Objective To construct an infectious full-length cDNA clone of enterovirus 71(EV71)and develop a technological platform for study on vaccine development as well as molecular virology of EV71.Methods According to the nucleotide sequence of EV71 strain 085 isolated in China,four pairs of primers were designed for amplification of four end to end overlapping subgenomic cDNA fragments,the cDNA fragments were directional cloned into pBluescript SK(+)vector,and the virus genome cDNA clone was obtained by ligation orderly.The rescued virus of parental strain 085 from RNA transfected host cells was identified by RT-PCR,IFA,titration as well as transmission electron microscope(TEM)after the transcription of the full-length cDNA clone in vitro.Results The full-length cDNA clone was constructed successfully,and the typical CPE was observed after its transcription into Vero cells.The rescued virus with 20-30 nm in diameter can not only be neutralized by EV71 special anti-serum but also react with anti-EV71 monoclonal antibody that virus infected cells stained with FITC can be detected by IFA.After amplification from the total RNA extraction of virus infected cells by RT-PCR with EV71 special primers,the 226 bp products can be detected.The growth curve showed that the rescued virus can propagate in Vero cells stably with a titer of 4.5 ～6.0 lgCCID50/ml during 8 passages.The plaque formed by rescued virus is identical as parental virus in morphology but smaller in size.Conclusion An infectious full-length clone of EV71 was developed successfully,which will be used for further study on pathogenesis and vaccine development of EV71.

Objective To explore the correlation between blood glucose and postoperative recovery of pancreatic cancer patients with diabetes.Methods According to the level of glycemic control after operation,68 cases of pancreatic cancer patients with diabetes were divided into two groups,including 34 cases of intensive control group (blood glucose 4.4 ～ 6.1 mol/L),34 cases of control group (blood glucose 6.1 ～11.1 mol/L).Fasting blood glucose (FBG),fasting insulin (FINS) and C reaction protein (CRP) in all patients were detected at 1st,3rd and 7th day after operation,and the recovery condition,postoperative complications and other clinical data were compared and analyzed.Results FBG,FINS and CRP in intensive control group were (6.94 ± 0.94) mmol/L,(17.38 ± 7.37) mmol/L,(108.33 ± 37.25) mg/L,and in control group were (7.81 ± 1.36) mmol/L,(23.73 ± 8.25) mmol/L,(131.51 ± 42.34) mg/L at 3rd day after operation.There were significant difference in serum FBG,FINS and CRP level between the two groups at 3rd day after operation (P ＜ 0.05).Compared with control group,postoperative duration of fever [(1.4 ± 0.8) vs (2.5 ± 1.1) d],duration of antibiotics [(3.1 ± 0.7) vs (4.6 ± 0.8) d] and time of anal exhaust [(2.5 ±0.5) vs 93.7 ± 0.8)d] in intensive group were significantly reduced (P ＜ 0.05).The wound infection rate in intensive control group was significantly decreased (5.9％ vs 23.5％,P ＜0.05),the rate of mortality and other postoperative complications between the two groups was not statistically significant (P＞0.05).Conclusions Intensive glycemic control can contribute to improve insulin resistance,reduce inflammation reaction and improve the prognosis of postoperative pancreatic cancer patients with diabetes,and it does not increase the postoperative complication rate.

Objective:To explore the effect of argatroban combined with Kallikrein on progressive cerebral infarction.Methods: One hundred and fifty two progressive cerebral infarction patients were randomized into groups observation (n=76) and control (n=76). Observation group were given treatment ofargatroban and Kallikrein, control only Kallikrein. NIHSS scores, Barthel index, Modified Rankin Scales(MRS) were used to evaluate the efficacy in two groups.Results: The difference of the effect was significant in two groups(x2=11.463,P>0.05).In both of the two groups, NIHSS scores were decreased, there was significant difference between the two groups (t=1.501,t=1.844,t=1.341;P<0.05). The Barthel index in argatroban combined Kallikrein group was higher than Kallikrein group, Modified Rankin Scales was lower than Kallikrein group, and there was significant difference between the two groups (t=2.121,t=2.332,t=2.219;P<0.05). The observation group and the control group patients don''t have bleeding gums,subcutaneous bleeding, gastrointestinal bleeding and other adverse reactions.Conclusion: Argatroban combined with Kallikrein, improve the neurologic impairment symptoms, clinical effect, improve the life quality of the patients, of a relatively good effect in treatment of progressive cerebral infarction, can improve obviously the cognitive ability and neural function and patients, activities of daily living. Moreover, its security and tolerability are good.

Part of undergraduates were recruited from the universities round Xueyuan Road, Haidian district in Beijing. Self-administered questionnaire were conducted, on the basis of the status quo of students in understanding the environmental and health knowledge, attitude, behavior (KAP), to analyse the need for environmental health educational courses setting among undergraduates and to afford related suggestions on contents and methods for the course.

