Objective To develop a vaccine for Wilm's tumor in order to lay a foundation for effective treatment and substitute for the radiotherapy and chemotherapy of this tumor. Methods DNA fragments encoding the WT1 antigens (including, HLA-A * 2402 and HLA-A * 2402x), and DNA fragments encoding the couple antigens HLA-A * 2402-HBc and HLA-A * 2402x-HBc were cloned and inserted into the eukaryotic expression vector pcDNA3.1(+) in sense orientation and baculovirus expression pFactbac respectively, then transfected into COS-7 cells and Sf 9 cell respectively. The expression of the target proteins in the cells were identified by CPE, SDS-PAGE and electron microscopy. Virus-like particles were observed under electron microscopy. Results The fusion gene of HLA-A * 2402 and HLA-A * 2402x and HBc express virus-like particle protein. Conclusions This preliminary result shows a hopeful future in developing an effective immunotherapy to Wilm's tumor.

Objective To investigate the expression of interleukin (IL)-34/colony stimulating factor(CSF)-1R in the process of transforming growth factor ( TGF)-β1 inducing epithelial-mesenchymal transition ( EMT) of human alveolar epithelial cells A549 cells.Methods A549 cells were cultured in vitro.CCK 8 was used to test the influence of the proliferative rate of A549 cells which were stimulated by TGF-β1 at different concentrations and time points .A549 cells were stimulated by 5μg/L TGF-β1 at 0 hour, the 12th hour, the 24th hour, and the 48th hour.Western blotting was adopted to detect changes of the following proteins: α-smooth muscle actin (α-SMA ) , E-cadherin ( E-Cad ) , matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1). Real-time PCR was adopted to detect changes of the following genes: IL-34 mRNA, CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA.Results TGF-β1 had no significant influence in the proliferation of A 549 cells compared with the control group(P>0.05).TGF-β1(5μg/L)stimulated A549 cells at different time point (0 hour, 12, 24, 48 hours), compared with the control group .The epithelial phenotype E-Cad protein was gradually down-regulated ( P <0.01 ) , while the mesenchymal phenotype α-SMA protein was gradually up-regulated ( P <0.01 ) and the protein of MMP-2 increased gradually (P<0.01).The protein of MMP-9 increased firstly and then was reduced (P<0.01),the peak was at the 24th hour.The protein of TIMP-1 was firstly transiently increased and then reduced (P<0.01), the minimum was at the 48th hour.Compared with the control group , the gene of IL-34 mRNA increased gradually (P<0.01), and the genes of CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA increased firstly and then decreased ( P <0.01), which were peaked respectively at the 24th hour, the 24th hour, the 12th hour, respectively.Conclusion In the process of TGF-β1 inducing A549 cells transition,there is accompanied with the expression of IL-34/CSF-1R.

OBJECTIVE:To determine the plasma concentration of Phencynonate hydrochloride(PCH)in dogs,and to calcu-late pharmacokinetic parameters. METHODS:6 Beagle dogs were given PCH tablets(2 mg and 4 mg)intragastrically. The blood samples were collected 5 min before medication and 0.17,0.25,0.5,0.75,1.0,1.25,1.5,2,3,4,6,8,10,12,14,16,18, 24 and 36 h after medication,2 ml each time. Using penehyclidine hydrochloride as internal standard,HPLC-MS/MS method was adopted to determine the plasma concentration of PCH. The medication plans were interchanged 2 weeks later. The pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS:The linear range of PCH was 0.1-15 ng/ml(r=0.999 6);the low-est limit of quantification was 0.1 ng/ml;the methodology recovery were 97.30%-103.20%;the extraction recovery were 52.30%-60.11%(RSD<11%,n=5). The main pharmacokinetic parameters of low and high doses were as follows as t1/2α of (0.678±0.525)and(0.405±0.465)h,tmax of(1.042±0.401)and(0.900±0.418)h,cmax of(14.063±6.29)and(31.580±9.673) mg/L,AUC0-36 h of(48.186±14.776)and(79.269±34.649)mg·h/L. CONCLUSIONS:The method is simple,sensitive and speci-fic,and can be used for pharmacokinetic study of PCH in dogs.

