Objective To explore the difference of DNA methylation levels between normal Schwann cells (NSCs) and activated Schwann cells (ASCs) in rats. Methods The adult Wistar rats were received sciatic nerve ligation and fed for 7 days. The ASCs and NSCs were separated from ligated sciatic nerves and brachial plexus respectively. Immunocytochemical staining of S-100 antibody was used to identify the cells. The growth condition of cells was detected by CCK-8 method. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) was applied to filter the differentially methylated regions in ASCs and NSCs. The distribution of differentially methylated genes related with axonal regeneration in chromosome was analyzed, and Gene ontology(GO)and PATHWAY analysis were also conducted. Results High purity of ASCs and NSCs were obtained successfully, which were both positive for S-100 antibody. In the same culture condition, ASCs showed a faster proliferation than that of NSCs. A total of 177 176 differentially methylated regions were found by MeDIP-Seq. Among them, 1 097 were located in the promoter (≤1 kb), 1 136 in the promoter (1-2 kb) and 567 on the CpG. After functional annotation of differentially methylated genes, 214 differentially methylated genes related with axonal regeneration were found in ASCs and NSCs. Compared with NSCs, 191 genes were up-regulated and 23 genes were down-regulated in ASCs. These genes were located on different chromosomes, most of which on chromosome 12 (22 genes) and the least on chromosomes M (2 genes). GO analysis indicated that the differential methylated genes were involved in axon growth, axon formation, axon elongation and axon guidance. The MAPK, cell adhesion molecules, Ras signaling pathway may be related with the differential methylated genes. Conclusion The methylation levels between ASCs and NSCs are significantly different, which are probably related with axon regeneration.

Objective:To analyze the chemical constituents of Rhizoma belamcandae by using high-performance liquid chromatography-diode array detector/electrospray ionization-mass spectrometry (HPLC-DAD/ESI-MS).Methods: Dried Belamcanda chinensis powder was extracted with 70% ethanol by sonication.The chromatographic separation was performed on a YMC ODS-C18 column (250 mm?4.6 mm I.D.,5 ?m) with a mobile phase composed of 20%CH3OH(A)-70%ACN(B) (0→30→45→65 min,20→30→90→100 B),eluted at a flow rate of 0.45 ml/min,and the UV detection wavelength was set at 265 nm.Positive ionization mode with a needle voltage of 5 000 V,a capillary voltage of 20 V,a gas(N2)press of 20 psi and a temperature of the drying gas of 300℃ was selected.Relative molecular mass data acquisition was performed from m/z 250 to 550 in full MS scan mode.Results: Nine major isoflavones were identified from Rhizoma belamcandae based on their retention behavior obtained on-line by their UV spectra and the HPLC-DAD/ESI-MS.Conclusion: A rapid and efficient HPLC-DAD/ESI-MS method for identifying the chemical constituents of Belamcandae chinensis has been established,which provides more scientific information for quality control of Rhizoma belamcandae.

Objective To observe the role of baicalin on the expression of phosphorylated protein of signal transduc-ers and activators of transcription signaling proteins (STATs) during the process that neural stem cells (NSCs) differentiating into neurons. Methods NSCs were isolated from the embryonic cerebral cortex of the 14-15-day pregnant SD rats, which were cultured and passaged in vitro. The 3rd generation of NSCs was used in the experiment. NSCs were randomized into nat-ural differentiation control group, three different doses of baicalin groups (7.5μmol/L, 15μmol/L and 30μmol/L), leukemia inhibitory factor (LIF)+basic fibroblast growth factor (bFGF) group and baicalin+LIF+bFGF group. After 6 d culture in vi-tro, the immunohistochemical method was used to observe the expressions of microtubule-associated protein 2(MAP-2) and glial fibrillary acidic protein (GFAP) in different groups. The expression levels of phosphorylation protein of STAT 3 in NSCs were detected by Western blotting method after 2 h and 6 d of culture. Results The expression of MAP-2 in NSCs was in-creased by baicalin, but the expression of GFAP in NSCs decreased. The expression of GFAP in NSCs was enhanced in LIF+bFGF group, which was inhibited by baicalin+LIF+bFGF. The phosphorylation level of STAT3 in NSCs was downregulat-ed by baicalin, but the phosphorylation level of STAT3 was upregulated in LIF+bFGF group. The upregulated phosphoryla-tion level of STAT3 was inhibited in baicalin+LIF+bFGF group(P＜0.05). Conclusion Baicalin can induce NSCs to dif-ferentiate into neurons, which may be caused by the downregulation of the phosphorylation level of STAT3 in NSCs.

