Child ; Humans ; Nephrology ; Pediatrics

Child ; Humans ; Vesico-Ureteral Reflux ; therapy

In surgery operations, wound should be cleaned with warm sterilized saline solution. In order to reach rapidly warming the washing solution from the room temperature during the surgery, we designed a thermostatic medical infusion pump. The present paper mainly presents researches on the two temperature control methods in the standby mode and in the flushing mode of the system. In the standby mode, the traditional proportional-integral-derivative (PID) control algorithm was adopted. In the flushing mode, dynamic characteristics of the system was changed in real time, which made the thermostatic control process more complex, and the fitted control function combined with the PID control algorithm was adopted in this mode. The temperature control parameters were adjusted in real time according to the initial temperature and the flow rate of the washing solution to obtain a constant temperature of the washing solution, no matter how the initial temperature and the flow rate are changed. The experiment results showed that this kind of control system performed well with a high accuracy.
Algorithms ; Equipment Design ; Infusion Pumps ; Sodium Chloride ; Solutions ; Temperature

G) was relatively rare. Conclusion: DHPLC is a rapid and reliable method for polymorphism screening. Eight polymorphisms in the NR3C1 gene are detected in Chinese population with the technique of DHPLC , of which two haplotypes have been identified for the first time.

Objective To compare the sensitivity and specificity of Leptospira interrogans using loop-mediated isothermal amplification (LAMP) and real-time PCR technology, then looking for a rapid,sensitive and specific methods for the detection of Leptospira interrogans. MethodsIn accordance with lipL41 gene from Leptospira interrogans, primers for LAMP and real-time PCR were designed and used to detect Leptospira interrogans in cultured 15 reference strains of 15 serogroups in China, then compared the sensitivity and specificity of the two methods in the detection of Leptospira interrogans. ResultsThe LAMP reaction could be completed within 30 min, and whole process of it less than 60 min. The whole real-time PCR reaction could be finished at about 60 min. Both of them had the same detection sensitivity and specificity,the lower detection limits in the reactions was approximately 100 copies and there was no false positive occurred. ConclusionBoth LAMP and real-time PCR were time-saving and had the same sensitivity and specificity. But LAMP reaction could be done under a constant temperature conditions, and need not a special expensive equipment. Therefore, as a sensitive and specific method for quantifying Leptospira interrogans,the LAMP assay was more rapidly and convenient than conventional methods.

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 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 investigate the distribution of ompA gnne in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogate, and to express recombinant OmpA ( rOmpA ) and to identify immunogenicity and immunoprotection of rOmpA. Methods Genomic DNAs from different leptospiral strains were extracted by phenol-chloroform method. Entire ompA gene fragments from the strains were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of ompA gene from L. interrogans strain 56601 was constructed, and the expression and yield of rOmpA were determined by SOS-PAGE plus Bio-Rad Agarose Image Analyser. Rabbits were immunized with rOmpA for obtaining antiserum, and immunodiffusion test was used to measure the antiserum's titer. Western blot assay was performed to determine the immunoreaetivity of rOmpA with the antiserum against rOmpA and antiserum against whole cell of L. interrogans strain 56601, while mi-croscopic agglutination test (MAT) was applied to detect the cross agglutination to the 15 L. interrogans strains. A leptospire adhering cell model and a leptospire infecting guinea pigs model were used to determine the adhesion-bloc-king effect of rOmpA antiserum and immunoprotection of rOmpA. Results All the 15 L. interrogans strains, but not L. biflexa strain Patoe Ⅰ , had sequence conserved ompA genes. The yield of rOmpA was approximate 20% of the total bacterial proteins, rOmpA could induce rabbits to produce antibody and immunodiffusion titer of the anti-serum was 1:4. Both antisera against rOmpA and against whole cell of L. interrogans strain 56601 were able to pro-duce positive Western blot signs to rOmpA, and the former offered 1 : 20-1 : 320 MAT titers to the 15 L. interrogans strains. 1: 10-1:160 dilutions of rOmpA antiserum could efficiently block L. interrogans strain 56601 adhering to J774A. 1 cells, and 100 μg and 200 μg rOmpA displayed 50.0% and 75.0% immunoprotective rates in the infee-ted guinea pigs. Conclusion ompA gene only exists in genomes of different pathogenic L. interrogans serogroups. rOmpA has relatively stronger antigenicity, cross immunoreactivity and certain immunoprotection, implying that this recombinant protein may be used as a candidate antigen for developing universal genetic engineering vaccine of L. interrogans.