Objective To explore the diet features of diarrhea predominant irritable bowel syndrome (IBS-D) with small intestinal bacterial overgrowth (SIBO).Methods IBS-D patients were enrolled in outpatient department of Peking University Third Hospital from March 2015 to April 2016.Healthy volunteers were recruited as controls (HC).All the subjects completed screening examinations,clinical and food investigation,and lactulose methane and hydrogen breath test (LMHBT).The high fat diet is defined as the daily total calories supplying from fat is more than 50％.Results Eighty-eight IBS-D patients and 32 HC were finally enrolled.The positive rate of LMHBT in IBS-D was significantly higher than that of HC [39.8％ (35/88) vs 12.5％ (4/28),P=0.005].The 28 HC with negative LMHBT were enrolled in the follow-up analysis.(1) The BMI of IBS-P (IBS-D with positive LMHBT) was significantly lower than IBS-N (IBS-D with negative LMHBT) [(21.57 ±0.54) vs (23.30 ±0.53) kg/m2,P=0.032].IBS-D patients with SIBO had higher scores of abdominal pain assess.(2) The proportion of dietary protein and carbohydrate in IBS-D was significantly higher than that of HC (14.39％ vs 12.22％,P =0.001;53.94％ vs 46.25％,P =0.003,respectively).The proportion of diet fat was significantly higher in IBS-P than IBS-N[(47.19 ± 2.62) ％ vs (40.74 ± 1.66) ％,P =0.038].(3) The baseline of breath methane in IBS-P was significantly higher than that of in IBS-N [(8.69 ± 0.39) × 10-6 vs (6.39 ± 0.47) × 10-6,P =0.002].IBS-D patients with high fat diet had higher LMHBT positive rate than that of non-high fat diet patients[54.2％ (13/24) vs 17.2％ (11/64),P =0.001].Breath methane peak value was positively correlated with the fat proportion of diet (r =0.413,P =0.022).Conclusions About 39.8％ IBS-D patients diagnosed by Rome 11Ⅲ are combined with SIBO.SIBO may affect IBS-D patients' nutritional status.High fat diet might be one of the risk factors for IBS-D with SIBO.Proper diet structure might reduce the prevalence of IBS-D,especially for IBS-D with SIBO.

OBJECTIVE: To establish a method for analysis of proteome of human peripheral leukocytes and to explore the difference between the healthy volunteers and patients with acute lymphocyte leukemia(ALL) in leukocyte proteomics and to study the ALL-related abnormal proteins.METHODS: Peripheral blood samples taken from 10 healthy volunteers and 10 ALL patients were separated for the extraction of leukocyte proteins.The leukocyte proteins were separated by two-dimensional electrophoresis(2-DE).2-DE patterns were analyzed by PDQuest 2D software and the differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight-mass spectrometer(MALDITOF-MS) and bioinformatics.RESULTS: The 2-DE patterns of peripheral blood leucocytes from patients with ALL versus those from healthy volunteers were analyzed by PDQuest 2D software and the differentially expressed proteins identified by matrix assisted laser desorption ionization-time of flight-mass spectrometer(MALDI-TOF-MS) were confirmed to beALL-related proteins: AnnexinA5 and cytoskeleton 14 etc.CONCLUSION: Using MALDI-TOF-MS and bioinformatics,the AnnexinA5 and cytoskeleton 14 proteins had been proved to be of close relationship with ALL.The results provided a satisfactory basis for the searching for treatment targets and the molecular targets for the early diagnosis of ALL.

BACKGROUND: How to deal with bone defect is a big problem to surgeons. In recent years, the development in the technology of molecular biology and tissue engineering provides broad prospect for the clinical treatment of bone defect, which is one of the important study directions in department of orthopedics. The transforming growth factor-beta 1(TGF-β1),one of the important factors in bone formation, plays an important role in bone metabolism and recovery.OBJECTIVE: To observe the therapeutic effects of bone defects with osteoblasts transfected . By TGF-β1 combining with biomimetic biodegradable polymer scaffolds.DESIGN: Randomized controlled trial.SETTING: Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Siderophilin, trypsin, 3H-proline and sirius red, etc.MATERIALS: The experiment was completed in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from March to August in 2003. Twenty healthy adult Sprague-Dawley(SD) rats of SPF grade, weighing 200-250 g, were provided by the Experimental Animal Center of Tongji Medical College.METHODS: The osteoblasts transfected by TGF-β1 gene, combining with poly-DL-lactic acid scaffolds modified with poly-L-lysine, were transplanted into rat tibia defect. Radiographs and histological analysis were performed to evaluate the repair effects.MAIN OUTCOME MEASURES: The X-ray evaluation and histology observation were performed at the 4th and 8th weeks after the operation.RESULTS: Totally 20 SD rats were included in result analysis without one rat missing. ①In the experiment group, X-ray image indicated callus formation, while histology observation showed osteoid tissue and new bone formation, and osteoblasts attached to the surface of the materials after 4 weeks. Eight weeks later, the defect was essentially repaired, and the bone density of new bone was similar to that of the autogenous bone. ②In the control group, there was no formation of callus and osteoid tissue, and few osteoblasts attached to the surface of the materials, and a lot of lymphocytes infiltrated and blood capillary grew in the lacune of materials after 4 weeks. Eight weeks later, the imbedded materials were substituted mostly by fibrous tissue.CONCLUSION: The ideal repair effect of bone defect can be obtained through the combination of molecular biology with tissue engineering.

