The fused gene (PTH-HSA) of parathyroid hormone (PTH) gene and Human Serum Albumin(HSA) gene was amplified without linker by Overlapping PCR technology. The spliced gene was clone into Pichia pastoris secretory vector pPIC9K. With the help of promoter AOX1 and mat ? signal peptide, the PTH-HSA gene was designed to secretory expression.Linearized by restriction enzyme SalI, The recombinant plasmid pPIC9K/PTH-HSA was transformed into Pichia pastoris KM71 by electroporation. The recombinant strains which were identified by G418 and PCR analysis were induced by methanol to express protein PTH-HSA. The target protein was expressed in fermentation supernatant. Western blot analysis of the fusion protein showed that the expressed fusion protein PTH-HSA had the antigenicity of HSA.adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis The specific activity of broth was about 318IU/ml.

Extremities ; Humans ; Joint Prosthesis

Objective To explore the two different anesthesia methods on hemodynamics and inflammatory cytokines in elderly patients during peroperative period.MethodsFifty elderly patients with Knee Replacement( ASA Ⅰ,Ⅱ )were randomly divided into general anesthesia group( group A,n =25 ) and combined general and epidural anesthesia group( group B,n =25 ).The changes of mean arterial pressure(MAP) and heart rate ( HR ) were monitored before induction of anesthesia ( T1 ),at intubation ( T2 ),during skin incision ( T3 ) and at the time of extubation ( T4 ),at 30 min after extubation ( T5 ).Blood samples were taken from artery for determination of plasma TNF-α,IL-6,IL-10 concentrations before tourniquet inflation ( T5 ),10 min after tourniquet deflation(T6),30 min after tourniquet deflation (T7)and 30 min after operation (T8)by enzymelinked immunosorbent assay(ELISA).ResultsThe MAP and HR of patients in two groups at T2,T3,T4 were all increased when compared with T1 [ group A:HR:( 94.3 ± 10.4 ) bpm,( 96.4 ± 12.7 ) bpm,(93.3 ± 11.1 )bpm vs(62.6 ±7.3)bpm;MAP:( 18.8 ±3.4)kPa,( 19.6 ±3.4)kPa,( 17.8 ±2.0)kPa vs ( 14.5 ± 1.5)kPa,P＜0.05;group B:HR(76.2 ±6.5)bpm,(70.1 ± 9.7) bpm,(71.5 ± 8.3) bpm vs(64.6 ± 8.4) bpm;MAP:( 16.3 ± 2.5 ) kPa,( 15.3 ± 1.2) kPa,( 14.8 ± 1.4) kPa vs ( 14.1 ± 1.3 ) kPa,P ＜ 0.05 ].There was significant difference on MAP and H R between group A and group B( F =11.957,9.745;P ＜ 0.05 ).The level of plasma TNF-α,IL-6 and IL-10 were significantly increased at T6 to T8 compared with T5 in both groups[ groupA:TNF-α:(4.36 ±0.18) ng/L,(7.54 ± 1.23) ng/L,(10.35 ±2.21 )ng/L vs (2.26 ±0.16) ng/L; groupA:IL-6:(4.32 ±0.21 ) ng/L,( 8.35 ± 1.26 ) ng/L,( 10.23 ± 2.23 ) ng/L vs ( 1.36 ± 0.08 ) ng/L; groupA:IL-10:(5.32±1.10) ng/L,(7.56 ± 1.36) ng/L,(8.63 ± 2.21) ng/L vs (1.25 ± 0.03) ng/L; groupB:TNF-α:(3.43 ±0.06)ng/L,(5.24 ±2.10) ng/L,(7.68 ± 1.43) ng/L vs(2.22 ±0.15) ng/L;groupB:IL-6:(3.41 ±0.08 ) ng/L,(5.34 ± 1.34 ) ng/L,( 8.54 ± 2.03 ) ng/L vs ( 1.28 ± 0.04 ) ng/L; groupB:IL-10:( 7.28 ± 1.22 )ng/L,( 10.53 ± 2.14)ng/L,( 12.45 ± 2.03 )ng/L vs( 1.31 ± 0.04)ng/L,P ＜ 0.05 ].And there was significant difference on TNF-α,IL-6 and IL-10 between group A and group B( F =7.889,3.554,5.443,respectively,P ＜0.05).ConclusionCompared with general anesthesia group,combined general and epidural anesthesia group can ensure hemodynamic stability of elderly patients during peroperative period very well and can reduce the releasing of inflammatory cytokins,it is a viable and an ideal method.

