Objective To investgate role of TLR2 in the activation, the innate immune and inflammation of human platelets. Methods Human washed platelets were separated from healthy people(n=5) and were stimulated with different concentrations(1μg/ml, 5μg/ml, 10μg/ml) of TLR2 agonistPam3CSK4(a synthetic bacterial lipoproteins). Then the platelet aggregation rate, the expression of CD62p and TLR2 on the platelet surface were measured. Results The platelet aggregation rate were (28.32±5.67)%, (52.56±8.54)% and (76.24±11.23)%, respectively, at concentration of 1μg/ml, 5μg/mland 10μg/ml of Pam3CSK4, more than (12.83±2.43)% at 0μg/ml of it. In addition, the expression of CD62p were (18.45±2.66)%, (22.45±2.04)%, (29.53±4.08)%, respectively at above concentration of Pam3 CSK4, more than (11.20±1.67)% of CD62p at control group(P<0.01). The expression of TLR2 was not significantly increased at a lower concentration of Pam3CSK4(1μg/ml) with (16.85±6.10)% compared with(10.81±3.99)% at the control group. However, it were (21.15±9.90)% and (22.52±9.26)%, respectively, at a higher concentration(5μg/ml, 10μg/ml)more than(10.81±3.99)% at the control group(P<0.05). Conclusion Pam3CSK4 induce aggregation, activation and the up-regulation of TLR2 of platelet by stimulating TLR2 receptor of it. Thereafter, TLR2 play an important role in the innate immune of platelet.

Objective To investigate the clinical efficacy of liver resection combined intraoperative choledochoscope for intra‐hepatic biliary calculi .Methods A retrospective analysis of clinical data in seventeen patients with intrahepatic biliary calculi ,who have been received liver resection combined intraoperative choledochoscope in the department of hepatobiliary surgery during 2005 to 2014 was conducted .According to the distribution of intrahepatic bile duct stones ,six cases located in left liver lobe ,five cases lo‐cated in left half liver ,three cases located in liver section Ⅵ ,one case located in liver section Ⅶ ,one case located in liver section Ⅷ , one case located in left liver lobe associated with right posterior lobe lower segment .Seventeen cases were treated with hepatolobec‐tomy or segmental liver resection (single clamp method combined first hilar occlusion) ,among which six cases received hepatic left lateral lobectomy ,five cases received left hemihepatectomy ,three cases received partial hepatic resection in paragraph Ⅶ ,one case received partial hepatic resection in paragraph Ⅶ and one in Ⅷ ,one case received the left lateral lobe combined right posterior lower segmental resection ,ten cases at the same time received choledocholithotomy and T tube drainage .Results All patients were cured without serious complications ,no long term stone recurrence .Conclusion Liver resection combined intraoperative choledochoscope is positive and effective treatment for intrahepatic biliary calculi patients .

Objective To investigate the lipoteichoic acid(LTA) induced apoptosis and the expression of inflammatory cytokines in human alveolar macrophage (AM) and the anti-apoptotic and anti-inflamatory effect of moxifloxacin (MXF).Methods Obtained human AM from bronchoalveolar lavage and used MTT assay to observe the effects of LTA and MXF on cell activity,optical microscope to investigate the change of the cell morphology,flow cytometry to assess cell apoptosis,RT-PCR to detect the mRNA levels of TLR2,IL-1 β,IL-8 and TNF-α,ELISA for the production of IL-8 to exam RT-PCR.Results LTA showed cytotoxicity on AM in a dose-dependent manner ( P＜0.05 ) ; MXF inhibited the effect of LTA without cytotoxicicy ( P＜0.05 ).LTA promoted apoptosis ( P＜0.05 ) and the mRNA expressions of TRL2,IL-1 β,IL-8 and TNF-α significantly in AM (P＜0.05),the peaks and peak time ofthe above factors were (3.56±0.03) at 12 h,(46.63±7.06) at 6 h,(28.07±1.24) at 12 h and (2.34 ±0.50) at 3 h respectively and increased the release of IL-8 protein level at 24 h (P＜0.05).MXF inhibited the cell apoptosis and the above mRNA expression at 12h ( P＜0.05 ),and inhibited the IL-8 protein level at 24 h( P＜0.05 ).Conclusion LTA showed cytotoxicity on AM,induced AM apoptosis and increased the expression of TLR2,IL-I β,IL-8 and TNF-α of AM ; MXF could protect AM through inhibiting of the above effects and may play a key role beside bactericidal effect in gram-positive bacteria pneumonia.

