Objective To establish a real-time quantitative PCR assay for the identification of Campylobacter jejuni in fecal samples.Methods Specific primers of the PCR were designed according to the conserved sequences of Campylobacterjeju-ni,and the real-time quantitative PCR assay was established.150 cases of fecal samples were tested by both culture and PCR methods.With the culture testing results as the reference standard,the sensitivity,specificity,accuracy and repetition of the real-time quantitative PCR were validated.Kappa test was used to estimate the difference between the two detection meth-ods.Results The standard carve of the real-time quantitative PCR assay fitted the equationY=-3.51Log(X)+37.09 (R2=0.996)well.The sensitivity,specificity,and accuracy of the established method were 92.4%,95.8% and 94%,respective-ly.The theoretical detection limit of the PCR method was 102 CFU/ml,and its reproducibility was good (CV<5%).Statisti-cal analysis demonstrated that the results of the two methods were consistent,and the consistent strength was very strong (Kappa=0.88,P<0.05).Conclusion The established real-time PCR method can assay the Campylobacterjejuni in human fecal samples rapidly and accurately.

Objective To establish a real-time fluorescence PCR method to detect the drug resistance genes of pathogenic Campylobacter jejunum in human stool samples,and investigate the relationship between quinoloneantibiotic resistance and the related genes in Campylobacter jejuni .Methods According to the gyrA and gyrB gene sequences that related with the fluoroquinolone resistance in Campylobacter jejuni ,the primers of the PCR method was designed and synthesized.A rapid real-time fluorescence PCR method to detect the drug resistance genes in Campylobacter jejuni samples was established,and the optimum reaction system and conditions were screened through an optimized approach.The developed method was com-pared with the classical drug susceptibility assay.Results It was found in the compared results that,there were 8 inconsis-tent strains of Campylobacter jejuni ,2 of the 8 strains were drug sensitive but contented the drug resistance gene,while 6 strains were drug resistant but had no drug resistant gene.Conclusion The established method can be applied to detect the drug resistance relative genes of gyrA and gyrB in Campylobacter jejuni .There was some correlation between the drug re-sistance representation and its genotype,but this point requires further studies.

Animals ; Apoptosis ; Cell Cycle ; Cell Movement ; Cell Proliferation ; Connexin 43 ; genetics ; metabolism ; Down-Regulation ; Gap Junctions ; metabolism ; physiology ; Humans ; Neoplasms ; metabolism ; pathology ; Tumor Suppressor Proteins ; genetics ; metabolism

Objective To study the expressions of cysteinyl aspartate specific proteinase 3 ( Caspase 3 ) and proliferating cell nuclear antigen ( PCNA ) in intestinal tissue of neonatal rats with necrotizing enterocolitis ( NEC ) , and the protective effect of glutamine ( Gln ) on NEC. MethodsThirty-six neonatal Sprague-Dawley ( SD) rats were randomly assigned into 3 groups at 48 h after birth (12 in each group). The control group were fed with milk replacer. The NEC group were fed with milk replacer and experiencing cold exposure after hypoxic-reoxygenation twice a day for 3 days, The Gln+NEC group were fed with milk replacer plus Gln and experiencing cold exposure after hypoxic-reoxygenation twice a day for 3 days. All the rats were sacrificed and intestinal tissues obtained at day 3 of the establishment of model. The histological changes of ileal tissues were studied using hematoxy lin-eosin ( HE ) staining. The expressions of Caspase 3 and PCNA were detected using immunohistochemical(IHC)method.Results Caspase3expressioninNECgroup(77.3±8.6)℅was significantly higher than the control group (18. 9 ± 3. 4)℅ and Gln+NEC group (50. 3 ± 6. 2)℅ ( P<0. 05). Also, Caspase 3 in Gln+NEC group was significantly higher than the control group (P<0. 05). PCNA expression in the NEC group ( 15. 0 ± 1. 9 )℅ was significantly lower than the control group (34. 2 ± 5. 8)℅ and the Gln +NEC group ( 24. 0 ± 3. 9 )℅ ( P <0. 05 ) . PCNA expression in the Gln+NEC group was significantly lower than the control group ( P<0. 05). The pathological score of the intestinal tissues was significantly correlated with Caspase 3 expression ( r = 0. 769, P = 0. 005 ), Caspase3/PCNA ratio (r=0. 835,P=0. 002) and PCNA expression (r= -0. 698, P=0. 014) in the NECgroup.Conclusions Up regulation of Cas pase3 and down regulation of PCNA might be correlated with the process of NEC. Gln might be effective in prevention and healing of NEC by inhibiting apoptosis and promoting cell proliferation.