ObjectiveTo observe the changes of the respiratory dynamics during expand thymectomy,and to explore the protection of ulinastatin on pulmonary function.MethodsSixty patients with myasthenia gravis( Ossermann Ⅰ,Ⅱ b)undergoing expand thymectomy were randomly divided into control group( group C,n =30)and ulinastatin group( group U,n =30).Patients in ulinastatin group received intravenous injection of ulinastatin 4000 U/kg in 20 ml physiological saline immediately after entering operating room and pumped ulinastatin 2000 U/( kg · h)to the end of the operation continuously.Patients in control group received the same volume of normal saline.Heart rate ( HR ),mean arterial pressure ( MAP ),lung compliance,airway peak pressure,plateau airway pressure,inspiratory and expiratory resistance were monitored before induction of anesthesia( T1 ),during skin incision ( T2),at 30 min after operation ( T3 ) and at 60 min after operation (T4),at the end of operation before extubation(T5).ResultsCompared with T1,HR and MAP at T2 in two groups were increased obviously [ group U HR:( 90.2 ± 13.5 ) bpm vs ( 78.6 ± 10.4 ) bpm,MAP:( 15.5 ± 2.3 ) kPa vs ( 12.1 ± 1.5)kPa;group C HR:(94.3 ± 15.4)bpm vs(81.6 ± 12.2)bpm,MAP:( 16.8 ± 2.6) kPa vs( 12.6 ±1.8)kPa,P ＜ 0.05 )].There was no significant difference on HR,MAP at each time between the two groups (P ＞0.05).At T3,T4,T5,the lung compliance was significantly decreased when compared with T1 [ group U:T3,T4,TS(51.23 ± 12.33) ml/cm H2O,(50.35 ± 13.29) ml/cm H2O and(50.65 ± 13.16) ml/cm H2O vs T1 (53.69 ± 14.34) ml/cm H2O;group C:T3,T4,T5(41.56 ± 11.20)ml/cm H2O,(42.02 ± 10.12) ml/cm H2O and(39.85 ± 10.31 ) ml/cm H2O vs T1 ( 53.45 ± 15.21 ) ml/cm H2O; P ＜ 0.05 ) ].Airway peak pressure,plateau airway pressure,inspiratory and expiratory resistance at T3,T4,T5 were obviously increased compared with T1 in two groups [ airway peak pressure:group U:( 13.04 ± 2.14 ) cm H2O,( 13.12 ± 2.42 ) cm H2O,(13.22±2.48)cm H2O,vs(12.04 ±2.12)cm H2O;group C:(16.25 ±3.27)cm H2O,(15.56 ±4.34)cm H2 O,( 16.64 ± 3.45 ) cm H2O,vs ( 13.12 ± 2.32 ) cm H2O; plateau airway pressure:group U:( 10.54 ±2.46) cm H2O,( 11.76 ± 3.11 ) cm H2O,( 12.02 ± 3.25 ) cm H2 O,vs ( 9.48 ± 2.13 ) cm H2O; group C:(15.02 ±3.87)cm H2O,( 15.51 ± 3.13) cm H2O,( 15.67 ± 3.02) cm H2O,vs (9.25 ± 1.26) cm H2O;inspiratory resistance:group U:( 8.56 ± 2.52 ) cm H2O,( 9.31 ± 3.06 ) cm H2O,( 8.44 ± 2.45 ) cm H2O,vs (8.25 ±2.20)cm H2O;group C:(11.52 ±3.06)cm H2O,(12.16 ±3.02)cm H2O,(12.83 ±3.14)vs ( 8.31 ± 2.24 ) cm H2O ; expiratory resistance:group U:( 10.22 ± 2.24 ) cm H2O,( 10.34 ± 2.66 ) cm H2O,(10.27 ± 2.22) cm H2O,vs(8.46 ± 2.37) cm H2O; group C:(14.43 ±3.18)cm H2O,(14.56 ±3.32)cm H2O,( 14.46 ± 3.52 ) cm H2O,vs ( 8.55 ± 2.18 ) cm H2O; P ＜ 0.05 ) ].The increased degree of lung compliance and the decreased degree of airway peak pressure,plateau airway pressure,inspiratory and expiratory resistance at the time of T3,T4,T5 and T1 in ulinastatin group were all significantly higher than those in control group(F=6.167,3.138,4.137,5.217,4.361,respectively,P ＜0.05).ConclusionUlinastatin can improve respiratory dynamics,reduce lung injury,and play a protective role in patients with myasthenia gravis.

60 strains which have antimicrobial activity had been isolated from nutritious soil in China in the study.We have further selected 1 strain which product broad-spectrum antibiotics using agar well-diffusion method.The strain was identified Brevibacillus laterosporus after physiological biochemical characteristic experiments,sequencing of 16S rDNA and cluster analysis,named S62-9.

