OBJECTIVE:To study a simple method for the isolation and purification of thrombin-like enzyme (TLE) from the venom of Agkistrodon halys Pallas. METHODS:DEAE-Sephadex A-25 vs. Sephadex G-25 chromatography in the isolation and purification of TLE from the venom of Agkistrodon halys Pallas was analyzed. RESULTS:TLE was isolated from the venom of Agkistrodon halys Pallas. SDS-PAGE electrophoresis appeared as a strap with its molecular weight at about 35.5 kDa, meeting electrophoresis purity standard. Physical-chemical character assay showed that the TLE has hemostasia activity in vitro with its specific activity at about 12.57IU?mg-1, while the arginine esterase activity was about 137.65IU?mg-1 measured by BAEE method. PMSF and EDTA were used in the inhibition experiment on this enzyme, and the enzyme was proved to be serine protease other than metalloproteinase. CONCLUSION:The methods can be used for the isolation and purification of thrombin-like enzyme (TLE) from the venom of Agkistrodon halys Pallas.

Objective To explore the feasibility and clinical value of the trans-umbilical single-port laparoscopic operation with routine apparatus in treatment of hepatobiliary diseases. Methods From Mar 2010 to Sep 2012, 415 patients with hepatobiliary disease, splenic disease, abdominal cavity disease or combine hysteromyoma were performed by trans-umbilical single-port laparoscopic operation with routine apparatus. The clinical data of 415 cases were analyzed respectively.Result All patients except one patients who was added one port for hysterectomy, were performed successfully trans-umbilical single-port laparoscopic operation with routine apparatus without conversion to laparotomy or conventional laparoscopic surgery. Conclusions The trans-umbilical single-port laparoscopic operation with routine apparatus is safe and effective in treatment of hepatobiliary diseases. Limited by the anatomy position, the trans-umbilical single-port laparoscopic operation with routine apparatus can cure most hepatobiliary diseases. So the operator must have the experience both in single-port laparoscopic operation and traditional laparoscopic operation. With the improvement of rotatable equipment, the practical procedure is expected to be generalized in the future.

Objective To study the main risk factors related to the incidence of tuberculosis in China and to provide di -rections and basis for the protection of tuberculosis .Methods The results of 25 studies on the main risk factors of tubercu-losis of Chinese people from 2000 to 2012 were analyzed by meta-analysis method .Results The pooled odds radio values and 95%CI of history of exposure to pulmonary tuberculosis , smoking, marriage, contact with people, Bacille Calmette-Guerin (BCG)vaccination scar, BCG vaccination, low body mass index(BMI) , family history of tuberculosis, exposure to dust and to chemical fumes under working conditions were as follows:3.14(2.74-3.59),1.23(1.14 -1.33),3.05 (2.10-4.45),2.08(1.76-2.26),0.39(0.32 -0.47),0.58(0.46 -0.73),2.95(2.40 -3.64),2.56(1.82 -3.59),2.58(2.04-3.26),and 4.81(1.99-11.60).Conclusion Then History of exposure to pulmonary tuberculosis , smoking, marriage, contact with people , low BMI, family history of tuberculosis , exposure to dust and to chemical fumes under working conditions are considered to be the risk factors of pulmonary tuberculosis .While BCG vaccination scar and BCG vaccination are considered to be the protective factors of pulmonary tuberculosis .

Neutrophils are the first to reach the site of infection as they are the most numerous and dominant pathogen-killing cells in the circulatory system. They are both involved in initial immunity and act as effector cells in destructive inflammatory responses. The abundance of neutrophils in the human body and their inherent properties give them the ability to target inflammatory sites for drug delivery, and their important role in the inflammatory response has led to increasing attention to the therapeutic potential of neutrophils. In this review paper, the research progress in neutrophil-mediated drug delivery systems is reviewed, including the use of neutrophils as carriers, neutrophil exosomes as carriers, and in vivo targeted delivery of drugs using neutrophil properties.

Objective To investigate the effect of acidic tumor microenvironment on invasion and migration and its mechanism in glioma cells. Methods (1) The pH value of the medium was adjusted by acid-base titration. Human glioma cells U87 and U251 were cultured in the acid group and the normal group with pH values of 6.4 and 7.4, respectively; and 3 d after cultivation, the expressions of hypoxia-inducible factor-2α (HIF-2α) and CD44 were detected by Western blotting; Transwell assay was used to examine the invasion and migration of U87 and U251 cells; immunofluorescence was employed to examine the CD44 expression. (2) The U87 and U251 cells were divided into small interfering RNA (siRNA) -nonsense sequence group and siRNA-CD44-1 group, and the siRNA nonsense sequences and siRNA-CD44-1 interfering fragments were transfected by lipofectin-3000, respectively; three d after transfection, the migration and invasion abilities of cells from the two groups were detected by Transwell assay. (3) U87 and U251 cells were divided into acid group (cultured with a pH value of 6.4), blank control group, siRNA nonsense sequence group, siRNA-CD44-1 group, and siRNA-CD44-2 group; and cells from the later four groups were cultured with a pH value of 7.4; after culture for 4 d, the siRNA-nonsense sequence group, siRNA-CD44-1 group and siRNA-CD44-2 group were transfected with siRNA-nonsense sequences, siRNA-cd44-1 interfering fragments and siRNA-CD44-2 interfering fragments, respectively; three d after transfection, the expressions of CD44, N-Ca, Vimentin, and matrix metalloproteinase (MMP)-2 proteins in these 5 groups were detected by Western blotting. Results (1) As compared with the normal group, the expression levels of HIF-2α and CD44 in U87 and U251 cells of the acid group were significantly increased; both Transwell and invasion experiments showed that the number of transmembrane cells in the acid group was significantly larger than that in the normal group (P<0.05); immunofluorescence staining showed that the CD44 expression in acid group was significantly higher than that in normal group (P<0.05). (2) Both Transwell and invasion experiments showed that the number of transmembrane cells in the siRNA-CD44-1 group was significantly smaller than that in the siRNA nonsense sequence group (P<0.05). (3) Western blotting showed that the expression levels of CD44, N-Ca, Vimentin and MMP-2 in U87 and U251 cells of the blank control group, siRNA nonsense sequence group, siRNA-CD44-1 group, and siRNA-CD44-2 group were obviously decreased as compared with those in the acid group; the expression levels of CD44, N-Ca, Vimentin and MMP-2 in U87 and U251 cells of the siRNA-CD44-1 group and siRNA-CD44-2 group were obviously lower than those in the siRNA nonsense sequence group. Conclusion Acidic tumor microenvironment enhances the capabilities of invasion and migration of glioma cells through increasing CD44 expression.