Objective:To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor (CTGF) by RNA interference and verified its function. Methods:A specific sequence, which was verified to be able to silence CTGF gene with high efficiency, was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF. The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF. After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation. The recombinant adenovirus was packaged. The titer of the Ad-siCTGF was increased after three times of cross-infection. 4T1 cells were infected with the adenovirus. The silencing efficiency was tested by real-time fluorescence quantitative (RFQ)-PCR and Western blotting.Results:Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed. The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10~(10) pfu/mL after amplification and purification. The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%, respectively, compared with the control groups.Conclusion:The recombinant adenovirus which can silence the expression of CTGF was successfully constructed. It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.

Objective To explore the significance of the plasma procalcitonin (PCT) level for directing antibiotic therapy in elderly patients with ventilator-associated pneumonia (VAP).Methods The 50 elderly patients with VAP were randomly separated into the regular therapy group and the PCT-directed therapy group. The regular therapy group was given regular antibiotic therapy, while the antibiotic therapy was decided according to the plasma level of PCT in the PCT-directed therapy group. The used time and utilization rate of antibiotics, as well as inflammatory indicators including white blood cells, neutrophils, C-reactive protein (CRP) and clinical pulmonary infection score (CPIS) were compared between the two groups. Results After treatment, there were no significant differences in white blood cells, neutrophils and CRP between the PCT-directed therapy group and regular therapy group [(8.9 ± 3.5 ) × 109/L vs. (9.4 ± 3.7) × 109/L, 0.62 ± 0.04 vs.0.60±0.04, (18.7±8.5) mg/Lvs. (21.6±6.0) mg/L, t=0.47, 1.84 and 1.37, allP＞0.05],but the CPIS was markedly lower in PCT-directed therapy group than in regular therapy group [(4.0± 1.4) scores vs. (4.7± 1.0) scores, t= 2. 18, P＜0.05]. The neutrophils, CRP and CPIS were significantly lower after treatment than before in the both groups. The concentration of PCT was decreased after treatment than before [(0.5 ± 0.9) mg/L vs. (1.7 ± 0.7) mg/L]. Meanwhile, the time using antibiotics was longer in regular treatment group than in PCT-directed therapy group [(8.72±1.32) d vs. (5.17±0.72) d, t=11.96, P＜0.01], the utilization rate of antibiotics was higher (95.2 % vs. 55.2 %, χ2 = 12.41, P＜0.01) in regular treatment group. Conclusions Using PCT levels for directing treatment in elderly patients with VAP can achieve better curative effect and reduce the use of antibiotics.

Objective To investigate the clinical values of central venous pressure (CVP) versus stroke volume variation (SVV) in patients with severe sepsis after early goal-directed therapy (EGDT).Methods Totally 30 mechanically ventilated patients with severe sepsis who underwent goal-achieved EGDT were enrolled and randomly divided into CVP group (study group) and SVV group (control group) according to the data detected by pulse contour continuous cardiac output (PiCCO) analysis device.The differences in 28-day survival,3-day APACHE Ⅱ score,time of ICU stay,duration of mechanical ventilation,number that need CRRT,entral venous pressure (CVP),heart end-diastolic volume index (GEDVI),intrathoracic blood volume index (ITBVI),extravascular lung water index (EVLWI),cardiac index (CI),central venous oxygen saturation (ScvO2),lactate clearance rate and APACHE Ⅱ score were compared between the 2 groups.Results The death rate had no difference between the 2 groups(x2=0.240,P=0.624).Among survival patients in the CVP group,the time of ICU stay and duration of mechanical ventilation were shorter in study group than in control group(t=2.166,P=0.041;t=2.104,P=0.046),APACHE Ⅱ score at 3th day was decreased(t=2.20,P =0.038).The values of ITBVI,GEDVI,CI,lactate clearance rate were higher in study group than in control group (t=2.759,2.146,2.199,2.654,3.362,P=0.011,0.043,0.038,0.014,0.003).EVLWI and APACHE Ⅱ score were not different (P＞0.05) between the two groups.Conclusions SVV as a recovery target for fluid resuscitation can reach a better recovery results and improvement of prognosis than CVP goal-achieved EGDT.

Objective To compare the diagnostic yield of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for solid pancreatic masses performed with three different needle types through the cytological results.Methods All patients with solid pancreatic masses larger than 2cm from December 2010 to May 2011 were enrolled,and divided into two groups according to different access of EUS-FNA,trans-gastric approach with 19-,22-and 25-gauge needles (n =42) and trans-duodenal approach with 22-and 25-gauge needles (n =10).In both groups,EUS-FNA was performed with randomization of needle types.During the puncture,the suction,the number of movements,and the depth of insertion were fixed.At the end of the puncture,a liquid-based cytological (LBC) preparation was used to fix the specimen.One cytopathologists was assigned to make the diagnosis and comparison.Results Technical success was 100％ and no procedure related complications occurred.No statistically significant differences were observed in different needles in terms of all cytological parameters between two groups (P ＞ 0.05).However,the 25-gauge needle showed a trend towards a higher sensitivity,specificity,positive predictive value,negative predictive value and accuracy.Conclusion There is no significant difference in yield of cytological results between different needle types,although 25-gauge needle shows a relative superiority.