Objective To investigate the expression and effect of secondary lymphoid tissue chemokine(SLC)in experimental ulcerative colitis(UC)in rats.Methods Thirty Spague-Dawley rats were divided into control group and UC group.SLC expression in colon tissues was detected by RT-PCR and immunohistochemistry,respectively.Results The transcriptional level of SLC in UC tissues was significantly higher than that in the control group(0.846?0.07 vs 0.312?0.12,P＜0.01).The positive expression of SLC was concentrated mainly on submucosa,and the positive rate of SLC protein expression in UC group significantly higher than that in control group(P＜0.01).Conclusion SLC overexpression could contribute to the pathological processes in UC rats,thus SLC may be an ideal ther- apeutic target for UC.

The validity of HbA1C and fasting plasma glucose(FPG)for screening of diabetes in community population was investigated.A total of 1 794 subjects from two sub-districts of Yangpu District,Shanghai,underwent a 75 goral glucose tolerance test(OGTT)and HbA1C determination.Based on 1999 World Health Organization criteria,there were 1 411 subjects with normal glucose tolerance(NGT),111 impaired fasting glucose(IFG),73 impaired glucose tolerance(IGT),33 IFG+IGT,and 166 diabetes.According to the receiver operating characteristic curve,the optimal cut-point of FPG for diagnosing diabetes was 6.15 mmol/L with sensitivity of 89.0%,specificity of 92.8%,and area under the curve of 0.959.The cut-point of HbA1C for diagnosing diabetes was 6.5% with a sensitivity and specificity of 71.1% and 80.2%.The area under the curve was 0.822.The screening model using FPG ≥6.1mmol/L and HbA1C≥6.5% had sensitivity of 66.9% and specificity of 97.0%.When the model was FPG ≥6.1 mmol/L or HbA1C ≥6.5%,the sensitivity and specificity was 96.3% and 76.7% respectively.The results suggest that both FPG and HbA1C have good value for screening diabetes in community,and FPG combined with HbA1C may further promote diagnostic efficacy.

Objective To explore the impact of metabolic syndrome (MS)in bone mineral density (BMD) in elderly male type 2 diabetics (T2DM). Methods One hundred and fourty cases were divided into MS group and non-MS(NMS)group according to with or without MS. Height,weight were measured, body mass index(BMI) were calculated and the total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), glycated hemoglobin (HbA1c)were tested. The bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXA). The differences among these groups were compared and the risk factors for MS were analyzed. Results The MD, BMI, TC,TG in MS group was higher than that in NMS group (P < 0.05 vs. P < 0.01),and HDL-C was lower than NMS group (P < 0.05). Correlation analysis showed that BMD was negatively correlated with age , TG , HDL-C , and was positively correlated with BMI.The results of multiple stepwise regression analysis showed that age ,HDL-C,BMI were independent risk factors for BMD. Conclusion The BMD significantly increases in T2DM with MS.Obesity and low HDL-C have positive effects in bone mass.

OBJECTIVETo investigate the detection limit of multicolor combinational probe coding real-time PCR (MCPC-PCR) in detection of Salmonella and Staphylococcus aureus suspended in the food samples, and to apply MCPC-PCR to detect the samples of food poisoning.

METHODSSeries concentration of bacterium suspension (10(1) - 10(9) CFU/ml) was prepared by using 22 simulated samples including fresh meat and cakes and then MCPC-PCR was applied to detect Salmonella and Staphylococcus aureus in 22 samples. Enrichment broth of 101 frozen samples and 5 early patients' anal swabs in food poisoning cases were detected after the DNA samples were extracted.

RESULTSThe limits of MCPC-PCR assay in detecting Salmonella and Staphylococcus aureus were about 10(2) copies/test; 101 frozen enrichment broth of samples in food poisoning cases were detected by MCPC-PCR assay, of 23 positive samples, 18 were confirmed by bacteriology techniques; 96 samples detected by MCPC-PCR and bacteriology techniques had the same results, and the coincidence rate was 95.05%. Anal swabs, collected from 5 of early patients in a food poisoning case gave a clue to be Vibrio parahaemolyticus by MCPC-PCR assay and then were perfectly consistent with bacteriology assay.

CONCLUSIONAs a method of high sensitivity and good specificity, MCPC-PCR assay can quickly and conveniently detect multiple pathogens existing in food samples, therefore we recommend it to be used in rapidly screening or simultaneous detection of food-borne diseases.

Bacteriological Techniques ; methods ; Food Contamination ; analysis ; Food Microbiology ; Molecular Probe Techniques ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Salmonella ; genetics ; isolation & purification ; Sensitivity and Specificity ; Staphylococcus aureus ; genetics ; isolation & purification