AIM:Panton-Valentine leukocidin(PVL)is a pore-forming toxin secreted by Staphylococcus aureus epidemiologieally associated with the often-lethal necrotizing pneumonia.Until now,the mechanisms of pathogene-sis of PVL leading to the fatal pulmonia remains undefined and also acquired plenty of the toxins is difficult.In the present study，we obtain recombinant staphylococcal F and S components of the Panton-Valentine leukocidin by gene engineering and evaluate its biological activity in vitro,which provides an experimental basis for the further studies of its biological func-tion and its toxicity in pneumonia.METHODS:The full-length of F and S components of PVL gene amplified from the strain of Staphylococcus aureus DNA by hiSh-fidelity PCR was cloned into prokaryotic expression vector pET22b(+),and the vector was transformed into BL21(DE3)plysS to construct a prokaryotie expression system.The integrity of the opening-reading frame of each construct was verified by DNA sequencing.The recombinant PVL(rPVL)was induced by1.0 mmol/L IPTG.The expressed products were identified by SDS-PAGE and the fusion proteins(6His-LukS-PV and 6His-LukF-PV)were purified from lysates of transfeeted E.coli cells by affinity chromatography on nitrilotriacetic acid columns.The eytolytie activity was tested by incubation of rPVL with human polymorphonuclear neutrophils(PMNs)in vitro.RESULTS:The nueleotide sequence of the cloned PVL gene was the same as that of reported in GenBank.E coli BL21(DE3)plysS containing recombinant vectors grow at 37℃causes some proteins to accumulate as inclusion bodies.while incubation at 30℃led to a significant amount of soluble active proteins which accounted for about 31.7％ of the total bacterial protein.The relative molecular weight showed on SDS-PAGE profile was consistent with the expected value which the LukS-PV protein was about 34 kD.and the LukF-PV protein was about 35 kD.The purified rPVL was obtained and its cytolytic activity to PMNs was demonstrated.CONCLUSION:The genes of 1ukS-PV and lukF-PV are successfully cloned into plasmid pET22b(+)and expressed in E coli respectively.which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.

Objective To study influencing factor of mechanical ventilation (MV) time in patients withchronic obstructive pulmonary disease (COPD). Method Totally 128 patients with COPD and respiratory failurerequiting ventilation support were retrespectively investigated. The clinical characteristics of patients before and af-ter intuhation during the respiratory intensive care unit (RICU) stay were recorded and analyzed, t- test, X2 testand stepwise logistic regression analysis were used for statistical analysis. Results In the 128 patients, 61%,20%, and 9% of the patients required MV longer than 7, 14 and 21 days, respectively. There were no significantdifferences in the history of COPD, smoking, lung function, and complications between patients with MV>7 d, 14d, 21 d and patients with MV<7 d, 14 d, 21 d. Multiple regression analysis showed APACHE Ⅱ score (OR:2.3; 95% Ca: 1.2-5.7, P = 0.02) was an independent factor for MV > 7 days, while shock (OR: 0.7;95% CI: 1.0-1.9, P = 0.04) and decreased serum albumin (OR: 0.4, 95% CI: 0.2-0.8, P = 0.003)were an independent factors for MV > 21 days. The ventilator-associated pneumonia (VAP) was the most impor-rant risk factor for the time of MV. Conclusions APACHE Ⅱ score, serum albumin levels, hock and VAP arethe main factors affecting MV time in patients with COPD.

[Objective] To reduce misdiagnosis and underdiagnosis rate of pulmonary embolism,the prediction of the revised Geneva score and Wells score for pulmonary embolism were compared and analyzed by receiver operating characteristic curves.[Methods] Sixty-five cases with suspected pulmonary embolism (PE) were collected in the Third Affiliated Hospital of SUN Yat-sen University from January 1998 to October 2008.Of which 44 cases with PE were clinically confirmed.Relevant clinical data were recorded,summarized and the analysis variables were input to SPSS11.0 for statistical analysis.ROC curves was used to evaluate the probability of PE predicted by the Wells and the revised Geneva scores.[Results] Twenty-four patients had a low clinical probability of PE (Wells score ＜ 2 points ),of which 8 (33.3%) had proven PE.The prevalence of PE was 87.2% in the 39 patients with intermediate probability (2-6 points) and 100% in the 2 patients with high probability (＞ 6 points) (P ＝ 0.000).The confirmed PE was 22.2% in the 18 patients with a low probability (Geneva score 0-3 points),82.1% (32/39) in intermediate probability (4-10 points),100% (8/8) in high probability (score ≥11 points) (P ＝ 0.000).The area under curve (AUC) of the ROC curve in the Wells and Geneva scores was 0.785 ± 0.060 and 0.900 ± 0.038,respectively (P ＝ 0.000).The optimal cutoff value was 2 points in the Wells score and 6.5 points in the Geneva score.The Wells score more than 2 points predicted PE with a sensitivity of 81.8% and specificity of 76.2%.The Geneva score more than 6 points predicted PE with a sensitivity of 72.7% and specificity of 100%.The comparison of the area under curve between the Wells and the Geneva score had a significant difference statistically (P ＜ 0.05).[Conclusion] The Wells score and the revised Geneva score are beneficial to predict pulmonary embolism.The revised Geneva score is roughly superior to the Wells score both in sensitivity and specificity.