Flocculation of fermented broth with 1,3 -propanediol using component B of natural clarifier-Ⅱ was studied. Firstly, single factor tests and orthogonal tests were carried out. The results showed the main factors which influenced the flocculation and the optimal conditions were: pH6.0, flocculant B 0.01g/L , NaCl 0g/L, and the FR was 95.97% . The following results by filtration experiments, compared with the sample without pretreatment, indicated that the filtration speed of the flocculated sample was improved dramatically, and the weights of net and dry filter cakes were increased by 41.13% and 51.88% , respectively. Electrodialysis experiments showed that flocculation could accelerate desalting process, and could take the place of centrifugal pretreatment when it combined with the filtration.

Objective To explore the matrix effect on cyclosporine A (CsA) test by fluorescence polarization immunoassay (FPIA) and enzyme-multiplied immunoassay technique (EMIT), explain the discrepancy of external quality control results between these two methods and find the corrective action.Methods One hundred whole blood samples with various concentrations were adopted and CsA levels were detected by FPIA and EMIT.The results were compared with each other.Moreover, the influence of residual metal ions upon immunoreactions was assessed by adding Cu2+ and Zn2+.The effect of non-whole blood matrix on extraction efficiency for quality control materials and CsA calibrator was evaluated by adding identical volume of Hb-rich reagents followed with re-extraction.Results There is good correlation between results measured with FPIA(X) and EMIT(Y) methods ( Y=0.926 8X -8.115,R2 =0.996 9).Neither FPIA nor EMIT was affected by residual metal ions ( P ＞ 0.05 ). Non-whole blood matrix decreased the extraction efficiency of two methods, but it could be corrected by supplementation of the Hb-rich reagents (≥30 g/L).Conclusions Non-whole blood matrix may be the main reason for the inconsistent results measured by FPIA and EMIT methods.It could be corrected by using Hb-rich reagents.In addition,we should consider the influence of low lib on CsA test,espocially for organ transplant patients with lower Hb ( ＜30 g/L).

Objective To explore the prevalence,subtype,distribution characteristics and risk factors of IBS met ROME Ⅲ criteria in local adult residents of Shanghai Songjiang area.Methods With multi-stage,stratified cluster random sampling method,from April to May in 2010,residents of45 shanghai Songjiang communities were interviewed by questionnaire survey.IBS diagnosis was based on Rome Ⅲ criteria,and the subtypes were according to Bristol stool scale.Self-rating anxiety scale (SAS),self-rating depression scale (SDS) and Athens insomnia scale (AIS) were also simultaneously finished. Results There were 7648 individuals who completed the survey with qualified questionnaires; the efficient rate was 90％. The sample size accounted about 1.62％ of total population in Shanghai Songjiang area.Total 970 patients were detected,who met the Rome Ⅲ criteria,of those 492 males and 478 females.The adjusted prevalence of IBS was 13.1％,13.8％ in males and 12.7％ in females.The ratio of male to female was 1 ∶ 0.92.There was no significant difference in detection rate between males and females (P＞0.05).The prevalence was highest in the 18 to 29 years age group (18.2％,P=0.000).IBS unsubtyped (IBS-U) was the most common type in patients who met the Rome Ⅲ criteria (45.3 ％),IBS with diarrhea (IBS-D) type was more common in male (27.0％) and IBS with constipation (IBS-C) was more common in female (19.5％).The IBS prevalence of obesity group (x2 =4.046,P=0.044),higher education group (x2=31.210,P=0.000),mental group (x2 =8.409,P =0.015),unmarried group(x2 =26.933,P =0.000) and no abdominal surgery history group(x2 =5.894,P=0.015) was higher than that of control group.The risk of IBS prevalence in those who had the history of gastrointestinal infection,abdominal surgery,taking antibiotics or analgesics increased by 5.105,3.388,2.949 and 2.811 times respectively(P=0.000).Multivariate analysis indicated that the risk of IBS prevalence was increased by 1.69 times if took more spicy food,however high-protein diet was a protective factor (OR=0.900,P=0.000).Anxiety (OR=2.452,P=0.000) and insomnia (OR=1.923,P=0.000) were also risk factors of IBS.Conclusion IBS is a common and frequently occurring disease in Shanghai Songjiang community.The history of gastrointestinal infection,abdominal surgery,taking antibiotics or analgesics,intake of more spicy food,anxiety and insomnia may be risk factors of IBS.

