Objective To investigate the molecular epidemiology of CRAB isolated from children in wuhan. Methods Forty non-repetitive strains of CRAB were collected from hospitalized children of emergency department, neonatal medicine, cardiothoracic surgery, bone surgery, respiratory medicine and renal medicine in Wuhan children's hospital during December 2008 and May 2009. MIC values were PFGE; KPC, IMP, GIM, SPM, SIM, OXA-23, VIM genes and integrase gene were amplified by PCR and then sequenced to confirm the genotypes.; Plasmid conjugation experiment was used to study the transfer method of bacterial resistance and southern blot hybridization was used to target the resistance genes. Results Susceptible rates of 40 strains to gentamicin, tobramycin, amikacin, ciprofloxacin, levofloxacin, trimoxazole were 20%, 5%, 93%, 93%, 95%, and 23% respectively. Eleven types of clone were detected by PFGE,including 29 strains of type A clone, 2 strains of type B clone, and 1 strain for each type of C to K clone. Eleven isolates produced both IMP-4 and OXA-23 carbapemase. Twenty-six isolates only possessed OXA-23 carbapemase. Thirty-six strains carried class Ⅰ integron. The results of southern blot hybridization showed that Intl, IMP-4 and OXA-23 type were located on chromosome. Conclusions Type A clone of CRAB is the most common. OXA-23 and IMP-4 type are the major acquired carbapemases, especially the OXA-23 is the most common type. The horizontal transmission of OXA-23 and IMP-4 gene mediated by Int1 and the spread of type A resistant clone is the major way of the spread of carbapenem-resistant Acinetobacter baumannii in the region.

Objective To investigate the distribution of acquired carbapenemases in carbapenemresistant strains of Klebsiella pneumoniae, and explore its role in epidemiology of nosocomial infection. Methods From November 2008 to March 2009, twenty clinical isolates of carbapenem-resistant Klebsiella pneumoniae were collected from children hospitalized in Wuhan children's hospital. MICs of antibiotics were tested by DNA of Klebsiella pneumoniae. Modified Hodge test was used to screen strains producing carbapenemases,combined imipenem(IPM)-EDTA , meropenem(MEM)- EDTA and ceftazidime(CAZ) - EDTA double-disk synergy test (DDST) were used to detect metallo-β-lactamase-producing. PCR amplification of the carbapenemase and integrase genes, and sequencing were performed. Plasmid conjugation transfer experiments and Southern hybridization were applied to study the mode of drug resistance transmission. Results Four types of Klebsiella pneumoniae were detected by PFGE, type A consisted of 5 strains, including 3 strains of type Al and 2 strains of type A2), type B (2 strains), type C (12strains) and type D (1 strain). Type A and C were the main drug resistant clones. Eight strains of Klebsiella pneumoniae carried both KPC-2 and IMP-4 genes, 10 strains carried IMP-4 gene, 2 strains carried KPC-2 gene. None of NDM-1 ,GIM, SPM, SIM, OXA-23, and VIM carbapenemase genes was detected in 20 isolates. All of 20 isolates carried lntl which were found to be located on bacterial chromosome by Southern blot. Conclusions KPC-2and IMP-4 genes are the major carbapenemase genes in Klebsiella pneumoniae isolated in Wuhan.Transmission of drug resistance is mainly through vertical transmission of type C resistant clone and horizontal transmission of Intl on bacteria chromosome.

Objective To explore the effect of hepatitis B virus(HBV) on the expression of apolipoprotein B(ApoB) and its regulatory mechanism. Methods mRNA and protein expression of ApoB in HepG2 and HepG2.2.15 cells was measured by RT-PCR and Western blot, serum ApoB levels in patients with HBV infection and in healthy individuals were measured by biochemical analyzer Olympus 5400, the expression of ApoB difference among healthy individuals, patients with chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma were analyzed, HBV infectious clone pHBV1.3 was tranfected into HepG2 cells,and expression of ApoB and microsomal triglyceride transfer protein(MTP) was measured by RT-PCR and Western blot. Results Expression of ApoB mRNA and protein was lower in HepG2.2.15 cells than in HepG2 cells, serum apoB levels was much lower in patients with chronic hepatitis B and liver cirrhosis as compared to healthy individuals( P ＜0.05 ), HBV could inhibit the expression of ApoB and MTP at mRNA and protein levels. Conclusion HBV may downregulate the synthesis and secretion of ApoB via inhibits the expression of MTP.

