Objective To investigate the effects of tanshinol on the proliferation,apoptosis and NF-?B activation in rat hepatic stellate cells(HSCs) after IL-1? inducement,and to elucidate the anti-fibrotic molecular mechanisms of tanshinol.Methods The rat HSCs was isolated with collagenase in situ liver recirculation perfusion and cultured in vitro.The cells were divided into 5 groups: normal control,IL-1? treatment group(10 ng/ml),and tanshinol group 1,2 and 3.The later 3 groups were pretreated with tanshinol at the concentrations of 0.062 5,0.125 and 0.25 mmol/L respectively followed by 10 ng/ml IL-1? treatment 1 h later.MTT colorimetric assay was used to detect the proliferation of HSCs.AO/EB immunoflurorescence microscopy and combination Annexin-V-FITC/PI double-labelimmunofluorescence with flow cytometer were employed to examine the apoptosis of HSCs.Synthesis and secretion of collagen Ⅲ were detected by the quantitative immunocytochemical assay and ELISA respectively.The amounts of cytoplasm p-I?B? and NF-?B p65,and nuclear NF-?B p65 in HSCs were determined by Western blotting.Immunocytochemical staining and Western blotting were used to observe nuclear translocation of NF-?B p65.Results IL-1? increased the proliferation of HSCs(P

Objective To construct a new recombinant immunotoxin expression vector fused with a murine interleukin18(IL18) gene and a truncated pseudomonas exotoxin (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli (E. coli). Method Murine IL-18 (mIL-18) cDNA was cloned from murine liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was ligased with a PE38 gene carried by PRKL expression vector through T4 DNA ligase and constructed into fusion protein expression plasmid PRKL-IL18-PE38. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E.coli BL21 and induced by IPTG, the expressed product was obtained and the molecular weight and specificity were determined by SDS-PAGE and Western-blotting. Result The new recombinant immunotoxin expression vector was constructed successfully. DNA sequencing revealed that the mIL-18 and PE38 gene were consistent with NCBI Gene Bank. The IL-18-PE38 fusion protein was expressed in E.coli BL21, and Western-blotting analysis indicated that the molecular weight of the expression product is about 56 kDa, and could react with the specific antibody against mIL-18. Conclusion IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cells and may have some potential value in the treatment of Th1 cell-mediated autoimmune diseases.

Objective To study the effects of CD2-associated protein (CD2AP) on podocyte adhesion and extension ability and to explore its possible mechanism. Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. The podocytes were transfected with CD2AP small interfering RNA (siRNA) and serambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagen IV coated plates. The relative cell adhesion and cell area were examined 90 min later. Apoptotic rates of CD2AP siRNA transfected podoeytes and different PAN concentrations incubated podoeytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confoeal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot. Results The relative ceil adhesion of CD2AP siRNA transfected podocytes was apparently lower than that of control group[(41.72±6.07)% vs (64.46±8.53)%, P<0.05]. The cell area analysis had the similar result. The apoptotic rate of CD2AP siRNA transfected podocytes was significantly higher than that of the controls [(5.73±0.61)% vs (3.26±0.45)%, P<0.05]. 100 mg/L PAN could markedly induce podocytes to apoptosis and impair cell adhesion ability (P<0.05). Nevertheless, no significant difference was found in cell body spreading (P>0.05). The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level was conspicuously descended to some degree (P<0.05). Conclusions CD2AP depletion facilitates podocyte apoptosis and impairs cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness caused by CD2AP depletion may he partly responsible for the decline of cell adhesion and spreading.

Objective To investigate the efficacy of escitalopram combined with psychoanalysis in the treatment of refractory depression. Methods A total of 63 patients were randomly divided into escitalopram group ( n = 32) and escitalopram combined with psychoanalysis group( n = 31 ). All patients were evaluated with Hamilton depression Rating Scale(HAMD). Results After treatment,the scores of HAMD in two groups were both significantly lower than those before treatment. In the 8th ( ( HAMD ( 17.35 ± 2.98 ) ), 12th ( ( HAMD (9. 26 ±3.46) )weekend of treatment, the scores of HAMD in study group were significanlly lower than those in control group(8 th:21.97 ± 3.26; 12 th: 15.28 ± 3. 18 ). There were no significant differences in side effects between study group and control group. Conclusion Escitalopram angumented with psychoanalysis takes effects better than escitalopram single and doesn't increase side effects in the treatment of refractory depression.