AIM: To explore whether exogenous hydrogen sulfide (H2S) depresses high glucose (HG)-in-duced injury by modulating the Janus kinase/signal transducer and activator of transcription ( JAK/STAT) pathway in hu-man umbilical vein endothelial cells (HUVECs).METHODS:The protein levels of JAK2, STAT3 and cleaved caspase-3 were determined by Western blot.The cell viability was measured by CCK-8 assay.Mitochondrial membrane potential ( MMP) was detected by rhodamine 123 staining followed by photofluorography.The intracellular level of reactive oxygen species (ROS) was analyzed by DCFH-DA staining followed by photofluorography.The activity of superoxide dismutase (SOD) was also measured.RESULTS:Pretreatment of the HUVECs with 400 μmol/L NaHS (a donor of H2S) for 30 min prior to exposure to 40 mmol/L glucose ( HG) markedly attenuated HG-induced upregulation of the phosphorylation of JAK2 and STAT3.Pretreatment with 400μmol/L NaHS for 30 min or with 20μmol/L AG490 (inhibitor of the JAK/STAT pathway) for 30 min attenuated the injury of HUVECs induced by HG, as indicated by the increases in cell viability and SOD activity, and decreases in the protein level of cleaved caspase-3, ROS generation and dissipation of MMP.CONCLU-SION:Exogenous H2 S protects HUVECs against HG-induced injury by inhibiting JAK/STAT pathway.

Objective To explore an effective method in reconstructing of burn scar contracture of the chin and neck in children. Methods The blood in the transverse cervical artery might flow reverse into the suprascapular artery and the posterior intercostals perforators through anastomic branches. An axial skin flap in the cervico-scapulo-dorsal area could be adequately supplied by this artery, and we had therefore used this flap to repair burn scar in the neck. Results 30 cases obtained satisfactory clinical results with this flap. The follow-up for two years in 18 patients showed that the neck of the patients moved about freely, and the develpment of the chin was not influenced, and the color of flaps was fine. Conclusion The blood supply of the flap is reliable and stable. So it is a very useful method in reconstructing the burn scar of the chin-neck in children.

OBJECTIVETo discuss arthroscopic technique of single Kirschner wire and suture avoiding epiphyseal line fixation for tibial intercondylar eminence fracture and its clinical results.

METHODSFrom May 2008 to December 2012, 21 patients (13 males, 8 females, ranging in age from 6 to 14 years old) with tibial intercondylar eminence fracture were treated arthroscopically with single Kirschner wire and suture avoiding epiphyseal line fixation technique. According to Meyers and McKeever classification, 7 patients were type II, 10 patients were type III, and 4 patients were type IV. Active rehabilitation began at one week after operation. The patients were followed up for 10 to 30 months. X-ray films were taken to evaluate fracture healing at 1, 3, 6 months after operation; range of motion, the anterior drawer test, the lachman test and the Lysholm knee score were used to evaluate clinical effects.

RESULTSAll fractures were healed without displacement at 6 weeks after operation. Anterior drawer test and the lachman test were both negative in all patients at 3 months after operation. Lysholm knee score was 95.5 ±2.5 at 6 months after operation, and postoperative X-ray film did not find epiphyseal line broadening or narrowing.

CONCLUSIONArthroscopic treatment for tibial eminence intercondylar fracture with single Kirschner wire and 8-shaped suture avoiding epiphyseal line fixation technique has many advantages, such as firm fixation,early mobilization, less invasive, less injury of physis and satisfactory effect.

Adolescent ; Arthroscopy ; Bone Wires ; Child ; Female ; Fracture Fixation, Internal ; Humans ; Male ; Sutures ; Tibial Fractures ; surgery

