Objective The aim of the study was to develop a purification procedure on Pichia pastoris GS115/Fab expressing human anti-HBsAg Fab fragment. Methods Purity and yield ratio and conjugated activity of purified Fab fragment were analyzed with three purified ways of goat anti human Fab affinity chromatography and 14F7 monoclonal antibody affinity column, as well as Ion exchange Size exclusion column. Results 98% purity was reached through 14F7 monoclonal antibody column, and 95% purity was gained after goat anti human Fab fragment column. But yield ratio of the two affinity columns was low, being 35% and 55%, respectively. ForIon exchange Size exclusion column, purity and yield ratio of Fab fragment were very good, being 93.8% and 80% or more, respectively. Results of ELISA analysis showed that purified Fab fragment through three columns could bind to HBsAg specifically. Conclusion The purification process of recombinant anti-HBsAg Fab fragment was established. It lays a foundation for industrialization and clinical research of human anti-HBsAg Fab fragment.

Objective To study the expression, purification and bioactivity of a recombinant fusion protein consisting of anti-HBs single chain Fv and interleukin-2. Methods The engineering bacterium M15[pQE-ScFv-IL-2] which can express the fusion protein consisting of anti-HBsAg single chain Fv and interleukin-2, was induced by IPTG, then a 43kD recombinant protein was identified by SDS-PAGE and Western-blot analysis. Results The ratio of target protein to total protein of host reached 18%. Further analysis confirmed that the recombinant protein formed inclusion body in the cytoplasm of bacteria. 95% purity could be achieved after two-step purification of ScFv-IL-2, including Ni metal chelating chromatography (the first) and ion-exchange chromatogram (the second). The bioactivity assay of the purified product showed that the antibody-cytokine fusion protein could bind to HBsAg specifically and stimulate the proliferation of CTLL-2. Conclusion These results suggest that the fusion protein retains the bioactivity of its parental molecules, and may be a potential gene-engineering targeting drug for the treatment of chronic hepatitis B and other relevant diseases.

Objective To study the expression in high density fermentation of anti HBsAg Fab fragment in Pichia pastoris , and the purification and activity detection of expressed target protein. Method The high density fermentation of genetically engineered Pichia pastoris was proceeded in a 5L bioreactor using fed batch fermentation. The fermentation temperature was set at 28-30℃,the pH was 5 0-5 5, and the DO was kept over 20%. When the absorbance (OD 600 ) of the broth reached 400-450 (the first time of fermentation), 200-250 (the second time), and 300-350 (the third time), the induced phase was initiated, and the methanol concentration was 0 5%-1%. The fermentation ended after 96h's induction, the target protein was purified by affinity chromatography, and its activity was assessed by ELISA. Results It showed that the optimum initial cell density during methanol induced phase should be 300-350, which was good for control of the fermentation process and the expression of recombinant Fab. At the end of the fed batch phase, a yield of about 245mg/l of Fab was reached, and 98% purity could be reached as demonstrated by affinity chromatography. The results of ELISA showed that the supernatant of fermentation and the purified recombinant Fab could bind to HBsAg specifically. Conclusion The success of high density fermentation lays a sound foundation of mass production and clinical applications of recombinant humanized anti HBsAg Fab fragment

Objective To investigate the effect of preoperative oral midazolam on emergence agitation after sevoflurane anesthesia in children.Methods Sixty children,34 males,26 females, aged 2-7 years,ASA grade Ⅰ or Ⅱ,receiving sevoflurane for tonsillectomy/adenoidectomy were ran-domly divided into low dose midazolam group (group M1),high dose midazolam group (group M2) and control group (group C)(n=20 each).The 5 ml mixture of midazolam 0.5 mg/kg,0.75 mg/kg and 10% glucose was taken orally 30 min before anesthesia in group M1 and group M2,respectively. While 5 ml of 10% glucose was given in group C.Anesthesia was induced with inhalation of 8%sevoflurane and maintained with intravenous infusion of remifentanil and inhalation of sevoflurane. Scores of parental separation anxiety scale(PSAS),pediatric anesthesia emergence delirium scale (PAED scale),FLACC scale were compared.Times to extubation and discharge from PACU were al-so recorded.Results Group C showed significantly higher incidence of separation anxiety(P <0.05). The incidence of emergence agitation,peak PAED scores,FLACC scores,time to extubation were similar among three groups.Discharge time from PACU was longer in group M2 (P < 0.05). Conclusion Preoperative oral midazolam 0.5 mg/kg or 0.75 mg/kg can effectively reduce parental separation anxiety.This,however,dose not result in a reduced incidence of emergence agitation after sevoflurane anesthesia.Midazolam 0.75 mg/kg can extend the discharge time from PACU.