Objective To investigate the relationship between the serum concentrations of brain derived neurotrophicfactor (BDNF) and its G196A polymorphism in the amphetamine induced-psychosis inpatients.Methods The cross-sectional study included 233 amphetamine abuses and 110 healthy participants who served as controls.The serum concentration of BDNF was measured by sandwich ELISA,and the genotype of BDNF G196A polymorphism was determined used polymerase chain reaction (PCR) techniques.The data were analyzed using SPSS 12.0 statistics software.Results The serum concentration of BDNF in case group((205.81±75.36) pg/ml) were significantly higher than that in control group((95.04±31.63) pg/ml;t=15.02,P＜0.01).There was no significant difference about the BDNF serum concentrations between the inpatients with the amphetamine induced psychosis and the inpatients with the amphetamine abuse (P＞0.05).The BDNF serum concentration showed no significant difference in the genotype distributions and allele frequencies (P＞0.05).The genotype distributions and allele frequencies of BDNF G196A showed no significant difference among three groups(P＞0.05).Conclusion The BDNF serum concentration is correlated with methamphetamine abuse,while the BDNF G196A gene polymorphism may not be associated.

Objective To study the safety and feasibility of endobiliary intraductal radiofrequency ablation (RFA) to reopen occluded self-expandable metal stents in patients with malignant biliary obstruction.Methods 11 patients with malignant biliary obstruction and blocked metal stents were prospectively studied.During ERCP, after biliary cannulation, the blocked metal stents underwent RFA using a bipolar radiofrequency probe which was introduced into the stenotic bile duct via a guide wire.This was followed by a balloon to repeatedly remove debris and then endoscopic nasobiliary drainage.The patients were closely observed and followed up.Results RFA was successfully carried out in all the patients and patencies were achieved when compared with pre-RFA.The median post-RFA luminal diameter of the strictures showed significant improvement: 6 (4 ～ 10) mm versus 2 (0 ～ 5) mm, and the mean post-RFA total bilirubin level decreased sharply : (39.4 ± 8.7) μ mol/L versus (130.1 ± 38.2) μmol/L.Following this intervention, 3 patients developed fever, which were controlled with conservative therapy.There was no mortality, haemorrhage, bile duct perforation or bile leak.Of the 11 patients, 3 were dead and 6 were alive at a median follow-up of 187 (75 ～ 304) days.The median stent patency was 135 (75 ～ 203) days and the median survival was 278 (75 ～ 304) days.Four patients had their stents patent at the time of the last follow-up or death.Seven patients had their stents blocked on 113, 124, 154, 203, 96, 135 and 112 days post-procedure.Condusions Endobiliary intraductal RFA is technically feasible and safe to reopen occluded metal stents in malignant biliary obstruction.This efficacy needs to be confirmed by future randomized studies.

Objective:To conduct a clinical tretrospective study on the effect of Silymarin Meglumine Tab on acute and chronic hepatitis B and to explore the related clinical significance. Methods: 41 cases and 205 cases of hospitalized patients pathologically diagnosed as acute and chronic hepatitis B respectively by liver tissuse puncture were divided into three group: ① Silymarin Meglumine Tab group; ② Ganyanling group; ③ complex group consisting of Silymarin Meglumine Tab and Gangyanling and diammonium glycyrrhizinate. To detect the index of routine liver function test before and after the treatment and analyze the hospitalization treatment course. Results: Compared with before treatment, liver functions of acute and chronic hapatitis B were improved to certain extent within each group after treatment (P0.05). The hospitalization treatment time of acute hepatitis B of the three group were almost the same, but the hospitalization treatmeng time of chronic hepatitis B of the Silymarin Meglumine Tab group was 2-4days less than that of the Ganyanling group and the complex group. Conclusion: As far as Silybini Meglumine Tab was concerned, it was better for chronic hepatitis B than acute hepatitis B because it was less complicated to administer the medicine, relieving the liver's burden of metabolism, and hospitalization treatment time was shorter, relieving patients' financial burden,which suggested that it was important to administer proper amount of medicine while taking into consideration protecting the liver's compensation function. If consideration was given to stimulating liver microcirculation with small dosage of TCM, so as to promote the exchange of the liver metabolite in blood, or detoxication and expulsion of toxin, it was likely to be more advantageous to the restoration of liver function and the liver tissue structure.