Objective To explore the regulatory effect of LPS on the expression of interleukin 27 (IL-27),and uncover the relationships among cyclooxyenase-2(COX-2),prostaglandin E2(PGE2) and IL-27.Methods THP-1 cells were stimulated with different doses of LPS,IL-27 and PGE2 levels in the supernatants were measured by enzyme-linked immunosorbent assay(ELISA) at different time,expression of COX-2 was measured by RT-PCR and Western blot.Results IL-27 can be induced by LPS in THP-1 cells in a time and dose dependent fashion,and IL-27 induces COX-2 mRNA expression,COX-2 protein production,and PGE2 release in a time and dose fashion.Conclusion LPS can stimulate the relase of IL-27 at cell level,and IL-27 can induce the expression of COX-2 and production of PGE2.

Objective To analysis the risk factors and drug resistance of multidrug-resistant pseudomonas aeruginosa(MDRP) infection and to provide the basis for clinical anti-infective therapy .Methods Predisposing factors and drug resistance to clinical commonly used antibacterial drugs of MDRP which were separated from September 2010 to December 2011 were adopted for retro-spective analysis .Results A total of 235 hospital MDRP infection were collected ,97 multidrug resistant strains were concluded ,the separation rate was 41 .2% .The separation rate of ICU and neurosurgery were higher ,account for 35 .54% and 22 .31% respective-ly .The main resource of specimen is respiratory tract ,about 75 .21% .The resistance of MDRP was very serious .The resistant rate of Amikacin was the lowest ,account for 37 .11% ,followed by piperacillin/tazobactam ,ceftazidime ,account for 47 .4% and 48 .45%respectively .The resistant rate of other antimicrobial agents were greater than 50% .Advanced age ,serious underlying diseases ,long hospital stay ,long-term repeated application of broad-spectrum antimicrobial drugs ,admission to ICU ,invasive treatment operations were the risk factors for MDRP resistance .Conclusion The resistance is very serious ,in order to reduce the generation and spread of drug-resistant strains ,the hospital infection control ,drug resistance monitoring ,rational use of antimicrobial drugs and prevention of cross-infections should be strengthened .

Objective To investigate the effect of midazolam on human sperm motility in vitro.Methods Sperm samples were obtained from normal adults and prepared with discontinuous percoll gradient centrifugation technique.The samples were randomly divided into 3 groups (n =10 each):control group and 2 midazolam groups.The samples were incubated with normal saline in control group and with midazolam with the final concentrations of 5 or 1 μg/ml in 2 midazolam groups.The samples were incubated for 60 min in an airtight container at 37 ℃.Then human sperm motility was examined in vitro at 37 ℃ and analyzed by the computer-assisted sperm analysis at 10,30 and 60 min exposure to midazolam,including sperm motility (a + b)％,curvilinear velocity,straight line velocity,average path velocity,amplitude of lateral head displacement,beat-cross frequency,linearity,wobble,straightness,and mean angular displacement.Results There was no significant difference in the parameters of human sperm motility within each group and between groups (P ＞ 0.05).Conclusion Midazolam has no significant effect on human sperm motility in vitro.

Objective To investigate the effect of melatonin on ketamine-induced apoptosis in hippocampal neurons of fetal rats.Methods Sixteen to eighteen day pregnant Sprague Dawley rats were anesthetized.The fetal rats were obtained under sterile condition and decapitated.The hippocampal neurons were isolated and primary cultured for 5 days.The primary cultured neurons were randomly divided into 5 groups (n =6 each):control group (group C),ketamine group (group K),and 1.0,2.5 and 5.0 mmol/L melatonin groups (groups M1-3 respectively).Ketamine with the final concentration of 1 000 μmol/L was added to the culture medium and the neurons were incubated for 3 h in group K.In groups M1-3,1.0,2.5 and 5.0 mmol/L melatonin were added to the culture medium,respectively,at 60 min before the addition of ketamine,and the neurons were incubated for 3 h.While the equal volume of normal saline was added instead in group C.The neuronal viability during the developmental phase was assessed by MTT assay.The mitochondrial membrane potential (Ψm) was measured by flow cytometry.The expression of cAMP response element binding protein phosphorylation (p-CREB (Ser133)),Bcl-2,Bax,and cytochrome C was detected by Western blot.Results Compared with group C,the neuronal viability and Ψm were significantly decreased,and the expression of p-CREB and Bcl-2 was down-regulated,while the expression of Bax and cytochrome C was up-regulated in group K (P ＜ 0.05).Compared with group K,Ψm was significantly increased in groups M2 and M3,and the neuronal viability was significantly increased,the expression of Bcl-2 was up-regulated,while the expression of Bax and cytochrome C was down-regulated in groups M1-3 (P ＜ 0.05).Conclusion Melatonin can protect the hippocampal neurons of fetal rats from apoptosis triggered by ketamine via regulating the expression of Bcl-2 and Bax,stabilizing Ψm,inhibiting the release of cytochrome C from mitoehondria,and preventing apoptosome formation.

