Objective:To establish a method of multi-PCR to amplify the complete DNA sequence (CDS) of TCR ? and ? chain of the antigen-specific T lymphocytes in local pathologic specimen of active pulmonary tuberculosis patients, and to analyze ?/? T cell receptor gene rearrangement and CDR3 repertoire.Methods:The lymphocytes in bronchoalveolar lavage (BAL) of active pulmonary tuberculosis patients were separated. Following total RNA extraction, cDNA synthesis, Multi-PCR, recombinant clones construction, and sequencing, the CDS of TCR ? and ? chains from these lymphocytes were analyzed by using software of DNAstar and internet TCR resources.Results:24 of ? chain CDS and 13 of ? chain CDS from 3 samples of BAL were obtained. As for TCR ? chain, AV1S2 (54%), AV12S3 (41%), and AV12S2(5%) appeared frequently. BV2(38%), BV29S1(46%), BV14(3%), and BV4S2(3%) in TCR ? chain appeared more often. There were CDR3 diversities between samples and even in the same sample by amino acid sequence analysis, but there were a few identical or similar amino acid sequences. There was the same amino acid sequence of SVGTGTLHQETQY in CDR3 region of ? chain of BAL sample No.1 and No.2; The sequence of AVRDWAGNMLT appeared in two ? chains of BAL sample No.2 and No.3; Moreover, the sequence of AV…DNN…RLM appeared in ? chains of BAL sample No.2 and No.3.Conclusion:A method of Multi-PCR is used to amplify TCR ? and ? chain CDS of tuberculosis patients. There are characteristic T cell clones to proliferate,with TCR ? and ? chain repertiore skewing in local infective focus. The sequences of CDR3 in different TCR clones are mostly different but there are a few identical or similar sequences in the same patient or even between different patients. The identical amino acid sequences of CDR3 are possibly specific for recognizing MTB polypeptide.

Objective To discuss the protective effects and mechanisms of dexamethasone and Shenfu sepa-rate and joint administration of flap after ischemia-reperfusion injury. Methods 40-month-old fairly healthy rats were randomly divided into 4 group as A, B, C, D, and to product the abdominal island flap, then blocking the flow of blood of the pedicle artery respectively before 30 minutes when injecting with normal saline (1 ml/kg), dexametha-sone (1 ml/kg), Senate (10 ml/kg), joint injection with dexamethasone (1 ml/kg) and Shenfu (10 ml/kg). 4 groups of animals' blood samples were collected from the pedicle vein before the time of I hour when blocking vascu-lar pedicle and reperfusion after the time of 1 h, 6 h, 12 h, 24 h, respectively. Then the plasma concentration of TNF-α was measured. Results The concentration of TNF-α in the treatment group was significantly lower than the blank group(P <0.01). And the group D and the group B、C has significant differences either(P <0.01). Conclu-sion Using Dexamethasone, Shenfu injection in early can reduce the concentration of TNFα in repeffusion injury of flap and has a protective effect on the flap, but make better effect in combined.

Objective To examine the effects of oxidative stress induced damage to the Prestin expression in HEI-OC1 cells,and to study the mechanism of sensory deafness.Methods We used different concentrations (50μM,100μM,200μM)of hydrogen peroxide canister to cultivate HEI-OC1 cells,and to detect the activity of su-peroxide dismutase(SOD).The quantitative real-time PCR and immunofluorescence were used to detect the prestin expression of mRNA.Results The SOD activity decreased in the HEI-OC1 cells damaged by oxidative stress.The high concentration of the infected group decreased more significantly(F= 9926.293,P<0.01).The expressions of Prestin mRNA and Prestin protein were decreased obviously in the HEI-OC1 cells.The high concentration of in-fected group decreased more significantly (F= 4065.046and7657.217,P<0.01).Conclusion Oxidative stress in-ducing damage inhibits the expression of prestin.Prestin protein may be used as a molecular marker of sensory deafness.

Objective:To analysis the relationship of cephalometric measurements from intercuspal position(before and after treat-ment)and retruded contact position(before treatment).Methods:18 cases with Class III malocclusion were treated using MBT straight wire appliance technique,all the incisal relationships could be edge-to-edge when the mandible was retruded,X-ray films of intercus-pal position and retruded contact position before treatment and intercuspal position after treatment were taken.The cephalometric meas-urements were statistically analysed by ANOVA.Results:After 25 months treatment the anterior crossbite were corrected in all cases. The molar relationship were class Ⅰ.The measurements of SNB,ANB,MP-FH,MP-SN,U1-SN,L1-MP,Y-axis angle and ANS-Me were significantly changed.Conclusion:After the treatment of Class III malocclusion by MBT straight wire appliance,the sagittal and vertical mandibular location can be between intercuspal position and retruded contact position of pretreatment,and it is more close to the retruded contact position.