AIM:To investigate the specific anti-tumor effects of mature dendritic cells ( DCs) transfected with amplified mucin 1 ( MUC1) mRNA in vitro.METHODS:DCs separated and purified from the peripheral blood mononu-clear cells were induced in vitro and then identified by flow cytometry .pcDNA3.1(+)-MUC1 plasmid was constructed and was able to transcribe MUC1 mRNA in vitro.The MUC1 mRNA was transfected into DCs by electroporation .MUC1-trans-fected DCs were used to induce T cells to be cytotoxic T-lymphocytes .Quantitative real-time PCR was performed to assess MUC1 mRNA expression in transfected DCs .The proliferation of T cells was examined by MTT assay .The proportion of CD8 +cells in the T cells was determined by flow cytometry and the specific cytotoxicity was measured by LDH assay .The secretion of IFN-γwas detected by ELISA .RESULTS: The marker gene expression in the DCs transfected with MUC 1 mRNA was significantly increased compared with control group , peaking at 24 h.The transfection group showed the higher capacity to stimulate the proliferation of T cells compared with control group when the ratio of DCs to T cells was 1∶10.The proportion of CD8 +cells in transfection group was higher than that in control group .The lethal effect of special cytotoxic T-lymphocytes on target cells in transfection group was stronger than that in control group .The level of IFN-γin the cell su-pernatant of transfection group was higher than that in control group .CONCLUSION:DCs plus MUC1 mRNA by electri-cal transfection induces specific anti-tumor effects , which provides an experiment evidence of using MUC 1 as a target for immunotherapeutic strategy against non-small cell lung cancer .

Objective: To evaluate the effect of plasma exchange combined with high-dose continuous venovenous hemodiafiltration method (CVVHDF) in the treatment of patients with acute-on-chronic liver failure with stage III-IV hepatic encephalopathy and the feasibility of pre-operative preparation for liver transplantation. Methods: Clinical data of 14 cases of medical intensive care unit of our hospital with acute-on- chronic liver failure accompanied with stage III-IV hepatic encephalopathy that underwent plasma exchange combined with high-dose CVVHDF from March 2015 to September 2017 were retrospectively summarized. The indexes of liver and kidney function, blood coagulation function, arterial blood PH, lactic acid and blood ammonia were monitored before and after treatment. Heart rate, blood pressure, APACHE II score, and consciousness recovery time were observed. Student’s t- test was used to compare the mean values between the two groups. Results: Serum total bilirubin (t = 9.43, P < 0.01), serum creatinine (t = 3.40, P < 0.01), serum ammonia (t = 10.64, P < 0.01), prothrombin activity (t = 9.19, P < 0.01), serum lactate (t = 9.25, P < 0.01), heart rate (t = 4.47, P < 0.01), and mean arterial pressure (t = 4.41, P < 0.05) were significantly improved in 14 patients before and after treatment. In addition, respiratory rate (t = 6.01, P < 0.01) and APACHE II score (t = 7.19, P < 0.01) were significantly improved (P < 0.05). Eight patients with stage III hepatic encephalopathy were treated with intermittent plasma exchange combined with CVVHDF for 3 to 14 days, and six patients with stage IV were transformed to stage III to II. Liver transplantation was successfully performed on 14 patients with shortest time duration of 3days, and longest time duration of 1 month. Conclusion Plasma exchange combined with CVVHDF can significantly improve liver and kidney functions, reduce blood ammonia level and improve mental health in patients with hepatic failure accompanied with stage III-IV hepatic coma. In addition, it also effectively increases the average arterial pressure, maintain stability of vital signs, maintain fluids, electrolytes and acid-base balance, create a stable internal environment for liver transplantation before operation, and extend time for liver transplantation.