Objective: To isolate Mycobacterium tuberculosis H37Rv and H37Ra genes especially,and to construct two cDNA libraries by using suppression subtractive hybridization (SSH).Methods: We used suppression subtractive hybridization (SSH) to clone the differential genomic genes between Mycobacterium tuberculosis virulence strain H37Rv and attenuated strain H37Ra.After two times of subtractive hybridization and two times of PCR,the products of last PCR amplification were inserted into pGEM-T Easy vector and be transformed into the E.coli DH5α and screened of blue and white clones of the transformants.The subtracted cDNA library of differentially expressed genes were identified by RT-PCR.Results:High or especially expressed genes as tester had been obtained by SSH in correctitude reaction (H37Rv as tester) and reverse reaction (H37Ra as tester),the cDNA libraries of A and B of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed.90% of the colonies were white clones,the single band of the colonies was 75% and 80%.Conchision:The cDNA libraries of Mycobacterium tuberculosis H37Rv and H37Ra were successfully constructed using SSH technique,which lay a solid foundation for screening and cloning new specific differentially expressed genes in them.

Objective:To construct the highly efficient expression recombinant adenovirus that expresses elafin protein for further study.Methods:The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with pAdEasy-1 in BJ5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and sequence scanned. Then the recombined adenovirus plasmid was digested with Pac Ⅰ and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein(GFP) expression. The expression of elafin protein was detected by Western blot and ELISA to appraise the function of elafin protein for oppressing the activity of elastic protease.Results:Digestion with restriction endonuclease, PCR, Western blot and ELISA confirmed that elafin gene was cloned into the adenovirus vector successfully.Conclusion:The recombined adenovirus Ad-elafin is constructed successfully, which will be benefit for future study.

Objective:To investigate the role of vitamin D-induced autophagy in macrophages against Mycobacterium tuberculosis(Mtb).Methods:Induce U937 cells to differentiate into macrophages with phagocytic capacity by phorbol 12-myristate 13-acetate(PMA).The cells were randomly divided into negative control group,vitamin D group,autophagy inhibitor(3-MA)+vitamin D group,positive control(rapamycin)group.Infect all groups with Mtb for 6 hours.In the 4th day after infection,the mRNA expressions of autophagy-related genes ATG5, Beclin-1 and LL-37, LC3B were determined by semi-quantitative RT-PCR.LC3B-Ⅱ+and/or Mycobacterium tuberculosis antigen 85A+( Ag85A+)-cells were counted by flow cytometry.Results:Compared with the control groups, the mRNA expressions of ATG5,Beclin-1,LL-37 and LC3B were enhanced(P<0.01),the numbers of LC3B-Ⅱ+-cells increased,the numbers of Ag85A+-cells decreased, and the numbers of LC3B-Ⅱ+-Ag85A--cells increased ( vitamin D group 38.0% vs negative control group1.08%).Compared with the vitamin D group,the mRNA expressions of ATG5,Beclin-1 and LC3B were suppressed in the autophagy inhibitor(3-MA)+Vitamin D group,the mRNA expressions of LL-37 were reduced,and 3-MA inhibited the expression of LC3B-Ⅱ in cells with inhibition of LC3B-Ⅱ+-Ag85A--cells increase as well.Conclusion: Vitamin D can induce macrophage autophagy and further contribute to scavenging Mycobacterium tuberculosis.

Objective To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.Methods The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion,PCR,and sequencing.Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification.Infection titer and rate were monitored by green fluorescent protein(GFP)expression.Finally the primary airway epithelium was infected with Ad-elafin,and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection.The elafin protein was detected by ELISA in the supernatant.Results Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully.Twenty-four hours after transfection,the GFP expression was seen by fluorescence microscopy.The concentration of elafin protein was(2.2?0.4)ng/ml in supernatant of transfected group,while that of control group was(1.9?0.3)ng/ml,P

Objective:To research the anti-inflammatory effect of LL-37 in mycobacterium tuberculosis ( Mtb ) infected-macrophages,as well as its influence on the secretion of inflammatory cytokines. Methods:( 1 ) THP-1 cells were cultured and incubated with phorbol 12-myristate 13-acetate (PMA) to transform into an adherent macrophage-like state (macrophage,Mφ). Then the THP-1 cell derived macrophages were infected with mycobacterium tuberculosis,and then stimulated with different concentrations of LL-37. (2)The experiment was divided into following groups: ① Control group:THP-1+normal saline (NS);② Mtb group:THP-1+Mtb;③Mtb+LL-37 5 μg/ml group:THP-1+Mtb+5 μg/ml LL-37;④Mtb+LL-37 10 μg/ml group:THP-1+Mtb+10 μg/ml LL-37;⑤Mtb+LL-37 20 μg/ml group:THP-1+Mtb+20 μg/ml LL-37. (3) The mRNA expression levels of IL-12p40,TNF-α,IL-4,and IL-10 will be determined by Real-time PCR respectively at 6,12,24 and 48 hours. The secreted levels of IL-12p40,TNF-α,IL-4,and IL-10 will be determined by ELISA analysis respectively at 6,12,24 and 48 hours. Results:The mRNA expression levels and secreted levels of IL-12p40,TNF-α,IL-4 and IL-10 were increased in Mtb group than those in control group. The mRNA expression levels and secreted levels of pro-inflammatory cytokines IL-12p40 and TNF-α were decreased in the LL-37 groups than those in Mtb group. However,anti-inflammatory cytokines IL-4 and IL-10 mRNA expression levels and secreted levels were increased in the LL-37 groups than those in Mtb group. Conclusion:Exogenous LL-37 inhibited the secretion of inflammatory cytokines in macrophages during mycobacterium tu-berculosis infection. The effect is related to the concentrations of LL-37 and the stimulated time of macrophages which were infected with mycobacterium tuberculosis. The results will provide new insights into the treatment of Mtb infection.