Objective To evaluate the utility of CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay in detecting antigen‐specific CD14+ monocytes in the blood of tuberculosis (TB) patients .Methods CD4+ TCR tetramers were used to detect tetramer‐positive CD14+ monocytes in the peripheral blood (PBL ) samples of inpatients with advanced pulmonary TB (PTB) by flow cytometric analysis .The PBL samples obtained from non‐TB patients and umbilical cords were used as controls .These tetramers were also used to examine tetramer‐bound CD14+ monocytes and Mycobacterium tuberculosis (MTB) antigen‐specific and tetramer‐bound cells by cell climbing slice in situ staining .Results The median percentage of tetramer‐bound CD14+ monocytes in PBL samples from PTB patients ,non‐TB patients and umbilical cords were 1 .32% , 0 .50% and 0 .26% respectively by using CD4+ Vα21‐J39/Vβ29‐D1‐J2 tetramer , while the medians were 1 .05% , 0 .49% and 0 .19% respectively by using CD4+ Vα21‐J39/Vβ29‐D2‐J2 tetramer . The percentage of tetramer‐bound CD14+ monocytes in PTB patients group was significantly higher than the other two control groups .In cell climbing slice in situ staining ,tetramer‐bound CD14+ monocytes ,and MTB antigen‐specific and tetramer‐bound cells were positive in PTB tissue compared with negative in control tissues . Conclusions CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay could be used to sensitively detect M TB antigen‐specific CD14+ monocytes in the blood of TB patients ,and more accurately evaluate the changing profile and clinical significance of these cells in TB patients .

Objective To study the ability of standard strain and clinical isolates of Ureaplasma spp. to form biofilms in vitro and to compare the antibiotic susceptibility of sessile cells and their planktonic counterparts. Methods A total of 21 Ureaplasma wealyticum(Uu) isolates recovered from female patients diagnosed with cervicitis and Uu serovar 3 and Uu serovar 8( Uu3, Uu8) were included. Scanning electron microscope and confocal scanning laser microscopy were used to identify biofilm formation. Conventional antibiotic susceptibility tests and biofilm susceptibility assays for tetracycline, erythromycin and ciprofloxacin were carried out. The paired rank sum test and was applied to analyze the statistical differences between the MIC and the minimal biofilm inhibitory concentration. The x2 test was applied to analyze the statistical differences of global resistance percentages between planktonic cells and sessile cells. Results Uu3, Uu8 and 21 Uu isolates all can form biofilms in vitro. Minimal inhibitory concentration of sessile cells compared with planktonic cells were obviously higher for tetracycline, erythromycin and ciprofloxacin (P <0.001). Global resistance percentages between planktonic cells and sessile cells were different for erythromycin (9.52% vs 61.90% , P < 0. 001), ciprofloxacin ( 80. 95% vs 100% , P = 0. 035 ) and tetracycline (4. 76% vs 14.29% , P =0.293). Conclusion Uu isolates and Uu1, Uu8 all can form biofilms in vitro, and biofilm formation can strengthen resistance of Uu to antibiotics, even multidrug resistance was observed.

Objective To explore the relationship between histological chorioamnionitis(HC),fetal vasculitis(FV)and the morbidity of neonatal respiratory distress syndrome(RDS).Methods Three hundred and forty-seven cases of infants at the gestational ages of 28 +0 to 31 +6 weeks who were admitted to the Neo-natology Department of our hospital from October 2009 to June 2013 were analyzed retrospectively.They were divided into four groups according to the occurrence of HC and FV,namely,HC positive group and HC negative group,FV positive group and FV negative group.The patients in the HC positive group were further divided into FV positive group and FV negative group according to the occurrence of FV.The morbidity of RDS among above-mentioned groups was compared.Results The clinical characteristics including propor-tion of male,gestational age,birth weight,cesarean delivery,antenatal corticosteroid use,gestational hyperten-sion,gestational diabetes and cholestasis of pregnancy showed no statistically significant difference among all the groups(P 〉0.05).The incidence of RDS in the HC positive group(145 cases)was 49.6%(72 cases), which was significantly lower than that in the HC negative group(67.3%,126 /202,P 〈0.05).The incidence of RDS in FV positive group(64 cases)was 42.2%(27 cases),which was significantly lower than that in FV negative group(63.3%,179 /283,P 〈0.05)In the HC positive group(145 cases),the incidence of RDS in FV positive group (64 cases)was 42.2% (27 cases),and 55.6% (45 cases)in FV negative group (81 cases),which showed no significant difference(P ﹦0.01 ).Conclusion (1 )HC or FV can reduce the incidence rate of RDS in premature infants.(2)HC combined FV cannot furtherly reduce the incidence of RDS.