In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3.1(+) to generate pcDNA3.1(+)/HA and pcDNA3.1(+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1(+)/HA and pcDNA3.1(+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1(+)/HA or pcDNA3.1(+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

Aim To construct eukaryotic expressing plasmid of hi FGF2 ( high molecular weight isoform fi-broblast growth factor-2,hi FGF2) gene and to investi-gate its effect on apoptosis after its overexpression in HEK293 cells. Methods The DNA template primer was designed and synthesized. The pDsRed1-N1 plas-mids were digested by the restriction enzymes of Nhel and Hind III. The hi FGF2 was ligated with linearized pDsRed1-N1 by T4 DNA Ligase. The recombinant plasmid was identified by endonuclease digestion and sequenced. The recombinant hi FGF2 plasmid was transient transfected into HEK293 cells by Lipofectami-neTM 2000 Reagent. The transfection efficiency was de-tected by fluorescence inversion microscope. The cell apoptosis was detected by Annexin V-FITC/PI apopto-sis detection kit with flow cytometry analysis. Results The pDsRed1-N1 eukaryotic expression vector was consistent with the design. The recombinant hi FGF2 plasmid was transfected in HEK293 cells. The trans-fection rate was more than 70%. The FITC/PI dyeing rate in hi-FGF2 over-expression HEK297 cells was a-bout ( 29. 12 ± 2. 81 )%. Conclusions pDsRed1-N1 eukaryotic expression vector is successfully constructed and transfected into HEK293 cells. Over-expression of hi FGF2 induces cell apoptosis.

Objective To investigate the effects of positive end-expiratory pressure (PEEP) after recruitment maneuvers (RM) on respiratory mechanics and gas exchange during laparoscopy in obese patients.Methods Sixty-three ASA physical status Ⅱ or Ⅲ patients,aged 42-64 yr,with body mass index 30-40kg/m2,were randomly allocated into 3 groups (n=21 each):PEEP0 group,PEEP5 group and PEEP10 group.PEEP was not given after RM in PEEP0 group.In PEEP5 and PEEP10 groups,a recruiting maneuver was followed by PEEP 5 and 10 cm H2 O,respectively,until the end of pneumoperitoneum.The intraabdominal pressure was set at 12mmHg in the three groups.Parameters of respiratory mechanics including peak airway pressure (Ppeak),airway plateau pressure (Peat),chest wall plateau pressure (PplatCW),airway resistance (Raw),elastance of respiratory system (ERS),elastanc of chest wall (ECW) and elastance of lung (EL) and parameters of gas exchange including oxygenation index (PaO2/FiO2),arterial to end-tidal difference in carbon dioxide (Da-ETCO2),alveolar-arterial oxygen tension difference (DA-aO2),and dead space/tidal volume ratio (VD/VT) were measured before pneumoperitoneum (T0),at 20 min of pneumoperitoneum (T1),at 10 min after the end of recruitment (T2),and at the end of pneumoperitoneum (T3).Results Da-ETCO2,ERS and Raw were decreased at T2,ECW and EL were decreased at T3 in PEEP5 group,and Da-ETCO2,VD/VT,DA-aO2,Pplatcw Raw and EL were decreased at T2.3,and PaO2/FiO2 was increased at T2,ECW was decreased at T3 in group PEEP10 as compared with that in group PEEP0 (P ＜ 0.05).Da-ETCO2 and VD/VT were decreased and PaO2/FiO2 was increased at T2,3,Raw was increased and EL was decreased at T2 in group PEEP10 as compared with that in group PEEP5 (P ＜ 0.01).Conclusion PEEP after RM can improve respiratory mechanics and gas exchange during laparoscopy in obese patients and PEEP maintained at 10 cm H2O after RM provides better efficacy than PEEP at 5 cm H2 O.