. 40 cells/μl) or GVHD mitigation group (7. 36 ± 0. 16 cells/μl), while among three groups there was statistically significant difference at the day 9 (GVHD group: 153. 64 ± 35. 35 cells/μl vs bone marrow transplantation group: 10. 49 ±5. 61 cells/μl and GVHD mitigation group: 47. 82 ± 4. 69 cells/μl). The scores of pathological aGVHD had no significant difference at the day 5 between GVHD group (4. 33± 1. 53) and bone marrow transplantation group (3. 33 ± 0. 58) or GVHD mitigation group (4. 00 ± 1.73), while among three groups there was statistically significant difference at the day 9 (GVHD group: 10. 0 vs bone marrow transplantation group: 3. 33 ± 1.15 or GVHD mitigation group: 4. 33 ± 0. 58) and at the day 14 (GVHD group: 10. 33 ± 2. 58 vs bone marrow transplantation group: 2. 33 ± 1.25 or GVHD mitigation group 3. 33 ± 1.15). Conclusion Occurrence of GVHD causes endothelial damage again and injured endothelium worsens the GVHD.

Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneie bone marrow transplantation (allo-BMT)on graft-versus-host disease (GVHD) in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) converted CD4~+ CD25~- T cells from Balb/c mice into engineered Tregs in vitro.An allo-BMT model of Balb/c→C57BL/6 mice was established.Mice were randomly assigned into four groups:(1) The recipients in engineering Treg group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenoeytes plus 5×10~6 genetic engineering Treg;(2)The recipients in transplantation control group were iniected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytes;(3) The recipients in radiation group were injected with 0.2 ml RPMI 1640;(4)The recipients in empty vector control group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytas plus 5×10~6 empty vector transduced CD4~+ CD25~- T cells.Survival time,clinical GVHD Score or histopathological analysis(skin,liver and small intestine) were observed after allo-BMT.Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured Results The mean survival times in radiation group, transplantation control group,erIgineering Treg group and empty vector control group were (8.8±0.6),(36.7±2.5),(51.6±4.0) and (34.1±2.3)days respectively.The survival time in engineering Treg group was signiticantly prolonged as compared with other groups as judged by the log-rank test(P<0.05).Histopathological ahalysis 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 Treg group and clinical GVHD scores in this group were significantly decreased compared to transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD during allo-BMT in mice

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.