Aim To investigate the effects of rosmarinic acid on H2O2-induced apoptosis in rat VSMCs and its related mechanisms. Methods Flow cytometry was used to determine apoptotic rate of VSMCs. nuclear staining by acridine orange for morphologic change,MTT assay for cell viability and Western blot for expression of Bcl-2,Bax, fas and FasL. Results After being treated by H2O2(0,250,500,750 ?mol?L-1),apoptotic rate of VSMCs was 3.71%?0.56%,17.6%?6.92%,34.9%?2.55 %, and 85.6%?5.22% by FACS analysis,and VSMCs appeared nuclear condense and nuclear fragmentation after being treated by 500 ?mol?L-1 H2O2 for 24 hours,which were typical morphological changes of apoptosis. ① VSMCs treated with 500 ?mol?L-1 H2O2 for 24 hours had a significantly decrease on cell viability compared with control,and the apoptosis rate was increased to 35.7%?1.33%; Bcl-2 protein expression decreased,Bax protein expression increased, Bcl-2/Bax protein ratio decreased and Fas receptor and Fas ligand expression also increased.② Pre-incubation with rosmarinic acid(10, 20, 40 ?mol?L-1)for 30 min enhanced the cell viability, and decreased the apoptotic rate to 31.1%?1.38%,21.2%?1.18%,13.6%?0.51% in a dose-dependent manner. Moreover, Bcl-2/Bax protein ratio increased and Fas and FasL expression decreased. Conclusions ① Rosmarinic acid antagonizes H2O2-induced apoptosis of VSMCs. ② The antagonism of rosmarinic acid on apoptosis may be correlated with an increase Bcl-2/Bax protein ratio and a decrease expression of Fas and FasL protein.

Objective: To study if the immune-enhancing effect of Arg was mediated via liver.Methods: The direct effect of Arg on T cell proliferation was measured by 3H-TdR incorporation. Rat hepatocytes were primarily cultured in serum-free DMEM/F12 medium, and then cultured in medium containing Arg(?mol/L:0,7.5,75,750,7 500), and the supernatant was collected at 0, 24, 48, 72 hours, and added to splenocytes culture, and T cell proliferation, NK cell activity and IL-2 activity,〔Ca 2+〕i were measured respectively.Results: Arg had no direct effect on splenocyte proliferation. The hepatocyte culture supernatant significantly increased the lymphocyte〔Ca 2+〕i ,IL-2 activity and T lymphocyte proliferation; 7 500 ?mol/L was most effective. Conclusion: Arg may enhance immune function via secretion of bioactive molecules by hepatocytes.

Objective:To establish an LC-MS/MS method for the simultaneous determination of acetaminophen and its five metabolites in mice plasma,and investigate the metabolism of acetaminophen by using the method.Methods:Para aminobenzoic acid was used as the internal standard.The plasma samples were precipitated by methanol,and then separated on a C18 column with the mobile phase of methanol and 5 mmol·L-1 ammonium acetate buffer solution containing 0.1% formic acid (55∶45).The flow rate was 0.5 ml·min-1,and the column temperature was 25℃.An electrospray ionization source was applied and operated in a positive ion mode using MRM:APAP,-,m/z 152.0→110.0;APAP-cys,-,m/z 271.2→140.1;APAP-glut,-,m/z 457.0→328.0;APAP-NAC,-,m/z 313.4 →208.0;APAP-sulf,-,m/z 232.4→152.1;APAP-gluc,-,m/z 328.2→152.1;IS,-,m/z 138.2→120.0.Results:The method exhibited good linearity over the concentration range of 0.2-10 μg·ml-1for APAP,1.0-20 μg·ml-1 for APAP-gluc,1.0-20 μg·ml-1 for APAP-sulf,1.0-20 μg·ml-1 for APAP-glut,0.4-15 μg·ml-1 for APAP-NAC and 0.2-10 μg·ml-1 for APAP-cys (r≥0.990 0).The inter-day accuracy and precision of acetaminophen and its five metabolites were all below 15%.The average recovery was between 85% and 115%,and RSDs were all below 15%.Conclusion:The LC-MS/MS method is proved to be quick,sensitive and accurate,and suitable for the determination of acetaminophen and its five metabolites in mice plasma.

