Objective Vaccination is a most effective method for the prevention of severe diseases caused by pandemic influenza and microRNA ( miRNA) mediated gene silencing has offered a novel approach to the construction of new vaccines.Our study aimed to construct a recombinant influenza A ( H1 N1 ) virus with the PB1 gene that carries the target fragment of miRNA Let-7b. Methods After comparing the sequence of the A/Nanjing/108/2009 H1N1 viral fragments with that of Let-7b, we selected PB1 as the optimal gene sequence, inserted the Let-7b binding target gene into PB1, ligated the modified fragments with pDP 2000, and named the recombinant plasmids pDP-mu-PB1 and pDP-sclb-PB1, respectively.We co-transfected the MDCK and 293T cells with the recombinant and other seven plasmids and injected the supernatant into the allantoic cavity of the chickenembryo for virus propagation, followed by detection of the virus by hemagglutination ( HA) assay and measurement of the viral titer by TCID50 .We amplified the viral cRNA by RT-PCR and identified the viruses by agarose gel electrophoresis and nucleotide sequence analysis. Results PB1 was the optimal sequence ( 83 bp -107bp) for the attenuation of viruses.The HA-titers of miRT-H1N1 and scbl-H1N1 were 1∶32 and 1∶64, and their viral loads were 4.68 ×105 and 7.94 ×104 TCID50/mL, respectively.Nucleotide sequence analysis showed the expected fragment in the rescued virus. Conclusion A recombinant strain vaccine was successfully constructed, which has laid the foundation for fur-ther assessment of virulence.

Objective The use of in vivo cryotechnique (IVCT) in combination with electrocardiograph (ECG) to study cardiac microcirculation under different hemodynamic conditions in living mouse.Methods Living mouse heart monitored by electrocardiograph was suffered from IVCT and freezing substitution under normal blood flow,myocardial ischemia or cardiac arrest conditions.Hematoxylin eosin (HE)staining,Schiff's staining and immunofluorescence staining for serum albumin,immunoglobulin were utilized on continuous paraffin sections,respectively.Confocal microscopy and statistical analyses were used.Results Comparing with normal hemodynamics,microvascular red cell volume reduction,morphology changed,myocardial cell glycogen loss,serum albumin ectopic distribution to myocardial cytoplasm,T tubular network failure and spacing width were happened in myocardial iscbemia condition; different shapes of red blood cells,myocardial cells glycogen deficiency,T tubular network failure and interval narrowing were found under cardiac arrest conditions.Conclusions Cardiac microcirculation,pathological changes of myocyte and its surrounding microenviroument in living mouse heart can be immediately captured in situ by the application of IVCT and ECG.

AIM: To evaluate the specific cellular immune response in mice inoculated with the recombinant hepatitis B virus (HBV) surface vaccine (rHBs). METHODS: Spleen T lymphocyte reactivity to rHBs was assessed by a proliferation assay and cytokine secretion. BALB/c mouse were intraperitoneally inoculated with rHBs at doses of 0.65, 1.25, 2.5 or 5 ?g for once or twice. 4 weeks later, spleen lymphocytes were harvested and restimulated with rHBs in experimental group or with PBS as control. The spleen lymphocytes were labeled with [~3H]-thymidine for 3 days. The [~3H]-thymidine incorporation was measured, which expressed as the incorporation of [~3H] (counts?min~ -1 ) and stimulation index (SI) was calculated by the method of dividing the cpm obtained in the experimental group by that in control group. The content of IL-2 and IFN-? secreted from the spleen lymphocyte were measured by the method of ELISA. RESULTS: 2 weeks after primary vaccination, the SI in 0.65, 1.25, 2.5 and 5 groups was 1.55, 1.93, 2.41, 2.81 ng/L, respectively. IL-2 was 5.48?8.88, 9.28?6.98, 28.53?14.32, 64.69?20.88 ng/L, respectively. IFN-? was 8.22?8.61, 9.89?9.34, 20.27?15.50, 30.77?22.12 ng/L, respectively. 2 weeks after boost vaccination, the SI in 0.65, 1.25, 2.5 and 5 groups was 1.61, 2.05, 3.74, 3.62 ng/L, respectively. The IL-2 was 5.75?5.04, 102.53?67.52, 177.13?91.12, 332.10?124.31 ng/L, respectively. IFN-? was 3.63?4.42, 28.33?13.04, 59.66?25.75, 80.73?19.30 ng/L, respectively. CONCLUSION: Specific proliferation activity and IL-2, IFN-? secretion from the spleen lymphocytes of the mouse inoculated with rHBs are produced,that the strength is dependent on the dose of vaccination.

AIM To study the chemical constituents from Vigna umbellata Ohwi et Ohashi.METHODS The ethyl acetate fraction of 80％ ethanol extract from V.umbellata was isolated and purified by silica,Sephadex LH-20 and ODS column,then the structures of obtained compounds were identified by spectral data and physicochemical properties.RESULTS Twelve compounds were isolated and elucidated as (+) catechin (1),(-) epicatechin (2),3-furanmethanol-β-D-glucopyranoside (3),myricetin-3-O-β-D-glucopyranoside (4),quercetin7-O-β-D-glucopyranoside (5),(+) catechin-3-O-β-D-glucopyranoside (6),(+) catechin-5-O-β-D-glucopyranoside (7),quercetin-3 '-O-α-L-rhamnoside (8),(±) dihydroquercetin (9),quercetin (10),ethyl gallate (11),propanediol (12).CONCLUSION All the compounds are isolated from V.umbellata for the first time,and compounds 4,8,9,11,12 are first obtained from genus Vigna.