Objective To prepare recombinant plasminogen activator(Pla) protein in E.coli BL21 cells that can be used in studying interactions between Yersinia pestis proteins and immunologic diagnosis of plague.Methods The pla gene was amplified by PCR and cloned into the pET28a expression vector.E.coli BL21 competent cells were transformed with the recombinant vectors, and isopropyl-β-D-thiogalactopyranoside ( IPTG) was added to induce expression of Pla protein. The expressed protein was detected by SDS-PAGE electrophoresis.The inclusion bodies of Pla protein were denatured in 8 mol/L urea, and then refolded using gradient urea solutions.The purified protein was identified by SDS-PAGE electrophoresis and Western blot.Results and Conclusion The constructed expression vector was demonstrated to be correct through agarose gel electrophoresis and sequencing.The recombinant Pla protein was accumulated as an inclusion body in E.coli, and the overexpression product was mainly a target protein, the yield of which was very high.SDS-PAGE purity of the bioactive Pla protein was obtained by denaturing and refolding the inclusion bodies.This study provides a simple and quick method for highly efficient preparation of biologically active Pla protein.

Objective: To explore the antagonistic effect of quercetin on fine particulate matter (PM2.5)-induced embryonic developmental toxicity in vitro.Methods: PM2.5 was collected on glass fiber filters by PM2.5 samplers during the heating period of Dec.2015 to Mar.2016 in an area of Haidian District, Beijing City.The sampled filters were cut into 1 cm×3 cm pieces followed by sonication.The PM2.5 suspension was filtered into a 10 cm glass dish through 8 layers of sterile carbasus and stored at-80 ℃ until freeze drying.Frozen PM2.5 suspension was dried by vacuum freeze-drying.In vitro post-implantation whole embryo culture was used in this study.Pregnant rats with 9.5 gestation days (GD) were killed by cervical dislocation and the uteri were removed into sterile Hank's solution.The embryos with intact yolk sacs and ecto placental cones were induced by PM2.5, and then subjected to intervention of quercetin at the doses of 0.1 μmol/L, 0.5 μmol/L, 1.0 μmol/L and 5.0 μmol/L, respectively.At the end of the 48 h culture period, the cultures were terminated, and all embryos were removed from the culture bottles and placed in prewarmed Hank's solution for evaluation.Morphological evaluation of the embryos was conducted under a stereomicroscope using the morphologic scoring system by Brown and Fabro.The mitochondrial reactive oxygen species (ROS) level was detected by FACSCalibur flow cyto-metry using MitoSOXTM Red staining.Results: An obvious antagonistic effect was achieved through querce-tin at the dose of 1.0 μmol/L, which could result in an increase of visceral yolk sac (VYS) diameter, crown-rump length and head length, somite number, and the differentiation of visceral yolk sac vascular vessels.The scores of allantois, flexion, heart, hind brain, midbrain, forebrain, auditory system, visual system, olfactory system, branchialarch, maxillary process, forelimb bud and hindlimb bud also revealed a significant increase and the relative mitochondrial ROS level of embryonic cells was significantly decreased when compared with PM2.5 group.Although quercetin at the doses of 0.1 μmol/L, 0.5 μmol/L, 5.0 μmol/L also exhibited protective effects against PM2.5-induced embryonic developmental toxicity, the protective effect was weaker when compared with the dose of 1.0 μmol/L.Conclusion: Quercetin at proper dose may be of great benefit for the development of embryos exposed to PM2.5 in the uterus of the rats.Quercetin provides an effective strategy for the prevention of PM2.5-induced embryonic developmental toxicity.Clearance of mitochondrial ROS may be one of its mechanisms.