Objective To investigate the level of self-perceived burden(SPB) and analyze the influencing factors in continuous ambulatory peritoneal dialysis(CAPD) patients.Methods One hundred and ten CAPD patients were investigated by a self-designed demographic questionnaire and Self-Perceived Burden Scale (SPBS).Results The total score of SPBS was (30.27±7.88) points,being in a middle level,and 85.45％ of the patients showed different levels of burden.Single factor analysis showed the influencing factors of self-perceived burden were marital status,family per capita income,the main caregivers and selfcare ability.Conclusions Most CAPD patients have obvious self-perceived burden,nursing staff should use the relevant nursing measures to reduce the SPB with CAPD patients and improve their quality of life.

Aim To investigate the time-dependent effect of insulin on the expression of SREBP-1(sterol regulatory element binding protein-1),FAS(fat acid synthase)and lipid droplet formation in HKC cells(human renal proximal tubular epithelial cells line).MethodsHKC cells were respectively treated with 100 nmol·L~(-1) insulin for 0,2,4,6,12 h and 24 h.The analysis of SREBP-1 and FAS mRNA was performed by RT-PCR and the expression of SREBP-1 protein was detected by Western blot and immunocytochemistry.Furthermore,Oil Red O staining was used to determine cellular lipid droplet formation.ResultsCompared with HKC cells of 0 h group,there was no difference of SREBP-1 and FAS mRNA in HKC cells of 2 h group.However,the expression of SREBP-1 and FAS mRNA was significantly increased in HKC cells of 4,6 h and 12 h group.Further,the most expression of SREBP-1 and FAS mRNA was at 6 h group and was respectively increased by 3.578 and 4.272 times compared with 0 h group.The results of Western blot showed that the precursor and mature segments of SREBP-1 protein in 4,6 h and 12 h group HKC cells were increased and those of 6 h group HKC cells were the highest and about 4.106 and 5.167 times than those of 0 h group HKC cells.Immunocytochemistry presented the result that SREBP-1 protein was located in the plasma and the expression of 4,6 h and 12 h group HKC cells was significantly higher than that of 0,2 h and 24 h group HKC cells.The result of Oil Red O staining showed that lipid droplet markedly deposited in 6 h group HKC cells,contrarily,no lipid droplet was found in HKC cells of other groups.ConclusionAbove results suggested that insulin up-regulated SREBP-1 and FAS in time-dependent manner that led to cellular lipid droplet deposit,which may play an important role in the pathogenesis of renal lipid accumulation in metabolism syndrome.

