Child ; Humans ; Nephrology ; Pediatrics

Child ; Humans ; Vesico-Ureteral Reflux ; therapy

Objective To determine the effect of Vibrio vulnificus cytolysin (VVC) inducing ap-optosis in human umbilical vein endothelial cell(HUVEC) and its possible mechanism. Methods The en-tire vvhA gene that encoding VVC from V. vulnificus strain GTC333 was amplified by PCR and sequenced af-ter T-A cloning. E. coli BL21DE3pET-42a-vvhA, a prokaryotic expression system of the vvhA gene, was then con-structed. Ni-NTA affinity chromatography was applied to purify the target recombinant protein rVVC, and SDS-PAGE plus Bio-Rad Agarose Image Analyzor were used to measure the output of rVVC and to determine the purity of rVVC extract. The activity of rVVC dissolving rabbit erythrocytes was detected by hemolysis test. DPNH chromotometry and TphBNa chromotometry were performed to examine the contents of LDH and K+ in the supernatants of rVVC-treated HUVEC cultures, respectively. The effect of rVVC inducing apepto-sis of HUVEC was detected by flow cytometry, rVVC was labeled with FITC and the location of FITC-labe-ling rVVC in HUVEC was observed by laser canfocal microscopy. Results The cloned whA gene had 96.09% and 98.26% similarities of nucleotide and amino acid sequences compared to the corresponding se-quences in GenBank. rVVC, with a dosage of 1 μg/ml, could dissolve rabbit erythrocytes (P<0.01). 10 μg/ml rVVC was able to promote the increases of K+ content (P<0.01) but no change of LDH content could be found in the cell supernatants. HUVEC was apoptotic after the cell was treated with 1～100 μg/ml of rVVC for 2 h. In the 5～240 min duration of co-incubation of FITC-labeling rVVC and HUVEC, the rV-VC gradually moved from surface to inner side of the membrane and then entered the cytoplasms. When FITC-labeling rVVC treated HUVEC for 30 min, most of the rVVC was found to be intracellular location. Conclusion rVVC has cytolytic activity. VVC has an ability to enter HUVEC and causes injury of HUVEC via inducing apoptosis, which may be the major pathogenic mechanism of VVC.

Objective To determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Methods Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A. 1 and PMA-activated human monocyte line THP-1 and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A. 1 and PMA-activated THP-1 cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated. Results The leptospires in J774A. 1 cells were located in phagocytotic vesicles while the leptospires in THP-1 cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A. 1 and THP-1 cells in dose-dependent manner. The numbers of leptospires in J774A. 1 and THP-1 cells that pre-blocked with 10 μmol/L or above MDC and 1 μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05＝. After J774A. 1 and THP-1 cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased ( P<0.05 ).Conclusion Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in human and mice after infected with L. interrogans.

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.

Objective: To compare the effects of ultra-fast track anesthesia and traditional anesthesia on blood levels of high sensitivity C-reactive protein (Hs-CRP), C-reactive protein (CRP) and procalcitonin (PCT) in patients after pediatric cardiac surgery.
Methods: A total of 101 patients received pediatric cardiovascular surgery by a same anesthesiologist in our hospital from 2013-09 to 2014-05 were retrospectively reviewed. The patients were studied in 2 anesthesia groups:Ultra-fast track group, in which the extubation was conducted in operating room, n=40 and Traditional group, n=44. Blood levels of Hs-CRP, CRP and PCT at pre-operation (T0), 1st day post-operation (T1) and 2nd day post-operation (T2) were compared.
Results: ①Hs-CRP levels were higher at T1 and T2 than T0 in both groups, all P<0.05;Hs-CRP level in Ultra-fast track group was lower than Traditional group at T1 time point, P<0.05. ②CRP levels were similar among 3 time points in Ultra-fast track group;while in Traditional group, CRP level at T2 was higher than T1, P<0.05; CRP level was higher in Traditional group than Ultra-fast track group at T2, P<0.05.③PCT levels at 3 time points were similar in the same group;while PCT level in Ultra-fast track group was lower than Traditional group at T1 time point, P<0.05.
Conclusion: Compared With traditional anesthesia, ultra-fast track anesthesia could decrease the post-operative elevations of Hs-CRP, CRP and PCT in patients after pediatric cardiac surgery.

