Objective To determine the hemolytic activity of products of sphingomyelinase hemolysin encoding genes of Leptospira interrogaas, and the transcriptional level alterations in the infected host cells. Methods By using genomic DNAs of pathogenic L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai and serogroup Pomona serovar Pomona strain Luo, and non-pathogenic L. biflexa serogroup Sama-ranga serovar Patoc strain Patoc Ⅰ as templates, PCRs were performed to amplify entire sph1-sph4 genes. The amplified products were sequenced after T-A cloning. Prokaryotic expression systems of sph1-sph4 genes were re-spectively constructed, and the expressions of target recombinant proteins rSph1-rSph4 were examined by SDS-PAGE. Ni-NTA affinity chromatographic column was used to extract the expressed rSph1-rSph4. Hemolytic ac-tivities of rSph1-rSph4 on sheep blood agar plate were identified. Transcription alterations of sphl-sph4 genes in L. interrogans strain Lai after infected J774A. 1 cells were measured by real-time fluorescence quantitative RT-PCR. Results From genomic DNAs of both L. interrogans strain Lai and Luo, but not from that of L. biflexa strain Patoc Ⅰ , the target fragments of sph1-sph4 genes could be amplified. All the cloned sph1-sph4 genes had 100% nucleotide sequence identities compared to the corresponding reported sequences. The constructed pro-karyotic expression systems were able to efficiently express the target recombinant proteins rSph1-rSph4, respec-tively. All the rSph1-rSph4 had hemolytic activities, and among the four products rSph2 displayed the strongest hemolytic activity. After L. interrogaas strain Lai infecting J774A. 1 cells, the transcriptional levels of sph1-sph4 genes were remarkably up-regulated, especially for mRNA levels of sph2 and sph4 genes. Conclusion sph1- sph4 genes exist only in pathogenic L. interrogans species, and their products have hemolytic activity. The up-regulation of sph1-sph4 gene transcriptional levels in L. interrogans strain Lai after infected cells implies that the sphingomyelinase hemolysins may play important roles in the process of L. interrogans infection in hosts.

To investigate and analyze the factors which influence the public attitudes towards autopsy after accidental deaths.The study has carried on an investigation among 386 individuals in random,with a questionnaire named "The cognition of the public whether to carry on the autopsy after accidental deaths".Using the statistical methods of the Logistic Regression analysis and optimum regression analysis to analyze the recycling questionnaires.Through the diagnosis,we found there are four main factors which influence public opinion upon the autopsy after accidental deaths,the knowledge of autopsy,the believing in autopsy,years of schooling and family financial circumstances.

Objective To analyze the occurrence of insulin resistance ( IR ) and abnormal glucose metabolism in adolescent polycystic ovary syndrome (PCOS). Methods PCOS group included 141 patients aged 15-19 years old, who were diagnosed as PCOS according to criteria by reference to the European Society of Reproduction and Embryology/American Society for Reproductive Medicine proposed in 2003, at Rotterdam; and 266 age-matched female students,with regular menstrual cycles and no family history of diabetes, were enrolled in control group. Fasting insulin(FINS),fasting plasma glucose(FPC) ,and homeostasis model assessment for insulin resistance ( HOMA-IR) were measured in control group. 73% percentile value of control group was set as physical upper limits of FINS and HOMA-IR. PCOS patients were divided into obese ( OB-PCOS) and non-obese (NOB-PCOS) groups, and oral glucose tolerance test(OGTT) were performed. Results According to 75% percentile value of control group,the physical upper limits of FINS and HOMA-IR were 13.13 mIU/ L and 2.69, respectively. FINS and HOMA-IR values in PCOS group were higher than those in control group [ (17.68±16. 13 vs 10.40±5. 33)mIU/L,2. 64±2.01 vs 2. 01 ±1. 61,both P<0.01]. FINS and HOMA-IR values in OB-PCOS group were higher than those in the NOB-PCOS group [ (22.04± 18.01 vs 13.06± 12. 60) mIU/L,4. 62±3. 87 vs 2.38±2.26,both P<0.01]. In PCOS group,FINS of 75 cases(53.19% )and HOMA-IR of 67 patients(47.52% ) exceeded the physical upper limits. In 79 OB-PCOS patients, FINS of 56 cases (70. 89% ) and HOMA-IR of 52 patients (65.82% ) exceeded the physical upper limits while in 62 NOB-PCOS patients there were 19(30.65% ) and 15 (24. 19% )patients. In PCOS group,2(1.42% ) patients were diagnosed diabetes mellitus,and both FINS and HOMA-IR of these two cases increased. Meanwhile, 12 cases(8.51% ) were impaired glucose tolerance(ICT) ,of whom 11 patients FINS and HOMA-IR increased. Conclusion Pathological IR is prevalent in adolescent PCOS, more severe and popular in obese-PCOS, a part of them with abnormal glucose metabolism.

Objective To evaluate the effect of metformin in treating adolescent polycysfic ovary syndrome (PCOS).Methods Group A [ the metformin and medroxyprogesterone acetate (MPA) group ] was made of 135 adolescents with PCOS taking metformin,and MPA if no menstruation came,while group B ( the control group) consisted of 45 patients taking MPA every two months.Endocrinologic profiles and ovulation were evaluated before and after six months treatment.Results ( 1 ) All patients suffered from anovulation before treatment.( 2 ) Ovulation failure was successfully improved in 74.81％ ( 101/135 )of patients in group A after 6 months,which was significantly higher than that before treatment.In group B,no case recovered ovulation.(3) In group A,fasting serum insulin,homeostasis model assay for insulin resistance ( HOMA-IR),total testosterone,LH were significantly decreased.(4) The retrieval of ovulation was negatively correlated with fasting insulin and HOMA-IR ( both P＜0.05 ). Conclusions ( 1 ) Metformin effectively improves ovulation function and reproductive endocrine parameters in adolescent girls withPCOS.(2) Recovery of ovulation is associated with the decrease of serum insulin.

