Objective To explore a rapid diagnostic method in neonatal sepsis and bacterial me- ningitis. Methods The primers and TaqMan probes were designed and synthesized based on the sequences of bacterial 16S rRNA gene. Nineteen bacterial strains, 3 different viruses, fungus and human genomic DNA were tested by FQ-PCR assay. Blood specimens and CSF from 195 cases of suspected septicemia were detected with both TaqMan PCR assay and blood or/and CSF culture. Results The FQ-PCR showed very high sensitivity and specificity and was able to detect at least 10 copies of 16S rRNA gene equivalent to 1~2 copies bacterium. No cross-reaction was found with human genomic DNA, other fungus and viruses. Among the 195 cases, the positive rate by FQ-PCR was 12.8%(25 cases) and 7.1%(15 cases) by blood culture ( P

Objective To establish a method for rapid diagnosis and identification of seven species human herpesviruses infection.Methods Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase genes in human herpesviruses, namely herpes simplex virus type 1 (HSV-1),HSV-2,varicella-zoster virus (VZV),Epstein-Barr virus (EBV),cytomegalovirus (CMV),and human herpes virus 6 (HHV-6A/6B).DNA microarrays were made by printing the oligonucleotide probes on the special glass slides.A total of 282 blood specimens from children with suspected infection were analyzed by this DNA microarray technique,and the results were compared with those of TaqMan PCR.Results The products of the seven human herpesviruses after PCR amplification could be used to identify the virus species with DNA microarrays.The detection limits were 10 copies/?l for HSV-1,HSV-2,VZV,EBV,CMV,HHV-6A,and HHV-6B,respectively.The assay did not show cross-reaction to the DNA extract of hepatitis B virus,staphylococcus aureus,E.coli,Candia albicans and human genome.Among the 282 samples,59 were positive for human herpesviruses DNA.Compared with those of TaqMan PCR,the sensitivity and specificity of the microarray assay were 96.7% and 99.5%,respectively,and the index of accurate diagnosis was 0.962.Conclusions This DNA microarray for identifying human herpesviruses species is specific and sensitive,and may serve as an efficient technique for simultaneous detection and species identification of human herpesviruses in clinical specimen.

Objective To explore the feasibility of using human umbilical cord derived mesenchymal stem cells as seed cells to repair sciatic nerve defects of rats by tissue engineering methods. Methods Mesenchymal stem cells from human umbilical cord were cultured and induced into neuron-liked cells,which were co-cultured with acellular basal lamina tube to construct tissue engineering nerve;models of sciatic nerve defects 10 mm in length were set up with thirty healthy adult SD rats and were divided randomly into 3 groups:tissue engineering nerve group (group A, compound of human umbilical cord derived mesenchymal stem cells and acellular basal lamina tube), pure acellular basal lamina tube group (group B), and autogenous nerve bridging group (group C). Evaluation of electrophysiological and histological results was carried out 10 weeks after operation. Results The engineering nerve group had good result in nerve regeneration which was close to the effect of autogenous nerve transfer group (group A), and much better than the effect of pure acellular basal lamina tube group. Conclusion Engineering nerves from human umbilical cord derived mesenchymal stem cells can effectively repair 10 mm defects of sciatic nerve.

Objective To improve the speed and accuracy of bacteria detection, and develop the test of 16S rRNA genes PCR amplification plus gene chip hybridization to diagnose neonatal sepsis. Methods Bacterial 16S rRNA genes were detected in blood and CSF samples of 125 suspected neonatal sepsis, and the results were compared with blood culture, CSF culture, and non-specific diagnostic parameters (WBC, PLT, CRP). 30 non-infectious neonates were regarded as the negative control group. Gene chip test were performed by extraction of DNA, primers and probes design, PCR amplification, preparation of gene chip, hybridization, laser scan and reading of the results. 18 specific probes, including universal 1, universal 2, Gram positive bacterial probe, Gram negative bacterial probe 1, Gram negative bacterial probe 2, Staphylococcus aureus, Staphylococcus epidermidis, CoNS (Coagulase Negative Staphylococcus), Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Bacillus, Meningococcus, Corynebacterium, Propionibacterium, were used in the test. Results The positive rate of PCR test was 51.2% in 125 blood samples, and was significantly higher than the positive blood culture (25.6%), or the indicator of two abnormal non-specific parameters (32.8%) (P