Objective To investigate the clinical significance of nuclear factor ( NF)-κB activation in peripheral blood mononuclear cells (PBMCs) and the serum levels of correlated inflammatory cytokines in children with infant muggy syndrome(IMS).Methods Blood samples from 100 patients with IMS and those from 32 healthy infants( control group)were detected by ELISA for amount of NF-κB activation in PBMCs and for serum levels of interleukin ( IL ) -17,IL-6,tumor necrosis factor ( TNF ) -α and IL- 10 respectively from Jan 2008 to Jan 2011.At the same time,blood samples from 46 out of the above 100 patients with IMS and those from the 32 controls for positive rate of activation of NF-κB in PBMCs were detected by flow cytometry as well.The relationship between all the data and multiple organ dysfunction syndrome( MODS ) were analyzed respectively.Results As compared with that of control group,the percentage of activated NF-κB in PBMCs in 100 patients with IMS detected by ELISA [ ( 11.042 ± 6.792 ) ％ vs ( 4.528 ± 1.378 ) ％ ] and the positive rate of NF-κB activation in 46 patients with IMS detected by flow cytometry [ ( 28.780 ± 13.820 ) ％ vs (7.078 ±5.395)％ ] were both significantly higher ( P ＜0.01 ).The serum levels of IL-17,IL-6 and IL-10were also significantly higher in patients with IMS than those in control group( P ＜0.01 ).The serum level of TNF-α was higher in patients with IMS than that in control group but without significance( P ＞ 0.05 ).The percentage of activated NF-κB [ ( 14.591 ± 7.626) ％ vs ( 8.576 ± 4.851 ) ％ ],the positive rate of NF-κB activation [ ( 36.087 ± 12.056) ％ vs ( 23.590 ± 11.263 ) ％ ],and the serum levels of IL- 17,IL-6,TNF-α and IL-10 were all significantly higher in IMS patients with MODS than those in IMS patients without MODS ( P ＜ 0.01 ).Conclusion The inflammatory factors of NF-κB activation in PBMCs and the high serum levels of IL-17 and IL-6 are potent to cause inflammatory damage in IMS patients,and the serum level of IL-10 is not able to compensate the damage.The activation of NF-κB and high serum levels of IL-17,IL-6 and TNF-α are correlated with MODS.

Objective To observe the changes in CD4 + CD25 + regulatory T cells(Treg) and T helper 17 cells (Th17) cells' proportions in the peripheral blood in children with Kawasaki disease(KD) before and after the treatment,and to analyze the role of Treg/Th17 cell imbalance in the pathogenesis of KD.Methods Fifty-two children with acute KD(KD group) and 34 age-matched healthy children(healthy control group) were selected at Jiangxi Provincial Children's Hospital from April to December of 2014.Morning peripheral vein blood was collected from 2 groups:one before the treatment by Immunoglobulin and Aspirin,and the other 3 days after defervescence treatment.Flow cytometry was used to detect proportions of Treg cells and Th17 cells in the peripheral blood.The enzyme linked immunosorbent assay was used to detect the levels of interleukin(IL)-6,IL-10,IL-17,IL-23 and transforming growth factor(TGF)-β.Results Proportion of Treg cells in the acute KD group was remarkably lower than that in the healthy control group [(1.48 ± 0.21) ％ vs.(5.13-± 0.32) ％,t =28.41,P ＜ 0.05],but it was significantly increased after treatment,and there was a significant difference [(4.71 ± 0.36) ％ vs.(1.48 ± 0.21) ％,t =-23.32,P ＜ 0.05].Proportion of Th17 cells in the acute KD group was markedly higher than that in the healthy control group [(8.06 ± 0.48) ％ vs.(2.65 ± 0.50) ％,t =-23.47,P ＜ 0.05],which was significantly decreased after treatment [(3.04 ±0.35) ％ vs.(8.06 ± 0.48) ％,t =25.55,P ＜ 0.05].Compared with the healthy control group,the levels of serum IL-6,IL-17,IL-23 in the acute KD group were significantly increased before treatment,and there were significant differences [(34.53 ± 0.53) ng/L vs.(10.88 ± 0.83) ng/L,t =-72.36;(57.05 ± 0.78) ng/L vs.(14.29 ± 0.98)ng/L,t =-55.29;(45.18 ± 1.52) ng/L vs.(18.25 ± 1.08) ng/L,t =-43.27;all P ＜ 0.05],but after treatment the levels were significantly decreased [(14.94 ± 1.06) ng/L vs.(34.53 ± 0.53) ng/L,t =49.63;(27.64 ± 0.91)ng/Lvs.(57.05±0.78) ng/L,t =26.49;(24.50-±1.13) ng/L vs.(45.18-±1.52) ng/L,t =32.17;allP＜0.05].The levels of serum IL-10,TGF-β in the acute KD group significantly decreased than those of the healthy control group,and there were significant differences [(14.29-± 0.64) ng/L vs.(29.57 ± 0.87) ng/L,t =42.24;(16.88 ±-0.90) ng/L vs.(38.83 ±0.84) ng/L,t =53.51;all P ＜0.05],but after treatment the levels were significantly increased,and there were significant differences [(23.01-± 0.61) ng/L vs.(14.29-± 0.64) ng/L,t =-29.54;(33.47±-0.82) ng/Lvs.(16.88±-0.90) ng/L,t=-40.68;allP＜0.05].Conclusion Imbalance betweenTreg cells and Th17 cells may be an important cause for the immune disorder of KD,the changes in related cytokines are involved in the pathogenesis of KD.