Objective To explore the effect of IL-1? and IL-6 on MMP-3 gene expression in human coronary artery smooth muscle cells.Methods We used IL-1?(20 ?g/L) and IL-6(10 ?g/L) to stimulate human coronary artery smooth muscle cells,which were co-culture for 0,2,4,8,24,36 h.IL-1?(0,5,20,40 ?g/L) and IL-6(0,5,10,50 ?g/L) were used to stimulate human coronary artery smooth muscle cells,which were co-cultured for 6 h.Then we detected the gene expression by fluorescent quantitation PCR.Results In the same concentration of IL-1? and IL-6,gene expression was up-regulated at 2 h,at 8 h the expression reached the peak,then began to descend.In different concentration of IL-1? and IL-6,gene expression was up-regulated with the dose of IL-1? and IL-6(IL-1?: r=0.907,P=0.000;IL-6: r=0.919,P=0.000).There were significant differences in MMP-3 expression among different groups(IL-1?: F=24.047,P=0.000;IL-6: F=14.081,P=0.001).There were no significant differences in matrix metalloproteinase-3 between IL-1? 20 and 40 ?g/L groups(P=0.154) and between IL-6 5 ?g/L and 10 ?g/L(P=0.292).Conclusion It suggests that IL-1? and IL-6 can promote MMP-3 gene expression in human coronary artery smooth muscle cells,and it may be one of the mechanisms of inflammation effect in acute coronary syndrome.

Objective To construct and apply a cell line screening Mycobacterium tuberculosis (Mtb)-specific tetramers of CD4+α/β T cell receptor(TCR). Methods The β chains of HLA class Ⅱ (DR) were amplified from tuberculosis patients by PCR. The pMT-HLA-DRB expression vectors that carries the HLA-DR 13 chain and pMT-HLA-DRA-P expression vectors which carries the genes of HLA-DR α chain loaded with Mtb antigen were transfected into S2 cells with the method of calcium phosphate transfection. The expressed Mtb peptide/HLA-DR complexes were primarily identified by the method of cell immunohistochemistry. The cell lines expressing Mtb peptide/HLA-DR complexes were used to screen tetramers of CD4+ TCR by flow cytometry. Results S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface were obtained, two kinds of Mtb specific tetramers of CD4+α/β TCR were screened. Conclusion S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface provide the solid basis of the further research on the TCR tetramers and are helpful for exploring new diagnostic study methods about tuberculosis and developing new vaccines.

ObjectiveTo investigate the specificity of CD4+ Vα9-J27/Vβ29-D1-J2 tetramer in detecting Mycobacterium tuberculosis(MTB) infections.MethodsThe above TCR tetramer by using biotinylated monomers expressed and purified from constructed stable Drosophila Schneider 2 cell( S2 cell) lines was prepared.The PE-labled TCR tetramer was used to costain with S2 cell lines expressing MTB prptide/HLA-DR complexes on the cell membrane,and also was used to detect tetramer-bound CD14+ monocytes and macrophages in the peripheral blood mononuclear cells (PBMC) of pulmonary tuberculosis (PTB) patients and three control groups by flow cytometric analysis.And the FITC-labled tetramer was used to examine tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells by in situ staining.ResultsThe TCR tetramer was well binding with S2 cell lines expressing C14/HLA-DR *1504 on the cell membrane.By flow cytometric analysis,the percentage of tetramer-bound CD14+ monocytes and macrophages in PTB patients group was higher than the other three control groups( P＜0.001 ).By in situ staining,tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells were positive in PTB tissue and negative in control pneumonia tissue.ConclusionThe spcificity of TCR tetramer in monitoring MTB infections by flow cytometric analysis and in situ staining could be seen,which laid a laboratory foundation in the diagnosis and immune mechanism research of TB by using TCR tetramers.