Objectives To understand the effects of different doses of vitamin D supplementation on serum calcium,phosphorus,alkaline phosphatase and 25-hydroxyvitamin D levels in very low birth weight infants (VLBWI) and to provide guidance for early prevention of metabolic bone disease in VLBWI.Methods A total of 90 VLBWI hospitalized in the Department of Neonatology,Huzhou Maternal and Child Healthcare Hospital between January 2014 and January 2016 were enrolled and randomly divided into two groups:highdose group and low dose group.High-dose group was given vitamin D 900 U/d orally and low-dose group was given 400 U/d since the eighth day after birth.Serum calcium,phosphorus and alkaline phosphatase levels were detected at 1,7,21 and 42 days of age and serum 25-hydroxyvitamin D levels were detected at 7,21and 42 days of age.Two-sample t-test,Chi-square test,one-way analysis of variance and LSD or Dunnett's T3 test were used for statistical analysis.Results No significant differences in serum calcium,phosphorus and alkaline phosphatase levels were found between the two groups at 1 and 7 days of age,nor were found in serum 25-hydroxyvitamin D level at 7 days of age (all P＞0.05).At 21 days of age,high dose group had higher serum calcium,phosphorus and 25-hydroxyvitamin D levels than low-dose group [(2.38 ± 0.09) vs (2.04 ± 0.15) mmol/L,t=2.421;(1.80±0.50) vs (1.71 ±0.60) mmol/L,t-0.637;(45.58± 18.43) vs (42.53± 16.33) nmol/L,t=0.421],but lower alkaline phosphatase level [(505.12± 185.61) vs (588.32± 168.72) U/L,t=5.314] (all P＜0.05).The same trends were found at 42 days of age.In high-dose group,serum calcium level increased at 7,21 and 42 days of age as compared with that at 1 day of age [(2.43±0.13),(2.38±0.09),(2.39±0.08) vs (2.06±0.57) mmol/L];serum phosphorus level at 7 days of age was lower than that at 1,21 and 42 days of age [(1.31 ±0.26) vs (1.89±0.39),(1.80±0.50),(1.98±0.30) mmol/L];serum alkaline phosphatase level at 7,21 and 42 days of age was higher than that at 1 day of age [(475.18± 133.73),(505.12± 185.61),(538.43 ± 168.16) vs (296.15 ± 99.41) U/L] and a significant increase was observed at 42 days of age as compared with that at 7 days of age;serum 25 hydroxyvitamin D level at 21 days of age was higher than that at 7 days of age,and that at 42 days of age was higher than that at 7 and 21 days of age [(73.55±23.65) vs (30.63± 12.66) and (45.58 ± 18.43) nmol/L];the differences were all statistically significant (LSD or Dunnett's T3 test,all P＜0.05).Conclusions Vitamin D supplementation from the eighth day after birth can improve calcium and phosphorus metabolism in VLBWI and the dose of 900 U/d is more effective than 400 U/d.

AlM: To examine the retinal nerve fiber layer ( RNFL ) changes in early primary open angle glaucoma ( POAG ) by comparing the superior half of the optic disc with the inferior disc half.METHODS: lt was a clinical observational study. The study included 30 patients ( 39 eyes ) with POAG and 20 normal subjects ( 40 eyes ) . Visual field was tested by Zeiss Humphrey750 and the optic disc topographic parameters and RNFL thickness were measured by Zeiss Cirru HD-OCT. Collecting information included visual field mean defect ( MD ) , glaucoma hemifield test ( GHT ) , intraocular pressure ( lOP ) , C/D ratio and RNFL thickness. Data were analyzed by statistic software SPSS18. 0. The differences between two groups were assessed using t test.RESULTS: The differences of superior RNFL minus inferior RNFL thichness on the corresponding clock-hour locations were caculated in two groups. There was a significant difference in superonasal- inferonasal RNFL thickness between two groups (t=2. 526, P=0. 014), and there were no significant differences in the others ( all P>0. 05).CONCLUSlON: The asymmetry of optic disc hemifield RNFL is found in early primary open angle glaucoma patients. The superonasal RNFL ( 1 o’clock for right eye, and 11 o’clock for left eye ) is more fragile to loss than inferonasal RNFL (5 o’clock for right eye, and 7 o’clock for left eye) .

Objective To investigate the effect of human cytomegalovirus (HCMV) on proliferation of colony forming unit T-lymphocyte (CFU -TL)and its interference methods. Methods Normal CFU - TL culture was used as blank control. Normal CFU- TL culture system plus inactivated HCMV fluid as inactivated HCMV control. The dilution of 1:10,1:100,1:1000 were added into CFU -TL colonies culture system directly as infected group. Astragalus (AMI) and ganciclovir(GCV) were added into culture system with HCMV dilution of 1:10 as experimental group. By methylcellulose semi-solid culture, different concentrations of HCMV - AD1699 affect CFU-TL and interfered by astragalus AMI, GCV. CFU - TL were surveyed. The effect of HCMV on CFU-TL proliferation was measured by MTT; HCMV-AD169 DNA in CFU-TL was found by PCR. Results 1. Compared with control group, the numbers of CFU -TL in the HCMV infection groups decreased significantly(P