Objective To establish a reversed-phase high performance liquid chromatograph ( RP-HPLC ) method for determination of equilibrium solubility and oil/water partition coeficient of phloridzin in different solvents. Methods A RP-HPLC method was established to detect the concentration of phloridzin in water and different organic solvents. The partition coefficients in the n-octanol-water/buffer solution systems of phloridzin were determined by shaking flask method. The Inertsil ODS-3 (4.6 mm×150 mm, 5μm) column was used and the detection wavelength was 284 nm.The flow rate was 1.0 mL??min-1, and acetonitrile-0.05% phosphoric acid(30??70)was used as mobile phase. Results The equilibrium solubility of phloridzin was 2.07 mg??mL-1 in water and 838.63 mg??mL-1 in methanol at 25 ℃.A good linear relationship of phloridzin was obtained within the range of 0.054 9-1.098 0 μg.The regression equation was Y=2 152.9X+7.26 (r=0.999 9).The solubility values of phloridzin were higher in ethanol and propylene glycol than in other solvents. Conclusion RP-HPLC method is simple, quick and accurate for the determination of phloridzin.Phloridzin was almost insoluble in petroleum ether and poorly soluble in water.The equilibrium solubility is higher in methanol than in other solvents. The apparent distribution coefficient of phloridzin varies significantly with pH under the alkaline conditions but less in the acidic solution.

OBJECTIVE:To establish a method for determining the plasma concentration of paeoniflorin and phillyrin and phar-macokinetic study before and after intragastric administration of Qianliean granules. METHODS:LC-MS/MS method was adopted. The column was Waters C18 with mobile phase consisted of acetonitrile(A)-2 mmol/L ammonium acetate(containing 0.05% formic acid)(B)(0-9 min:15%A→50%A;9-11 min:50%A→90%A;11-17 min:90%A;17-19 min:90%A→15%A;19-20 min:15%A),at the flow rate of 0.6 ml/min;column temperature was 35 ℃ and the volume was 20 μl;quantitative ions were paeoniflorin m/z 525.2 → m/z 449.0,phillyrin m/z 552.3 → m/z 355.3. 7 SD male rats were docked to collect blood 0.5 ml from angular vein 0.25,0.5,0.75,1,1.5,2,3,4,6,8,10,12,24 h after administration Qianliean granule solution 1 g(medicinal materials)/kg to determine the blood concentration of drugs. DAS 2.1.1 software was employed to calculate pharmacokinetic parameters. RE-SULTS:The linear range of paeoniflorin and phillyri were 5.0-2500.0 μg/L(r=0.9979)and 2.0-2000.0 μg/L(r=0.9982),re-spectively;RSD of precision test was less than 5.5%(n=5);the method recovery were 96.0%-104.0% and 92.0%-107.0%,the extration recovery were 71.4%-83.5% and 81.5%-92.3% and RSD of stability test was less than 5.0%(n=3). The pharmacokinet-ic parameters of paeoniflorin and phillyrin were as follows as t1/2 of (2.206 ± 0.631) and (1.355 ± 0.317) h;cmax of (1504.069 ± 620.885) and (79.043 ± 15.568)μg/L;tmax of (1.000 ± 0.250) and (1.214 ± 0.267) h;AUC0-24 h of (4897.645 ± 2207.577) and (263.475±54.795)μg·h/L;CL of(5.025±2.773)and(76.253±13.986)L/(h·kg). CONCLUSIONS:The method is highly sensi-tive,exclusive,simple,accurate and reliable,and can be applied to study the pharmacokinetic characteristics of paeoniflorin and phillyrin in rats in vivo.

Objective Pressure Ulcer Scale for Healing ( PUSH) was published in 1998 by National Pressure Ulcer Advisory Panel ( NPUAP) as a tool to evaluate the effects of pressure ulcer care.This study aimed to verify the reliability and validity of the Chi-nese version of PUSH in order to provide an efficient and reliable tool for evaluating the effects of pressure ulcer care. Methods Using the Brislin translation model, we translated the English version of PUSH into Chinese and finalized the Chinese version through modifica-tion by an advisory panel, pretests, and verification of its reliability and validity in the care of 126 cases of stage-Ⅱ-Ⅳpressure ulcers. We analyzed the reliability and validity of the scale based on its item scores, content validity, construct validity, Cronbach′s αcoeffi-cient, and test-retest reliability. Results The correlation coefficient value of the total and individual item scores was 0.616-0.963 (P<0.01).Each individual item score was significantly higher in the 27%high-score group (n=35) than in the 27%low-score group (n=36).The total content validity coefficient was 0.965, the correlation coefficient of construct validity between the total and individual item scores was 0.750-0.954 (P<0.01), and that between individual items was 0.666-0.826 (P<0.01), with statistically signifi-cant differences between the total and individual item scores at 7 and 21 days after treatment (P<0.01).The Cronbach′s αreliability coefficient value of the total score was 0.823 and those of individual item scores were 0.770, 0.791, and 0.868, respectively.The inter-rater reliability coefficients were all >0.85 and the test-retest reliabili-ty coefficients of individual items were 0.826, 0.885, and 0.958, re-spectively ( P<0.01) . Conclusion The Chinese version of PUSH, with its high validity and reliability, can be used to evaluate interven-tion effectiveness of Chinese patients with pressure ulcers.