Objective To confirm that rat bone marrow mesenchymal stem cells (MSC) transfected with nerve growth factor (NGF) gene in the bladder tissue of diabetic rats bladder tissues can survive and stably express NGF. Methods A diabetic rat model was constructed. The BrdU-labelled MSC transfected with NGF gene were transplanted into the diabetic rats bladder tissues. BrdUlabelled immunohistochemistry was used to observe the growth of MSC transfected with NGF gene in the diabetic rats bladder tissues. The expression of NGF mRNA and protein were checked by RT-PCR and ELISA. Results A diabetic rat model was successfully built by a single intraperitoneal injectionof STZ. The blood glucose was still high after 8 weeks. NGF gene modified MSC could be detected in the bladder of diabetic rats by BrdU-labelled immunohistochemistry. The concentration of NGF in the control group, disease group and treatment group were ( 114 ± 3), ( 70 ± 2), ( 110 ± 2) pg/ml by ELISA and mRNA quantity by RT-PCR were 0. 183±0. 004, 0. 032±0. 139, 0. 130±0. 165, respectively. Compared with the control group, the expression of NGF gene was decreased (P＜0. 05) in the incidence group. The expression of NGF gene was increased (P＜0. 05) in the treatment group compared with the disease group. Conclusions The NGF gene-modified MSC could survive in diabetic rats bladder tissues. The NGF gene in MSC could stably express in diabetic rats bladder tissues.

Objective To study the clinical features, effects of therapeutic regimen and prognosis of patents with mantle cell lymphoma (MCL). Methods Clinical data of 27 MCL patients admitted in Tianjin Medical University Cancer Institute&Hospital from January 2008 to December 2014 were retrospectively analyzed. Cox regression analysis was used to analyze influencing factors of prognosis of MCL. Results The median age was 68 years old for 27 patients, and the male-to-female ratio was 4.4∶1. Ann Arbor staging showed that 25 cases were stageⅢ-Ⅳ(92.6%), 8 cases were heptosplenomegaly (29.6%), 7 cases showed extranodal involvement (25.9%). ECOG scoring showed that 4 cases with scores of 2-4 (14.8%), 8 cases were 0-3 (29.6%), 14 cases were 4-5 (51.9%) and 5 cases were 6-11 (18.5%). The Ki-67 index≤30%was found in 9 cases (33.3%), and>30%was found in 18 cases (67.7%). Patients with B symptom was found in 10 (37.0%). The elevated lactate dehydrogenase (LDH) was found in 17 cases (63.0%). The increased Beta 2- microglobulin was found in 8 cases (29.6%). Seven patients were found with bone marrow involvement. The total effective rate (ORR) was 81.8%in group with R-CHOP method, and the ORR was 68.8%in group with CHOP method. Multivariate analysis showed that age, LDH and Ki-67 were independent factors influencing the prognosis of MCL (P<0.05). Conclusion Most patients with MCL are found in advanced stage. Patients with age>60 years, elevated LDH and Ki-67 index>30%are with poor prognosis.

Objective To analyze the metabolic changes of myocardial tissue in rats under acute exposure to macleaya cordata by gas chromatography mass spectrometry(GC-MS),explore forensic identifications of its characteristic metabolites,and verify its toxicological mechanism in poisoning cases.Methods The rats in the exposure group were given 382 mg/kg macleaya extract solution by gavage,and the rats in the control group were given the same dose of solvent.The myocardial samples were analyzed by GC-MS,and pattern recognition was conducted through partial least squares discriminant analysis(PLSDA).The differential metabolites with characteristic changes were identified by variable importance projection(VIP value＞1)and Student's t test(P＜0.01).Results Compared with the control group,21 potential characteristic metabolites were identified.Through KEGG pathway enrichment analysis,it was found that these metabolites were mainly involved in the pathways of glycine,serine and threonine metabolism;pyruvate metabolism and glycerolipid metabolism.Conclusion Through the study of myocardial metabolism in rats exposed to macleaya cordata,we found the information on metabolites closely related to poisoning,which provides new insight and reference for studies on the mechanisms of macleaya cordata poisoning in the field of forensic medicine.