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 screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.

Objective To investigate the expression and clinical significance of ABCG2 protein in hepato-cellular carcinoma (HCC). Methods Specimens of HCC were collected at The First Aifiliated Hospital of Sun Yat-sen University from January 2005 to December 2006. The expression of ABCG2 protein in 165 samples of HCC tissue, 25 samples of normal liver tissue and 40 samples of cirrhotic liver tissue was detected using immunohisto-chemistry. The correlation between the expression of ABCG2 protein and clinicopathological characters was then analyzed. Enumeration data, survival rate and the difference between groups were analyzed with a chi-square test, the Kaplan-Meier method and Log-rank test, respectively. Results ABCG2 protein expression was weakly posi-tive in all normal and cirrhotic liver tissues. In HCC tissues, the expression of ABCG2 protein was strongly positive in 66 cases and weakly positive in 99 cases. The expression of ABCG2 protein was related to tumor diameter, tumor number, adjacent organ invasion and TNM stages (χ2 =8. 130, 14. 279, 4. 820, 21. 179, P <0. 05). Kaplan-Meier survival analysis revealed that patients with strongly positive ABCG2 protein had a significantly lower 3-year overall survival (24. 1%) compared with those with weakly positive ABCG2 protein (39. 4%) (χ2 = 15.716, P<0.05). Conclusions The expression level of ABCG2 protein is related to tumor invasiveness, TNM stage and prognosis. ABCG2 has the potential to become a new target for HCC treatment.

Objective To assess the efficacy of rociverine tablets combined with imrecoxib tablets and comfortable nursing in the control of bladder spasm after transurethral laser resection of prostate.Methods According to the digital table,120 patients who diagnosed as benign prostate hyperplasia (BPH) were randomly divided into four groups.The patients in combined drug group (group A) were administered rociverine tablets(10mg tid) and imrecoxib tablets (100mg bid) from the day of surgery to the day of catheter removal.The patients in rociverine tablets group (group B) were treated with rociverine tablets (10rag tid),and the patients in imrecoxib tablets group (group C) were administered imrecoxib (100mg bid) from the day of surgery to the day of catheter removal.The patients in group D were administered indometacin suppositories in anus when they felt pain.All patients were given comfortable nursing.The frequency and duration of bladder spasm,the time of bladder irrigation,time needed for washing solution to turn clear,and the time of catheter indwelling in the four groups were observed and compared.Results Compared with group D,group A,group B and group C all had statistically significant differences in the frequency and duration of bladder spasm,the time of bladder irrigation,time needed for washing solution to turn clear,and the time of catheter indwelling (Fa =9.469,Fb =13.443,Fo =12.157,Fd =26.609,Fe =33.717,Ff =30.524,Fs =100.836,Fh =89.985,P3 ＜ 0.001).These parameters were lower in group A than those in group B and group C,and the differences were statistically significant (P1 =0.030,0.002,0.012,0.003,＜ 0.001,＜ 0.001,＜ 0.001,＜ 0.001;P2 =0.030,0.004,0.035,0.001,＜ 0.001,＜ 0.001,＜ 0.001,＜ 0.001).There were no statistically significant differences between group B and group C (all P4 ＞ 0.05).Conclusion Rociverine tablets combinined with imrecoxib tablets for the prevention and treatment of bladder spasm after transurethral laser resection of prostate was effective,which was worthy of clinic application.

Objective To evaluate the reliability and validity of the Chinese version of Uncertainty Scale for Kids-22 (USK-22).Methods The Uncertainty Scale for Kids was translated and adapted according to Chinese culture.The reliability and validity of the Chinese version of USK were tested in 117children and adolescents with cancer.Results The rate of recovery was 100％.The rote of effective questionnaire was 95.7％.The patients were easily to understand and it cost them average 8 minutes to finish the questionnaires.Three factors were extracted by factor analysis and could explain 57.043％ of total variance.The correlations between each item and subscale ranged from 0.526 to 0.762.The Cronbach α of USK was 0.872 and the Cronbach α of each subscale ranged from 0.774 to 0.826.The testretest reliability was 0.835.Conclusions The Chinese version of USK is reliable and valid,and can be used to measure the level of uncertainty among Chinese children and adolescents with cancer.

Objective To investigate illness uncertainty state and explore its influencing factors in hospitalized children and adolescents with cancer.Methods A cross-sectional survey was performed in 112 children and adolescents with cancer aged 7～18 years old from two hospitals with the Chinese version of Uncertainty Scale for Kids-20 and the self-designed demographic questionnaire.Results The mean total scores of illness uncertainty was (45.16±6.67) points.The results of single-factor analysis showed that the factors influencing the uncertainty in illness of children and adolescents with cancer were as follows:age,personality characteristics,course of disease,the methods of treatment,current disease status,absence from school or not,the times of hospitalization,family monthly income,and the extent of parents' understanding about the disease-related knowledge.Multi-factor stepwise regression analysis showed that the main influencing factors of the uncertainty in illness were the current condition,family monthly income,absence from school or not and the extent of parents' understanding about the disease-related knowledge.Conclusions Illness uncertainty is common in children and adolescents with cancer.Nurses should attach importance to the impacts of illness uncertainty,and take effective interventions to decrease it,alleviate psychological pressure and improve quality of life in children and adolescents with cancer.