Objective To discussion the effect of endoscopic single nostril transsphenoidal pituitary tumor resection on improving clini-cal symptoms and related quality of life of patients. Methods Selected 114 cases of patients with pituitary tumors in our hospital from August 2013 to August 2015, and randomly divided them into the nasal endoscopic group and the microscopic group according to the random number table,with 57 patients in each group. Patients of the 2 groups were treated with nasal endoscopic and microscopic single nostril transsphenoi-dal pituitary tumor resection respectively. The operation situation,hormone decline,incidence of complications after surgery and SNOT-20 and VAS score of the two groups were compared. Results Operative time of nasal endoscopic group was significantly higher than that in the mi-croscopic group,whlie the blood loss,length of hospital stay in endoscopic group was significantly lower than the microscopic group,and the difference was statistically significant (P<0. 05). The total resection rate of pituitary tumor of the two groups were of no statistically signifi-cance (P>0. 05). One week after operation,cerebrospinal fluid cell count,cerebrospinal fluid protein,adrenocorticotropic hormone,serum chloride,sodium, growth hormone,and prolactin levels of the nasal endoscopic group were lower than the microscopic group (P<0. 05). The complication rate was 14. 9% in the nasal endoscopic group,which was obviously lower than 59. 6% in the microscopic group (P<0. 05). One week after operation,the SNOT-20 scores and VAS scores were increased in both of the two groups,but the microscopic group increased more significantly (P<0. 05). Conclusion Endoscopic single nostril transsphenoidal pituitary tumor resection surgery cost longer operation time,but it lead to less trauma and less postoperative complications,which may delay the decline of patients’ quality of life and promote the improvement of prognosis.

Objective To determine the incidence of avascular necrosis after closed reduction and cannulated compression screws fixation of intra-capsular femoral neck fracture and to investigate the multiple factors correlated to avascular femoral head necrosis.Methods All the patients of intra-capsular femoral neck fracture who accepted closed reduction and cannulated compression screws fixation between 2001 and 2010 in Jiangsu Province were reviewed in multi-centers retrospectively.The multiple factors were analyzed including age,gender,affected side,mechanism of injury,fracture classification,procedure delay,quality of reduction,time of full-weight-bearing,configuration of the screws and removal of the screws.Multiple correlation factors were analyzed with SPSS 13.0 statistic system.Results Complete case records were documented in 1849 cases who were followed up for an average 6.5±2.7 years (range,2-10 years).Avascular necrosis occurred in 246 cases (13.3％).The average time of diagnosis of avascular necrosis was 17±4.6months (range,8-72 months) after injury.The average Harris score of the hips which didn't develop to avascular necrosis was 93.8±8.9 (range,78～100) at the last follow up.Multiple correlation factors analysis indicated that displacement degrees of fracture and the quality of reduction were significantly correlated to the incidence of avascular necrosis (OR=2.078,3.423).Conclusion Closed reduction and cannulated compression screws fixation after the intra-capsular femoral neck fracture can get satisfactory results.Displacement degrees of fracture and the quality of reduction are significantly correlated to the incidence of avascular femoral head necrosis.