Objective To investigate the prognostic value of stroke volume variation (SVV) in patients with severe sepsis after early goal-directed therapy (EGDT). Method Thirty-eight mechanically ventilated patients with severe sepsis underwent EGTD were divided into high SVV (≥10%) group and low SW (< 10%) group according to the data obtained from pulse contour continuous cardiac output (PiCCO) analysis device. The differ-enees in the rate of 28-day survival, length of ICU stay, duration of meehanical ventilation and eomplieation of in-fection between two groups of patients were compared. The rate of 28-day survival of patients was analyzed by using Kaplain-Meier survival analysis, and the relationship between SVV and mortality within 28 days was analyzed by using logistic regression model. Results In comparison with low SVV group, the rate of 28-day survival of high SVV group was signifieantly increased (87.5 % vs. 57.1%, P = 0.032), the length of ICU stay was significantly shortened (27.1±9.2) vs. (41.6±10.0) (P = 0. 004) and duration of mechanical ventilation was significantly more brief (20.4±7.3) vs. (28.5±8.3) (P = 0.038). The rate of cumulative survival of patients in high SVV group was higher than that in low SVV group. In addition, logistie analysis showed SW < 10% increased the risk of 28-day mortality (OR = 3.97; 95% CI 1.63 - 9.21, P = 0. 014). Conclusions The SVV can be served as a prognostic indicator in patients with severe sepsis after EGDT.

OBJECTIVETo investigate the clinical effect of Erigeron injection (El) on positive expression rate of CD62p in platelet and content of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in serum of patients with acute cerebral infarction (ACI).

METHODSSixty-eight patients with ACI were randomly divided into the treated group (n = 35) and the control group (n = 33). Conventional treatment were given to both groups, and EI 40 ml/d were given additionally to the treated group, the treatment course for both groups was 15 days. The positive expression of platelet CD62p and the serum TNF-alpha and IL-6 in patients before and after treatment were determined with flow cytometric (FCM) and electrochemical-luminescence (ECL) techniques respectively.

RESULTSThe total curative effect in the treated group were significantly higher than that in the control group (P < 0.05). Levels of platelet CD62p and serum TNF-alpha and IL-6 in ACI patients before and after treatment were significant higher than those in the healthy group (P < 0.05), all the three parameters were significantly decreased after treatment, and the lowering in the treated group was more significant than that in the control group (P < 0.05).

CONCLUSIONThe effect of El on ACI patients may relate to its action in down-regulating the expression of platelet CD62p, alleviating the immune response and inflammatory injury of central nervous system induced by cytokines.

Aged ; Aged, 80 and over ; Asteraceae ; chemistry ; Blood Platelets ; metabolism ; Cerebral Infarction ; immunology ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Injections ; Interleukin-6 ; blood ; Male ; Middle Aged ; P-Selectin ; biosynthesis ; blood ; Phytotherapy ; Tumor Necrosis Factor-alpha ; metabolism

Objective To investigate the correlation between the indications,findings,interventions of fibrobronchoscopy(FB) in neonates and their correlative diseases with neonatal FB results and clinical data.Methods Retrospective case series of 243 consecutive patients of 28 days old or younger were investigated underwent FB for the first time from January 2010 to December 2014,at a tertiary care hospital.The common indications for FB and detection rate of respiratory tract diseases were collected.If the findings of FB had significant associations with premature birth and other diseases were analyzed.Associations between interventions and basic illnesses were also analyzed.Results Of the 243 patients undergoing 275 procedures of FB,201 cases were boys(73.1％).The age of FB was (13.34 ± 9.76) days and the weight was (3.08 ± 0.68) kg.Forty-five cases were premature infants (16.4％).A total of 254 procedures were found to have congenital diseases (92.4％),and 177 cases of them had congenital heart diseases (CHD) (64.4％).Common indications for FB were dyspnea(140 cases,50.9％),tachypnea(82 cases,29.8％),and stridor(71 cases,25.8％).A total of 188 upper airway lesions were found and the most common findings were laryngomalacia(56 cases,20.4％) and vocal cord paralysis(bilateral/unilateral,50 cases,18.2％).A total of 315 lower airway lesions were found and the most common findings were airway mucosal inflammation (98 cases,35.6％),trachea and main bronchial stenosis (73 cases,26.5 ％).A total of 21 cases (7.6％) underwent supraglottoplasty during or after FB,while 17 cases (6.2％) underwent tracheal dilation and 10 cases (3.6％) underwent tracheotomy.Compared with non-CHD neonates,neonates with CHD were statistically significantly less likely to have congenital lesions statistically,such as laryngomalacia(15.8％ vs.28.6％,P =0.012),bilateral vocal cord lesions(6.2％ vs.21.4％,P =0.000) and congenital laryngeal dysplasia(0 vs.7.1％,P =0.001).The tracheotomy(0 vs.10.2％,P =0.000) and supraglottoplasty(2.3％ vs.17.3％,P =0.000) were more rare.Nevertheless,they were more likely to have secondary lesions such as the left main bronchial stenosis caused by extrinsic compression (23.7％ vs.1.0％,P =0.000),abnormal bronchial anatomy(9.6％ vs.2.0％,P =0.018),left vocal cord paralysis(9.0％ vs.1.0％,P =0.008) and airway mucosal inflammation(41.8％ vs.24.5％,P =0.004).The tracheostenosis and main broncial stenosis (37.3％ vs.7.1％,P =0.000) with long-term intubation(78.5％ vs.58.2％,P =0.000) were more common.There was no significant difference between term neonates and premature infants in the detection rate of respiratory tract diseases (P ＞ 0.05),tracheotomy (0 vs.4.3 ％,P =0.322),supraglottoplasty (13.3 ％ vs.6.5 ％,P =0.205) or long-term i ntubation (80.0％ vs.69.6％,P =0.157).Complications caused by procedure were rare and mild.Conclusions FB can detect whether the neonates with dyspnea,tachypnea and stridor have laryngomalacia,vocal cord paralysis,airway mucous edema,tracheal and main bronchial stenosis and other signs,and FB may play an important role in diagnosis,treatment and prognosis evaluation of neonatal respiratory diseases.