Objective To evaluate the pharmacodynamics of oral chloral hydrate sedation for echocardiography in pediatric patients with congenital heart disease (CHD).Methods Two hundred ASA physical status Ⅱ-Ⅳ pediatric patients with CHD, aged 5-620 days,scheduled for elective echocardiography,were enrolled in the study.The dose of oral chloral hydrate was set at 50 mg/kg in the first pediatric patient.The oral dosage was determined by up-and-down sequential experiment.Each time the oral dose increased/decreased by 10％ in the next pediatric patient.The pharmacodynamics was analyzed based on the dose-response model to determine the 50％ effective dose (ED50),95％ effective dose (ED95) and 95％ confidence interval (95％ CI) of chloral hydrate for sedation.The covariates (age,gender,time period of administration,fasting time,sleeping at 2 h before sedation,premature and cyanotic CHD) were introduced into the dose-response model,and the effect of each covariate on the pharmacodynamics of chloral hydrate sedation was evaluated.Results The ED50 of chloral hydrate for sedation during echocardiography was 42.2 mg/kg (95 ％ CI 40.2-44.2 mg/kg), ED95 was 67.4 mg/kg (95％ CI 53.7-81.1 mg/kg) in the pediatric patients with CHD.Each covariate provided no effect on the pharmacodynamics of chloral hydrate sedation (P ＞ 0.05).When fasting time and premature were introduced into the dose-response model,95％ CI of the slope of dose-response curve included 0.When age which was stratified was introduced into the dose-response model,it was difficult to fit or the data seriously deviated from the clinical data.Conclusion The ED50 and ED95 of chloral hydrate for sedation during echocardiography were 42.2 mg/kg (95％ CI 40.2-44.2 mg/kg) and 67.4 mg/kg (95％CI 53.7-81.1 mg/kg),respectively,in the pediatric patients with CHD.Gender,time period of administration,sleeping before sedation and cyanotic CHD do not affect the pharmacodynamics of oral chloral hydrate sedation,while the effect of age,fasting time and premature needs further determination.

Objective: To determine tetrahydropalmatine in Juyanbaokang Granules (Rhizoma Corydalis, Radix Aconcti Lateralis Preparata, Radix Rauwolfiae, etc.) by SPE-HPLC. Methods : Samples were purified by C 18 SPE column. HPLC column was Dikma Diamonsil C 18 (250mm?4.6mm,5?m), mobile phase was 0.05M potassium dihydrogen phosphate-0.005M sodium heptanesulfonate-acetonitrile (1.2∶1.2∶1), flow rate was 1.2 mL?min -1 and detection wavelength was 230nm. Results : The calibration curve was linear in the range of 0.067 ～0.402?g for tetrahydropalmatine ( r =0.9997). The average recovery was 98.66%. Conclusion : The method with good separation is convenient, rapid, accurate and reliable.

AIM: To study the determination of dl-tetrahydropalmatine (THP) in Chi nese traditional patent medicines by TLC-Scanning from the viewpoint of the pro cess control of manufacture. METHODS: To select the 5 kinds of representative drugs and its prep arations contained, THP was extracted with solvent method. The mobile phase contains diethylamine. THP w as separated with high-efficiency silica gel plate or handcrafted silica gel p late. Iodine was used for chronogenic agent and fluorescence defectiion for dete rmination. TLC-Scanning results were compared with HPLC. RESULTS: The method is practicable, the result is correlative with the HPLC. CONCLUSION: TLC-Scanning is fit for the quality control of the Chi nese traditional patent medicine in the process of manufacture.

RBRVS assessment system has been carried out in hospital performance management,which meets the needs of the reform of public hospitals and hospital fine management.The one year practice at the hospital has built a new model of performance management based on the RBRVS assessment system.Calculation of the RVS point values and CF values of the operations and determination of such indexes as the indirect workload reference coefficient of the grades,will yield the amount of the performance bonus of individual departments and posts.The new model proved effective in improving staff incentives and efficiency,saving human resource cost and controllable materials.However,its design and implementation is a complex systematic engineering in need of measures suited to local conditions and steady progress.