Objective To investigate the effects of two weaning modes of mandatory rate ventilation (MRV) and SIMV+PSV on respiratory mechanics in patients with type Ⅱ respiratory failure. Methods A total of 30 patients with type Ⅱ respiratory failure were sem randomly divided into two groups. Patients with type Ⅱ respiratory failure received ventilation in CMV mode or CMV/ACMV modes and then SIMV+PSV. When patients could be weaned, MRV mode was adopted in MRV group, but SIMV+PSV modes were adopted in the control group. After continuous operation in each mode for 60 min and when the tidal volume (VT) and minute ventilation (MV) in MRV mode were the same as those in SIMV+PSV mode, PIP, PP, Pm, PEEPi, blood gas changes, and weaning success in the two groups were observed during ventilation. Results PIP, PP, and Pm in patients in MRV mode were significantly lower than those in SIMV+PSV modes ( P 0.05), but the synchronism in patients in MRV group was better. Conclusion MRV is a more adaptable weaning mode for patients with type Ⅱ respiratory failure.

Clinical teaching constitutes a critical content of clinical course system.To that aim,we formulate typical case archive for teaching and apply bed-side teaching combined with PBL(problem based learning) method.Still supported by personating,case investigation and casuistics as well as multimedia means,we teach students in accordance with their aptitude and in so doing,initiatives of students can be mobilized to ensure sound teaching effects.

Objective To observe the expression of immune substances secreted by peripheral bloodγδT cells after stimulated by early secreted antigenic target-6(ESAT-6),and explore its mechanism of signaling pathway.Methods Peripheral blood mononucle-ar cells(PBMC)were collected from health human blood with the ficoll density gradient centrifugation method,and theγδT cells were separated from the PBMC with flow cytometry;the inhibitor of TLR-4 signaling pathway(E5564)was used to cocultured withγδT cells to inhibit the function of TLR-4,and the change of TLR-4 was analyzed by the methods of PCR and Western blot.ESAT-6 were used to stimulate theγδT cells,and the control group without any stimulating factor was established,then the expression levels of IL-17,TNF-α,IFN-γwere determined by ELISA method after 0、1、3、6、9 and 12 days.Results The results of PCR and Western blot showed that ESAT-6 could increase the expression of TLR-4(P<0.01);The results of ELISA showed that ESAT-6 could enhance the expression of IL-17,TNF-αand IFN-γ(P<0.01),and the inhibitor of TLR-4(E5564)could decrease the expres-sion of IL-17,INF-α,IFN-γ(P<0.01).Conclusion ESAT-6 can induceγδT cells to produce more IL-17,TNF-α,IFN-γ,and the mechanism of which maybe concerned with TLR-4 signaling pathway.

OBJECTIVE:To determine the resistance of clinically isolated bacteria to meropenem METHODS:The resistance of 150 clinically isolated strains to meropenem and several other antibiotics was detected by K-B method and the drug resistant spectra to E coli were compared between meropenem and imipenem RESULTS:The sensitivity of 130 clinical isolates to meropenem was 100% No cross resistant strains of E coli was found to meropenem and imipenem CONCLUSION:Meropenem is highly sensitive to the clinical isolates,and its antibacterial spectrum is similar to that of imipenem

Background and purpose: Anti-tumor chemotherapy compromises normal immune function of the patients. There were many reports that chemotherapy for advanced colorectal cancer often inhibit the cellular immune function. The effect of FOLFOX regimen chemotherapy on immunity of the patients with colorectal cancer before and after therapy was studied, and healthy people were used as a control. Methods: Eighty colorectal cancer patients were treated by FOLFOX regimen, which consisted of 2-hour infusion of oxaliplatin(85 mg/m~2) and 2-hour infusion of leucovorin (CF)(200 mg/m~2) on Day 1, followed by 5-fluorouracil(5-FU) bolus (400 mg/m~2) on Day 1 and 46-hour infusion (2 400 mg/m~2). FOLFOX regimen was repeated at 2-week intervals. Two treatments of the above regimen were defined as one cycle. Flow cytometry was used to detect T lymphocyte subsets and NK cells in blood samples from patients with colorectal cancer before and after therapy. Data obtained fi'om healthy people was used as control. Results: CD3~+, CD4~+ T cells, NK cells and CD4~+/CD8~+ ratio in blood samples were not significant before and after chemotherapy in first day, second week and fourth week(P0.05). Lower CD3~+, CD4~+ T cells, NK cells and CD4~+/CD8~+ ratio were detected in blood samples from cancer group than that from the healthy control(P<0.05). CD8~+ T lymphocyte were reverse. This change was related to the TNM pathological stage. Conclusion: FOLFOX regimen was effective for patients with coloreetal cancer, which can improve patients' life quality and did not impact on the immune function of the patients. The immune function of lymphocytes in peripheral blood of the patients with colorectal cancer was low, and even worse in the patients with late TNM stage. It is valuable for estimating the function of cell immune of the patients, patients prognosis and the role of immune therapy in the treatment of the patients by detecting T lymphocyte subset and NK cell.