Objective To establish rSAG1-IgM-ELISA with purified rSAG1 fusion protein for immunodiagnosis of toxoplasmosis. Methods The rSAG1 fusion protein was purified by Ni 2+ column. The ELISA plate was coated with different concentrations of rSAG1, reacted with pooled positive and negtive human sera. Goat anti-human IgM conjugated to horseradish peroxidase was used as the second antibody. The appropriate detecting condition of the rSAG1-IgM-ELISA assay was determined by orthogonal experiment. The reproducibility, sensitivity and specificity of the assay were assessed. Thirty-five IgM-positive and 57 IgM-negative human sera detected by the imported IgM-ELISA kit were detected with the rSAG1-IgM-ELISA. Results The purity of rSAG1 was above 90%. The appropriate detecting condition was that the coated rSAG1 was 2 5 ?g/ml, the human serum was in 1∶100 dilution, and the second antibody was in 1∶4000 dilution. The coefficient of variation (CV) value of IgM-positive and IgM-negative pooled sera were 13 8% and 7 7% respectively. The inhibition rate of the assay was 62 0% The positive correspondence rate and negative correspondence rate were 82 9% (29/35) and 91 2% (52/57) respectively,the total correspondence rate was 88 0%, compared with the imported IgM-ELISA kit. Conclusions The rSAG1-IgM-ELISA has high sensitivity and specificity, and good correspondence rate with the imported IgM-ELISA kit. It indicates that rSAG1-IgM-ELISA has potential value for early diagnosis of toxoplasmosis.

BACKGROUNDImmunological sensitization remains a major problem following renal transplantation. There is no consensus for the management of sensitized renal allograft recipients. The patients become tethered to dialysis while waiting for compatible donors. This study was designed to evaluate the efficacy and safety of preoperative single-bolus high-dose antithymocyte globulin (ATG) as induction therapy in sensitized renal transplant recipients.

METHODSA total of 56 patients were divided into two groups according to the level of panel reactive antibody (PRA): non-sensitized group (PRA < 10%, n = 30) and sensitized group (PRA > or = 10%, n = 26). The characteristics of the recipients and donors were comparable between the two groups. Mycophenolate mofetil (MMF, 1 g) or ATG (iv. 9 mg/kg) were given preoperatively in the two groups as induction therapy. After the transplantation, the patients were treated with standard triple therapy regimen consisting of tacrolimus (FK-506) or cyclosporine A, MMF, and prednisolone. Acute rejection (AR) and infection episodes were recorded and renal function was monitored during a 12-month follow-up. Chi(2) test and t test were used to analyze the data.

RESULTSDuring the follow-up, 6 patients (20.0%) suffered AR episodes in the non-sensitized group and 4 (15.4%) in the sensitized group (P = 0.737); 8 patients (26.7%) experienced 11 infection episodes (average, 1.4 episodes per infected patient) in the non-sensitized group, and 6 (23.1%) experienced 10 infection episodes (average, 1.7 episodes per infected patient) in the sensitized group (P = 0.757, 0.890). The safety of the drugs, which was assessed by the occurrence of side effects, was comparable between the two groups. The hospital stay was 13 - 25 days (mean, 16.7 +/- 3.3) in the non-sensitized group and 14 - 29 days (mean, 16.2 +/- 3.1) in the sensitized group, respectively (P = 0.563). No delayed graft function (DGF) was observed in all the patients. Both the 12-month actuarial patient and graft survival rates were 100% in the two groups.

CONCLUSIONPreoperative single-bolus high-dose ATG is an effective and safe induction therapy yielding acceptable acute rejection rate in sensitized renal transplant recipients.

Adult ; Antilymphocyte Serum ; adverse effects ; therapeutic use ; Female ; Graft Rejection ; prevention & control ; Graft Survival ; Humans ; Immunosuppressive Agents ; Kidney Transplantation ; Male ; Middle Aged

China ; epidemiology ; Enterovirus ; genetics ; isolation & purification ; Genes, Viral ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans