OBJECTIVE:To study the influencing factors for the extraction of the genomic DNA from Paeonia Suffruticosa.METHODS:Taking Paeonia Suffruticosa(root bark of Chinese medicinal herb) as material to investigate the influencing factors including concentrations of the NaCl and beta-mercaptoethanol,temperature and time of water bath,RNaseA,PCR(polymerase chain reaction) system etc in the buffer solution on the basis of modified CTAB method.RESULTS:The DNA obtained by modified CTAB method was pure,integrated,with the value of A260/A280 ranged from 1.8 to 2.0,the ampl-ified bands of PCR were clear and bright,which lay a solid foundation for the following molecular biology experiments.CONCLUSION:The modified CTAB method is economical,rapid and efficient,and it can be served as an extraction of genomic DNA from root bark Chinese medicinal herb as well as a theoretical basis for full scale production.

OBJECTIVETo explore the molecule mechanism of Salidroside inducing directional differentiation of mouse mesenchymal stem cells (MSCs) into neuronal cells.

METHODSThe mouse multipotent mesenchymal precursor cell line (D1) was taken as the objective. Cultured MSCs were divided into the negative control group (complete culture solution), the positive control group (containing 1 mmol/L β-mercaptoethanol), the Salidroside induced group (20 mg/L Salidroside), and the blocked group (20 ng/ ml DKK1, a special inhibitor of Wnt/β-catenin signal pathway). All cells were inoculated in a 6-well plate (1 x 10(4) cells/cm2) and grouped for 24 h. The expression of p-catenin was detected by fluorescence Immunochemistry in the negative control group, the positive control group, and the Salidroside induced group. The expression of neuron-specific enolase (NSE), beta 3 class III tubulin (β-tubulin III), nuclear receptor related factor 1 (Nurr1), glial fibrillary acidic protein (GFAP) mRNA, Wnt3a, β-catenin, low-density lipoprotein receptor-related protein6 (LRP6), Axin mRNA were detected using reverse transcrip- tion PCR (RT-PCR). The expression of β-catenin and NSE protein were analyzed by Western blot in the negative control group, the positive control group, and the Salidroside induced group. Ca2+ chelating agents (EGTA), L-type Ca2+ channel blocker (Nifedpine), and IP3Ks special inhibitor (LY294002) were used to block Ca2+ signal pathway respectively. The expression of Wnt3a, LRP-6, Axin, glycogen syn- thase kinase (GSK-3), and β-catenin mRNA were detected by RT-PCR. The β-catenin protein expression was analyzed using Western blot.

RESULTSCompared with the positive control group, β-catenin protein was strong positively expressed; the expression of Wnt3a, β-catenin, LRP6, Axin, NSE, β-tubulin III, Nurr1 mRNA, and NSE protein were obviously up-regulated in the Salidroside induced group (P < 0.01). Compared with the positive control group and the Salidroside induced group, β-catenin, NSE, Nurr1, and β-tubulin III mRNA expression decreased; β-catenin and NSE protein expression were also down-regulated in the blocked group (P < 0.01). Compared with the Salidroside induced group, the expression of Wnt3a, LRP-6, β-catenin, and Axin mRNA were down-regulated in the Ca2+ signal blocked group and the salidroside induced group (P < 0.01, P < 0.05).

CONCLUSIONSalidroside affected directional differentia- tion of MSCs into neuronal cells through Wnt/β-catenin and Ca2+ signal pathway.

Animals ; Cell Differentiation ; drug effects ; Glucosides ; pharmacology ; Glycogen Synthase Kinase 3 ; Lipoproteins, LDL ; Low Density Lipoprotein Receptor-Related Protein-6 ; Mesenchymal Stromal Cells ; physiology ; Mice ; Neurons ; Phenols ; pharmacology ; Phosphopyruvate Hydratase ; RNA, Messenger ; Signal Transduction ; Wnt Signaling Pathway ; physiology ; beta Catenin ; metabolism

Objective To investigate the distribution of pathogenic bacteria,drug sensitivity,and the relationship between drug sensitivity and Escherichia coli(E.coli) induced enzymes in childhood diarrhea in the last 2 years in Chongqing area,so as to provide important evidence for pediatric clinical therapy.Methods Thirty-one E.coli induced enzymes,extended spectrum ?-laetamases(ESBLs),cephalosporinase(AmpC)detected in different phenotype methods,and drug sensitivity was measured in paper strip method,and the specimens were collected from children′s hospital affiliated to chongqing university of medical sciences from Jan.2005 to Dec.2006 were determined.Among the total,there were 18 enteropathogeic E.coli(EPEC) strains,8 enterotoxigenic E.coli(ETEC) strains and 5 enteroinvasive E.coli(EIEC) strains.In addition,drug resistance tests by paper strip included chloramphenicol(CHL),amikacin(AMK),gentamicin(GEN),norfloxacin(NOF),ciproflocacin(CIP),cefazolin(CEZ),cefoperazone(CPZ),ceftriaxone(CRO),ceftazidime(CAZ),cefotacime(CTX),cefepime(FEP),imipenem(IPM).SPSS 12.0 software was used to analyze the data.Results Three point two percent of the 31 E.coli were drug resistant to IPM,and 35.5%,38.7% to NOF,CIP individually,but more than 60% to AMK,GEN,even more than 67.7% towards cephalosporin(except ceftazidime and cefepime);the gross enzyme-produced rate was 87.1%,rate of single ESBLs,AmpC,and induction of both enzymes simultaneously presented 64.5%,6.5%,16.1% respectively;and there was marked difference in drug resistance when bacteria that produced single AmpC versus bacteria that produced single ESBLs or that produced both ESBLs and AmpC(Pa﹤0.05).Conclusions The relationships among enzyme′s quantity,sort and bacterial resistance are different.These data show E.coli infected by bacterial diarrhea children in Chongqing due to a high rate of induced enzymes,and their drug resistance vary according to the state of induced enzymes.

OBJECTIVETo explore the clinical efficacy of self-made Kirschner wire hook in the treatment of severely displaced proximal humerus fractures in children.

METHODSFrom January 2007 to February 2012,35 children with severely displaced proximal humerus fractures were treated with self-made Kirschner wire hook fixation,including 25 boys and 10 girls with an average age of 13.2 years old ranging from 5 to 17. The duration from injury to operation was 1 to 10 days with an average of 4.5 days. Preoperative diagnoses were confirmed by X-ray films as type III in 26 cases and type IV in 9 according to the Neer-Horwitz classification of the proximal humerus fractures. All fractures were close fracture without nerve or vascular injuries. Anatomical reduction was achieved by open reduction and the fractures were fixed by self-made Kirschner wire hook. Intra-operative and postoperative complications, postoperative radiographic examination, upper extremity length and range of shoulder motion were observed and recorded. Neer score system was used to evaluate shoulder function.

RESULTSAll patients were followed up from 6 to 18 months with an average of 12.1 months. The mean Neer score of the injured side was 94.2 +/- 4.8 (ranged, 84 to 99), 30 patients got an excellent result and 5 good according to the Neer scores. The X-ray showed all the fractures healed without shortening deformity or epiphyseal arrest at early stage. There were no complications related to the implant including loosening or breakage of the self-made Kirschner wire hook. The range of shoulder motion in the injuried side was similar to the normal side, except in abductor lift. All the patients could participate in normal physical activities.

CONCLUSIONThe method of open reduction and fixed with self-made Kirschner wire hook is a safe,effective,convenient treatment for severely displaced proximal humeral fractures in children.