Objective To investigate the function of JAK2-STAT3 signaling pathway in pancreatic injury and systematic inflammatory response in rats with acute necrotizing pancreatitis ( ANP) . Methods SD rats were randomly divided into the ANP group (n=48), ANP+JAK2 inhibitor Ruxolitinib group (ANP+R group, n=48), ANP+STAT3 inhibitot Stattic group (ANP+S group, n=48), ANP+Ruxolitinib+Stattic group (ANP+R+S group, n=48), and sham operation group (SO group, n=48). 5％ sodium taurocholate injection via retrograde pancreatobiliary duct was used to establish ANP model. Blood samples from abdominal aorta and pancreatic tissue were collected after 3 h, 6 h, 12 h and 18 h after modeling. Serum amylase (AMY) and tumor necrosis factor-α(TNF-α) and interleukin-4 (IL-4) were tested. JAK2 and STAT3 mRNA expression and protein expression of p-JAK2 and p-STAT3 in pancreas were examined by RT qPCR and western blot, respectively. Results AMY, TNF-α and IL-4 in plasma, and JAK2 mRNA, STAT3 mRNA, p-JAK2 protein and p-STAT3 protein at different time points in ANP group were all obviously higher than those in SO group(P<0. 05). Serum AMY, TNF-αand IL-4 in ANP+R group, ANP+S group and ANP+R+S group at different time points were lower than those in ANP group [12 h (5391 ± 1009),(6130 ± 1227),(4818 ± 992)U/L vs (8524 ± 1360) U/L;(147.25 ± 27.85),(156.25 ± 23.17),(127.87 ± 21.39) ng/L vs (187.58 ±20.09)ng/L;(45.89 ±16.95),(50.19 ±15.87),(38.87 ±14.03)ng/L vs (58.85 ±9.34)ng/L] . JAK2 mRNA and p-JAK2 protein,STAT3 mRNA and p-STAT3 protein in ANP+R group and ANP+R+S group at different time points were obviously lower than those in ANP group (12 h 0. 357 ± 0. 091 vs 0. 597 ± 0. 121,1. 115 ± 0. 203 vs 1. 217 ± 0. 213,0. 361 ± 0. 089 vs 0. 489 ± 0. 097,0. 965 ± 0. 189 vs 1. 128 ± 0. 217, 0. 362 ± 0. 092 vs 0. 597 ± 0. 121,1. 107 ± 0. 212 vs 1. 217 ± 0. 213,0. 297 ± 0. 087 vs 0. 489 ± 0. 097,0. 713 ± 0. 184 vs 1. 128 ± 0. 217). STAT3 mRNA and p-STAT3 protein in ANP+S group were obviously lower than those in ANP group(0. 319 ± 0. 107 vs 0. 489 ± 0. 097,0. 849 ± 0. 177 vs 1. 128 ± 0. 217), and the difference was statistically different (P<0.05). Conclusions The activation of JAK2-STAT3 signaling pathway in pancreas may play a key role in the pathogenesis of systematic inflammatory response in ANP.

OBJECTIVETo assess the value of array-based comparative genomic hybridization (array-CGH) for analyzing tissues derived from spontaneous abortion and stillbirth.

METHODSAgilent Human Genome CGH Microarray 4×44 K chip and Affymetrix Cytoscan 750 K Array were utilized to detect genome-wide copy number variations (CNV) in 43 fetuses with spontaneous abortion and stillbirth. All identified CNV were analyzed with references from Database of Genomic variants (DGV), database of DECIPHER, ISCA and OMIM, as well as comprehensive literature review to determine whether the identified CNVs were pathogenic. Parental DNA of two cases was also analyzed with the same arrays for pathogenic or unknown significant CNVs.

RESULTSAll of the 43 specimens were successfully analyzed. Clinically significant chromosomal aberrations were identified in 32 (74.4%) of the samples, which included 26 aneuploidies and 10 pathogenic CNV.

CONCLUSIONArray-CGH is a fast and effective method for analyzing tissues derived from spontaneous abortions and stillbirths which may be difficult to culture for karyotype analysis.

Abortion, Spontaneous ; diagnosis ; genetics ; Adult ; Chromosome Aberrations ; Comparative Genomic Hybridization ; methods ; DNA Copy Number Variations ; Female ; Fetus ; chemistry ; Humans ; Karyotyping ; Pregnancy ; Pregnancy Complications ; diagnosis ; genetics ; Stillbirth ; genetics ; Young Adult