Objective To study the etiology,diagnosis,treatment and prophylaxis strategy of the urogenital fistula caused by gynecological and obstetrical surgery.Methods Data of 64 cases with urogenital fistula,who were admitted into second hospital of Tianjin medical university and Tianjin first central hospital from January 1992 to December 2012,were analyzed retrospectively.In Tianjin first central hospital,those cases include vesicovaginal fistula in 10,ureterovaginal fistula in 7 and urethro-vaginal fistula in one case.In second hospital of Tianjin medical university,those cases include vesicovaginal fistula in 26,ureterovaginal fistula in 18,urethro-vaginal fistula in 1 and ureterouterine fistula in one case.The median age was 42 years old (range 21-53).The history of diseased ranged from 16 days to 30 years.All patients were diagnosed by methylene blue test,cystoscopy,ureteroscopy,intravenous urography,ultrasound,computed tomography (CT) and magnetic resonance urography (MRU).The primary fistula was diagnosed in 50 cases and the recurrence was found in 14 cases.Single fistula existed in 56 cases and multiple fistulas were found in 8 cases.In 36 patients with vesicovaginal fistula,transabdominal repair of vesicovaginal fistula (n =20),transpubic surgery (n=10) and transvaginal surgery (n=6) were chosen.In 25 patients with ureterovaginal fistula,ureterocystostomy (n =10),ureterotomy with holmium laser (n =8),ureteral stent placement (n =6) and ureteral stricture excision and bladder-psoas suspension (n=1) were used.Two patients with urethro-vaginal fistulae were cured by the Latzko technique.One patient had uretero-uterine fistula and cured by ureteral stricture excision,ureterocystostomy and bladder-psoas suspension.Results Fifty-five(86％) cases were cured by single-stage surgical treatment and nine patients experienced more than two times of surgical treatment.The incipient patients have a higher success rate of first surgery than recurrent patients (92％ vs.64％,P＜0.05).Single and multiple fistulas have no significant difference about the surgical successive rate (88％ vs.75％,P＞0.05).In cases with vesicovaginal fistula,the success rate of vaginal and abdominal approaches are the same 85％ (P＞0.05).In cases with ureterovaginal fistula,abdominal and endoscopic approaches were 100％ and 85％,respectively (P＞0.05).The mean duration of follow was 20 months (range 3-48).There was no recurrence during follow-up.Conclusions Urogenital fistula caused by gynecological and obstetrical operation can be cured by surgery.Recurrent fistula is a challenge for diagnose and treatment,preoperative need reasonable operation mode to improve the success rate of operation.Both open surgery and endourology approaches are effective treatment options in management the urogenital fistula.

Objective To establish HPLC method for the simultaneous detgrmination of 9 effective components ofTanreqing injection.Methods The Agilent TC-C18 (2) (250 mm×4.6 mm, 5μm) column was used, the mobile phase consisted of acetonitrile (A) acetonitrile-0.1% formic acid aqueous solution (B) in a gradient mode, the flow rate was 1.0 ml/min,the column temperature was 30℃, the injection volume was 10μl, and the detecting wavelength was at 280 nm.Results All the 9 effective components showed a good linear relationship. TheRSDof the precision, reproducibility and stability tests were less than 2%. The average recoveries of the 9 effective components were in the range of 99.3%-103.1%.Conclusion This analysis method is simple, accurate and has a good specificity, which can be suitable for controlling the quality of Tanreqing injection.

Objective To study the thrombolysis effect and safety of UK combined with tirofiban in young patients with ST segement elevation acute myocardial infarction.Methods 76 young patients with ST segement elva-tion acute myocardial infarction were selected as research objects,and they were divided into UK group(control group, n =34)and UK combined with tirofiban group(observation group,n =42)by random number table,then the recanali-zation rate of infarct -related artery and incidence of complications were compared.Results The recanalization rate of infarct -related artery in the observation group was 97.6%,which was 91.2% in the control group,there was no significant difference between the two groups(χ2 =1.564,P >0.05);The vascular infarction related to blood flowⅡlevel of the patients was 90.5% in the observation group,which was significantly higher than 70.6% in the control group(χ2 =4.945,P <0.05);The death,complications incidences had not significant differences between the two groups(χ2 =1.252,2.837,2.837,all P >0.05).Conclusion The thrombolysis effect of UK has a high recanaliza-tion rate of infarct -related artery in young patients with ST segement elevation acute myocardial infarction,and the thrombolysis effect of UK combined with tirofiban is better,and it does not increase the incidence of complications.