Adolescent ; Bone Wires ; Child ; Child, Preschool ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Humeral Fractures ; physiopathology ; surgery ; Male ; Range of Motion, Articular

Objective To investigate the methods of reducing radiation dose in CT coronary angiography through optimizing individualized scan dosage protocol.Methods Two hundred patients (group A)underwent coronary CTA examination which was performed with fixed 120 kV and variable mA according to their BMI.The mA was set as 150-300 mA(BMI ＜ 18.5 kg/m2),300-500 mA (18.5 kg/m2 ≤ BMI ＜ 25.0 kg/m2),and 500-800 mA(BMI ≥ 25.0 kg/m2).When all examinations were finished,a linear regression was employed to analyze the correlation between mA and BMI,body surface(Suf),image noise(SD)respectively.The results of the analysis were used to formulate a regression equation,which was further used to establish a table list for quick search on how much mA that individualized coronary CTA scan would need.Another 200 patients(group B)enrolled for the individualized scan were scanned under new protocol that previous study established.The tube voltage was 100 and 120 kV.The tube current was variable according to the data in the table list.One-way ANOVA and Kruskal-wallis H test were used for statistics.Results Regression equation between mA and BMI,Suf,SD was:mA =17.984 × BMI + 169.149 × Suf-2.282 × SD-361.039.The SD(group A:32.08 ± 5.80,group B:28.60±4.47),dose index volume(CTDIvol)[group A:(41.97 ± 11.37)mGy,group B:(33.18±10.07)mGy],effective dose(ED)[group A:(10.91 ±3.07)mSy,group B:(8.83 ±2.72)mSv]had significant differences between the two groups(F =43.45,63.71,49.07 respectively,P ＜0.01 for all).The SD and ED results obtained in group B were better than those in group A.Conclusion Better performances were obtained when BMI combined Suf was used as a new individualized protocol than when BMI was used only,which means good image quality and lower radiation dosage in coronary CTA examination.

The antitussive activity assay for the root extraction of Disporum cantoniense was carried out with coughing mice induced by ammonia liquor. The results showed that the ethanol and water extractions of D. cantoniense possess strong antitussive activity, and the high dose of the former was better than positive control, and then the constituents of the ethanol extraction were separated and purified by various modern chromatographic techniques. Their structures were identified by physico-chemical properties and spectroscopic data. As a result, eight compounds were isolated and identified as stigmast-4-en-3-one(1), (22E, 24R)-ergosta-5, 7, 22-trien-3beta-ol(2), obtucarbamate A(3), obtucarbamate B(4), neotigogenin(5), azo-2, 2'-bis[Z-(2,3-dihydroxy-4-methyl-5-methoxy) phenyl ethylene] (6),dimethyl {[carbonylbis (azanediyl)] bis( 2-methyl-5, 1-phenylene) j dicarbamate (7) , and quercetin-3-O-pB-D-glucopyranoside(8). All compounds were isolated from this plant for the first time, and the result of bioactivity-directed isolation showed that compounds 3, 4, and 6 had obvious effect on antitussive activity, and compound 6 had the same level as positive control.
Animals ; Antitussive Agents ; chemistry ; isolation & purification ; pharmacology ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Ethanol ; chemistry ; Female ; Liliaceae ; chemistry ; Male ; Mice