0.05) at any observed time points as compared with the controls.3.From day 7 after the adriamycin injection,VEGF protein reduced significantly(P

Objective To construct the T-cells and B-cells combined epitope peptide gene based on the LipL32,OmpL1 and LipL21 protein from Leptospira interrogans and E.coli expression system,and better understanding of the immunological activity of the recombinant protein. MethodsThe immunodomaint T- and B-cells combined epitopes of LipL32,OmpL1 and LipL21 were identified and used to synthetic a new gene and then construct its prokaryotic expression system.The expression of recombinant protein was determined by SDS-PAGE; MAT was used to determine the titer of the antiserum to L. interrogans standard strains of China ; Western blot and ELISA were used to identify the immunity activity of the recombinant protein.Results The synthetic gene was effectively expressed in E.coli BL21 ( DE3 ) strain and mainly presented in dissoluble protein.Western blot result showed that the expression protein react well with the antibodies from immunized rabbit by Leptospira or recombinant protein.ELISA and MAT results showed that the multiepitope protein could cross-react with different serogroup or serovar of Leptospira.Conclusion In this study,we successfully constructed the recombinant T- and B-cells combine epitope gene of leptospires and expressed it in E.coli.The recombinant protein had a good immune activity,and could cross-reacted with antibodies from different serogroups Leptospira infected patients.

To modify and improve the quality standard of Fufang Shilintong capsules. Methods: Lonicerae Japonicae Caulis and Pyrrosoae Folium in Fufang Shilintong capsules were identified by TLC, and the content of chlorogenic in the capsules was determined by HPLC. Results: Specific spots of Lonicerae Japonicae Caulis and Pyrrosoae Folium were shown in TLC. Chlorogenic showed a good linear relationship within the range of 0. 020-1. 530 μg(r=1. 000 0), and the average recovery was 99. 2%(RSD=0. 8%, n=6). Conclusion:The method is accurate and reliable, which can be more effectively used in the quality control of Fufang Shilintong capsules.

Objective To analyze the dynamic trends of physical development,constitutional fitness,and prevalence of obesity among Han children and adolescents in Ningxia aged 7-18 years from 1985 to 2010.Methods Data were collected from the National Survey on Students Constitution and Health in 1985,1991,1995,2000,2005 and 2010,respectively.Height,weight and chest circumference were used to evaluate physical development,vital capacity,50-meter running and vital capacity versus weight ratio for the evaluation of constitutional fitness.Results 1.Weight and chest circumference increased faster than height.The average annual increase of weight among the urban girls was significantly faster than those in rural areas.2.The tempo per year of vital capacity in both boys and girls decreased during 25 years,especially after 2005.The rural students had a faster tempo than those in the urban students,and the rate in boys was higher than those in girls.Vital capacity to weight ratio in boys decreased from 2005 much more than before,and had a decreasing trend in each age group except for a few groups in girls.There was a decrease in the mean time of 50-meter running in both boys and girls from 1985 to 2005,whereas the decrease became slow during 1995-2005.The prolonged trend in 50-meter-run time existed during 2005-2010.The average prolonged speeds per decade in boys and urban students was higher than that in girls and rural areas.3.From 1985 to 2010 year,the increasing prevalence of overweight and obesity among boys and girls was 15.0％,25.0％ and 28.1％,12.4％,respectively.The average increasing rate was much higher in the obese than in the over-weighted children,and there were more in boys than in girls.There was a similar positive trend of increasing prevalence of overweight and obesity with the increased gross domestic product.Conclusions The physical growth and development among students in Ningxia increased rapidly,along with the descending trend of average annual rate of physical fitness as well as the rising trend of prevalence obesity.Some measures should be taken by the government to tackle with the situation,and the healthy intervention should be applied to the high risk population.