Objective: To determine the molecular weight and p urity of porcine platelet-derived growth factor (pPDGF) and to investigate its effect on DNA synthesis of human umbilical vein endothelial cells. Metho ds: In the present experiment, the high performance liquid chromatograph y was used and the molecular weight and purity of pPDGF were studied. Human umbi lical vein endothelial cells was cultured and effects of pPDGF on DNA synthesis of endothelial cells was observed by 3H-TdR incorporation in vitro. Results: The findings of high performance liquid chromatography showed that the molecular weight of pPDGF was 29 120 and the purity was 89.46%, a nd pPDGF significantly promoted DNA synthesis of quiescent endothelial cells wit h a maximal response at a concentration of 40 ng/ml at 48 h. Conclusion: The molecular weight of pPDGF is 29 120, and it can promote DNA synthes is of cultured human umbilical vein endothelial cells.

Objective To develop a method of simultaneous detection and Gram classification for pathogens causing sepsis with gram-specific probes based real-time PCR. Methods A pair of universal primers and a set of probes including Gram-positive probe and Gram-negative probe were designed based on the bacterial highly conserved region of 16SrRNA gene. With the gram-specific probes based real-time PCR, 35 clinical frequently-isolated strains including 17 gram-positive and 18 gram-negative bacteria were identified correctly with the corresponding gram probe. The blood samples from 512 cases of suspected septicemia, who were hospitalized in our neonatal ward and the NICU and developed clinical signs suggestive of infection, were tested with routine culture and bacterial gram-specific probes based real-time PCR separately. Results The detection limit of the gram-specific probes based real-time PCR assay was 10 CFU of the bacteria. The 35 isolates could be detected and classified correctly by gram-specific probes based real-time PCR. The PCR results were all negative for Cytomegalo virus, EB virus, hepatitis B virus, Cryptococcns neoformans, candida albican, human genomic DNA and negative control. The gram-specific probes based real-time PCR appeared to be quite specific. For 512 blood specimens from the patients with suspicious neonatal sepsis, the positive rate of the gram-specific probes based real-time PCR array was 8.20% (42/512,), which is significantly higher than that of blood culture (32/512, 6.25%) (χ2=8.10, P<0.01). When blood culture was used as a standard, the sensitivity of the gram-specific probes based real-time PCR was 100%. The specificity was 97.92% and the accuracy was 98.05%. Canclusions Cram-specific probes based real-time PCR with universal primers and gram-specific probes are developed. This study suggests that the bacterial gram-specific probes based real-time PCR are very useful for the rapid and accurate diagnosis of bacterial infection.

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on cultured anoxic-reoxygenation human umbilical vein endothelial cells. METHODS:In the present experiment, an anoxic-reoxygenation model was established by using cultured human umbilical vein endothelial cells. The uptake rate of trypan-blue and calcium and magnesium contents of endothelial cells and lactic dehydrogenase (LDH) activity in medium and malondialdhyde (MDA) content of endothelial cells were measured 60,120 and 180 min after anoxia and 30 and 60 min after reoxygenation, and the effects of 1?10 -8 mol/L CGRP on anoxic-reoxygenation endothelial cells were studied. RESULTS: The findings showed that, as anoxia prolonged, the uptake rate of trypan-blue and LDH activity and MDA content gradually elevated and, during reoxygenation, these parameters sharply increased. Calcium and magnesium levels gradually declined as anoxia prolonged, and during reoxygenation calcium content significantly increased. Meanwhile, 1?10 -8 mol/L CGRP might significantly reduce the uptake rate of trypan-blue and LDH activity and MDA content during anoxia and reoxygenation and lessen the increase in calcium content and the loss of magnesium during reoxygenation. CONCLUSION: These results showed that CGRP might have a direct protective function to endothelial cells afflicted with anoxic-reoxygenation injury by inhibiting lipid peroxidation, attenuating calcium overload and loss of magnesium and enzyme.