Objective To compare 99Tcm-MIBI MPI with delayed enhancement MRI (DE-MRI) in patients with idiopathic dilated cardiomyopathy (IDCM). Methods Forty patients with IDCM were included. They underwent both rest 99Tcm-MIBI myocardial perfusion imaging and DE-MRI within 7 days. 99Tcm-MIBI MPI was performed to identify diffuse or segmental abnormal perfusion patterns including reduced or defect perfusion segments. DE-MRI images were divided into 4 categories: no delayed enhancement, septal, subendocardial and transmural delayed enhancement, x2 test was used for data analysis. Results Diffuse and segmental perfusion abnormality on 99Tcm-MIBI MPI were found in 19 (47.5%) and 21 (52.5%)patients respectively, while DE-MRI enhancement was simultaneously found in 5 patients of the former (5/19, 26.3%) and 18 (18/21, 85.7%) of the latter (x2 =14.401, P＜0. 001). For those (n=18) with both segmental perfusion abnormality and DE-MRI enhancement, the number of segments of the 4 DE-MRI respectively. A significant difference was found in the DE-MRI enhancement categories between normal and defect perfusion segments (x2 = 29. 183, P ＜0.001 ) and between reduced and defect perfusion segments as well (x2 =25. 110, P＜0. 001). Conclusions Both diffuse and segmental perfusion abnormalities on 99Tcm-MIBI MPI can be found in patients with IDCM. DE-MRI enhancement is more frequently found in patients with segmental perfusion abnormality.

OBJECTIVETo observe changes of gonad functions of Gan-qi stagnation (GS), Pi deficiency (PD), Gan-qi stagnation Pi deficiency (GSPD) model rats, and the effect of Chaishu Sijun Decoction (CSD) on them.

METHODSRats were randomly divided into 7 groups according to romdom digit table, i.e., the normal control group, the GS model group, the GS medication group, the PD model group, the PD medication group, the GSPD model group,and the GSPD medication group, 10 in each group. Rats in the GS model group, the PD model group, and the GSPD model group were treated with chronic restraint, improper diet +excessive fatigue, chronic restraint +improper diet +excessive fatigue. The model was established for 4 successive weeks. Starting from the 15th day of modeling, CSD at the daily dose of 3.57 g/kg was given by gastrogavage to them for 14 successive days. Equal volume of distilled water was given by gastrogavage to rats in each model group and the normal control group for 14 successive days. The blood contents of gonadotrophin releasing hormone (GnRH), follicular stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and testosterone (T) were detected in rats of each group.

RESULTSCompared with the normal control group, there was statistical difference in GnRH, T, E,, and FSH in the GS, PD, and GSPD model groups (P < 0.05, P < 0.01). The content of LH was elevated in the GS model group (P < 0.05) and declined in the GSPD model group (P < 0.01). Compared with the GS model group, the contents of FSH, LH, and T decreased and E2 increased in the PD model group (all P < 0.05); the contents of FSH and LH also declined in the GSPD model group (P < 0.05). Compared with the PD model group, the T content increased and FSH decreased in the GSPD model group (all P < 0.05). Compared with each corresponding model group, the FSH content decreased (P < 0.01) and LH increased in the GS medication group; the T content increased, E2 and LH decreased (P < 0.05, P < 0.01) in the PD medication group; the T content decreased (P < 0.01), GnRH, E2, FSH, and LH increased (P < 0. 05, P < 0.01) in the GSPD medication group.

CONCLUSIONSThere exist different degrees of abnormal function of the gonad axis in the GS, PD, and GSPD models. CSD had certain regulatory effect on the 3 syndromes. Of them, it showed a more comprehensive role in improving the gonad function axis. Results of this experiment had provided the experimental evidence for higher correlation between CSD and GSPD syndrome.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Gonadal Steroid Hormones ; blood ; Gonads ; drug effects ; Male ; Rats ; Rats, Wistar

OBJECTIVETo explore the effect of qidong huoxue decoction (QHD) on inflammatory factors and Toll-like receptor (TLR4) mRNA expressions in acute lung injury (ALI) rats.

METHODSTotally 50 healthy male SD rats were randomly divided into the blank control group, the lipopolysaccharide (LPS) model group, low, middle, high dose QHD groups according to body weight, 10 rats in each group. Rats in low, middle, high dose QHD groups were intragastrically administered with QHD at 4, 8, and 16 mL/kg 24, 12 h before modeling and 12 h after modeling, respectively. Normal saline was intragastrically administered to rats in the blank control group and the LPS model group. An ALI rat model was established using intratracheal instillation of LPS. Rats were killed after 24-h modeling. Then the bronchoalveolar lavage fluid was prepared. Contents of TNF-α, IL-1β, and L-10 were detected using ELISA. TLR4 mRNA expressions were determined byreal time PCR.