Objective: To establish a method for determing the trichloroethylene(TCE)and trichloroethanol(TCOH)in blood samples by liquid-liquid extraction-gas chromatography with electron capture detector. Methods: With this method,ether was used as extraction solvent and trichloromethane was used as an internal standard. The whole blood sample was extracted with ether, and dehydrated by anhydrous sodium sulfate. Then the analytes were separated on HP-5 capillary column(30m×0.32mm×0.15μm)and detected byECD.The retention time was for qualitative analysis and the internal standard was for quantitation. Results: The standard curves of TCE and TCOH showed significant linearity between 95.5μg/L-7640.0μg/L(r=0.9997)and 19.0μg/L-1520.0μg/L(r=0.9992). The average recovery was 95.5%-103.6%.The intra-day and inter-day precisions(RSD)were 2.5%-6.8%(n=6)and 1.6%-4.3%(n=6) respectively. The detect limit of TCE and TCOH were 2.10 μg/L and 0.56μg/L(S/N=3)respectively.The blood can be kept 7 days at-20℃ refrigerator without significantly loss. Conclusion This method is proved to be simple,practical and highly sensitive. It can satisfy the request for the determination of blood samples of humans exposed to TCE.

Objective: To establish a method for determination the S-phenylmercapturic acid in urine by dispersive solid-phase extraction using Humic Acid/Fe₃O₄ magnetic nanocomposite as adsorbent. Methods: The 5 ml of urine samples were adjusted to pH 1.0 and extracted by Fe3O4@HA. Then the analytes were separated on EC-C₁₈ capillary column and detected by HPLC-VWD. The S-phenylmercapturic acid was characterized by the retention time and quantified by peak area and external standard method. Results: The standard curves of SPMA showed significant linearity between 0.04~1.00 mg/L (r=0.999 7) . The average recovery was 94.2%~102.4%. The intra-day and inter-day precisions (RSD) were 2.9~6.7% (n=6) and 3.1~7.5% (n=6) respectively. The detect limit of SPMA was 0.012 g/L (S/N=3) . Conclusion This method is simple, rapid, sensitive and accurate. It is applicable for determination of SPMA in the urine of works who were exposed to benzene.

Objective:To establish a method for determination of the butanone in urine by gas chromatography (GC) with pre-column derivation.Methods:For detecting of butanone in urine, potassium iodide and potassium dichromate were added into urine under acidic condition, sample derivatization was undertaken in 50 ℃ water bath for 60 min and the iodine butanone was extracted with n-hexane. After the sodium thiosulfate solution was used to remove excess iodine, urine samples were centrifuged at 10000 r/min for 5 min, then the supernatant was analyzed using temperature rising programming with the Agilent Hp-5 column (30 m×0.32 mm, 0.25 μm) and electron capture detector (ECD) as the detector. The detector temperature was 300 ℃, the inlet temperature was 200 ℃, and the carrier gas was nitrogen.Results:For detecting of butanone in urine, potassium iodide and potassium dichromate were added for derivatization under the acidic condition. After extraction and centrifugation, the supernatant directly put through column and detected by ECD. In present study, the sample pretreatment condition was optimized, the relative standard deviations of intra-day and inner-day, the spiked samples and its recovery were evaluated for analyzing the accuracy of the proposed method.Conclusion:This method has proved to be simple, efficient and highly sensitivity, it can be utilized for butanone detection in occupational population.

Objective:To establish a method for determination of the butanone in urine by gas chromatography (GC) with pre-column derivation.Methods:For detecting of butanone in urine, potassium iodide and potassium dichromate were added into urine under acidic condition, sample derivatization was undertaken in 50 ℃ water bath for 60 min and the iodine butanone was extracted with n-hexane. After the sodium thiosulfate solution was used to remove excess iodine, urine samples were centrifuged at 10000 r/min for 5 min, then the supernatant was analyzed using temperature rising programming with the Agilent Hp-5 column (30 m×0.32 mm, 0.25 μm) and electron capture detector (ECD) as the detector. The detector temperature was 300 ℃, the inlet temperature was 200 ℃, and the carrier gas was nitrogen.Results:For detecting of butanone in urine, potassium iodide and potassium dichromate were added for derivatization under the acidic condition. After extraction and centrifugation, the supernatant directly put through column and detected by ECD. In present study, the sample pretreatment condition was optimized, the relative standard deviations of intra-day and inner-day, the spiked samples and its recovery were evaluated for analyzing the accuracy of the proposed method.Conclusion:This method has proved to be simple, efficient and highly sensitivity, it can be utilized for butanone detection in occupational population.

How to give full play to the advantages of rehabilitation institutions, and to improve service quality and administration efficiency are some of the complex issues faced by the high-level administrators of rehabilitation institutions. This paper studied a total of 67 institutions to investigate the operation and management model of rehabilitation institutions in China, as well as their advantages and disadvantages.

The establishment of an efficient performance management system is crucial for a rehabilitation institution. This article discussed the concept of performance, performance management and ways to establish an efficient performance management system.