RESULTSCompared with the blank control group, contents of TNF-α, IL-1β , and IL-10 increased (P <0. 01), TLR4 mRNA expressions also increased in the LPS model group (all P <0. 01). Compared with the LPS model group, contents of TNF-α and IL-1β decreased (P <0. 05, P <0. 01), IL-10 levels increased (P <0. 01) , TLR4 mRNA expressions were also reduced (P <0. 01), in high and middle dose QHD groups. Compared with the high dose QHD group, con- tents of TNF-α and IL-1β increased in middle and low dose QHD groups (P <0. 05); IL-10 levels decreased (P <0. 05) in the low dose QHD group(P <0. 05), TLR4 mRNA expressions also increased in the low dose QHD group (P <0. 05). Compared with the middle dose QHD group, IL-10 levels was reduced, but TLR4 mRNA expressions increased in the low dose QHD group (P <0. 05).

CONCLUSIONSQHD had the protective effect on LPS induced ALI rats. Its mechanism might be associated with inhibiting TLR4 mRNA expressions, leading to decreased pro-inflammatory cytokines such as TNF-α and IL-β, elevated anti-inflammatory cytokine IL-10, and thereby, correcting unbalanced inflammation.

Acute Lung Injury ; genetics ; metabolism ; Animals ; Anti-Inflammatory Agents ; Bronchoalveolar Lavage Fluid ; Drugs, Chinese Herbal ; pharmacology ; Inflammation ; Interleukin-10 ; metabolism ; Interleukin-1beta ; metabolism ; Lipopolysaccharides ; Male ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDCorneal neovascular leakage can lead to edema and secondary scarring. Previous studies have shown that pericytes play a key role in maturation of angiogenesis. The present studies investigate the relationship between vascular permeability and pericyte coverage of endothelial cells in rat corneal neovascular induced by alkali burns.

METHODSCorneal neovascular vessels induced by alkali burns was performed in Sprague-Dawley rats. Corneas were excised on 1, 2, 3, 5, 7 and 10 days after cauterization. The vascular permeability rate was measured by the Evans blue method. The microvessel pericyte coverage index (MPI) was applied to quantify the pericyte coverage through double immunofluorescent staining of frozen sections of corneas with CD31 as the endothelial and alpha-smooth muscle actin (alpha-SMA) as the pericyte markers. The correlation between permeability rate and MPI was analyzed. Pericyte coverage was confirmed ultrastructually using transmission electron microscopy.

RESULTSThe vascular permeability rate was (1.14 +/- 0.17), (0.24 +/- 0.08), (0.29 +/- 0.16), (0.14 +/- 0.10), (0.09 +/- 0.06) and (0.05 +/- 0.04) microg x ml(-1) x mm(-2) respectively on 1, 2, 3, 5, 7 and 10 days after cauterization. The MPI was 0, 16.07%, 11.95%, 43.84%, 73.97% and 86.21% respectively at the above mentioned time points. The correlation coefficient between MPI and the permeability rate was -0.943 (P = 0.005).

CONCLUSIONSPericyte recruitment was significantly correlated with the permeability of corneal neovascularization induced by alkali burns in rats. Therapeutic strategies aiming at anti-leakage should be most effective if they promote pericytes proliferation in the course of corneal neovascularization.

Alkalies ; Animals ; Burns, Chemical ; physiopathology ; Capillary Permeability ; Cell Movement ; Cornea ; blood supply ; ultrastructure ; Corneal Neovascularization ; physiopathology ; Eye Burns ; chemically induced ; physiopathology ; Female ; Fluorescent Antibody Technique ; Pericytes ; physiology ; Rats ; Rats, Sprague-Dawley