Objective To observe the effects of menopause on prothrombotic profiles in ovariectomized rats .Methods Thirty two healthy female SD rats of 9 to 10 months were divided into 4 groups, the control shamed and the observation groups ovariectomized .Rats in the baseline group and the early menopause group were sacrificed one week later , and the control and late menopause group 10 weeks later.The prothrombotic profiles were detected including plasma FIB , ATⅢ activity, PAI-1 levels, D-D level, vWF levels and NO concentration, TXA2 and PGI2 levels.Results In early menopause, plasma FIB increased dramatically while ATⅢactivity remained lit-tle changed.PAI-1 demonstrated an increasing trend .vWF significantly increased but NO significantly decreased .In later menopausal stage, PAI-1 increased dramatically but FIB somewhat decreased .Plasma ATⅢactivity significantly increased and vWF still remained a high level.NO increased a little.In both early and later stage, TXA2 and PGI2 simultaneously increased while D -D showed little change between groups .Conclusion Menopause plays different roles in different aspects of thromoembolism , resulting in increased risk in early menopause due to prothrombotic state and decreased risk in later menopause when new balances between profiles were established .

Objective To study effects of raloxifene (RLX) with different doses of conjugated equine estrogen(CEE) on prothrombotic profiles in the ovariectomized rats model.Methods Total of 32healthy female SD rats at age of 9 to 10 months were equally divided into every 8 rats at 4 groups randomly.One week after ovariectomized,they were treated by drugs,including control group with placebo(0.9％ Nacl intragastric administration),RLX group with RLX 6 mg/(kg · d),RLX and low CEE group with RLX 6 mg/(kg · d) + CEE 0.07 mg/(kg · d)and RLX and high CEE group with RLX 6 mg/(kg · d) + CEE 0.5mg/(kg· d)for 10 weeks before death.Thrombin turbidimetry method was used to evaluate the plasma fibrinogen(FIB),transmitting substrate method for antithrombin Ⅲ (AT Ⅲ) activity,double-antibody sandwich ELISA for plasminogen activator inhibitor 1 (PAI-1),D-dimer (D-D) and yon Willebrand factor (vWF) and nitrate reductase method for nitric oxide(NO).Results (1) Coagulation and anticoagulation indicators:it was observed (1.62 ± 0.22) g/L FIB at control group,(2.02 0.54) g/L at RLX group,(1.97 ±0.16) g/L at RLX and low CEE group,(2.00 0.18) g/L at RLX and high CEE group.There was a statistically significant difference between control group and any one of treatment groups(P ＜ 0.05) and no statistical significance among those three treatment groups (P ＞ 0.05).No significant change was observed in plasma AT Ⅲ activity among groups (P ＞ 0.05).(2) Fibrinolytic and anti-fibrinolytic indicators:it was observed (14.1 2.8) μg/L PAI-1 at control group,(20.0 ±3.3) μg/L at RLX group,(41.5 ±5.5) μg/L at RLX and low CEE group,(38.9 ± 6.0) μg/L at RLX and high CEE group.A remarkable increase was observed between control group and any one of treatment groups(P ＜0.05).But there was no significant difference of D-D among groups (P ＞ 0.05).(3) Endothelial function:it was (43 ± 7) ％ vWF at control group,(49±5)％ at RLX group,(46±6)％ at RLX and low CEE group,(36±5)％ at RLX and high CEE group.The vWF of RLX and high CEE group was the lowest among all groups (P ＜0.05).There was no difference of NO among groups (P ＞ 0.05).Conclusions In the different links of thrombosis,RLX gives different fuction and may increase the risk.CEE plays a synergism role in the matter of fibrinolysis and anti-fibrinolysis with RLX,further giving rise to thrombosis effect of RLX.And it also has a protective role in the function of vascular endothelium to some extent.But this only works with high dose.

Objective To determine the contents of organic acids in different processed products and different parts of Schisandra Chinensis Fruits by HPLC;To discuss the influence of different processing methods on contents of organic acids.Methods Ecosil C18-AQ Column (250 mm×4.6 mm, 5μm) was used with mobile phase of methanol-0.5% acetic acid (15∶85), at a flow rate of 0.8 mL/min, and the detection wavelength was 260 nm for protocatechuic acid;mobile phase of acetonitrile∶15 mmol/L potassium dihydrogen phosphate buffered saline solution=3∶97 (phosphoric acid adjusting pH=3), at a flow rate of 1.0 mL/min, and the detection wavelength was 210 nm for citric acid.Results The contents of protocatechuic acid and citric acid in Schisandra Chinensis Fruits, wine steaming and vinegar steaming Schisandra Chinensis Fruits were 0.0098%, 0.0124%, 0.0121% and 14.8293%, 14.1694%, 14.3650%, respectively. The contents of protocatechuic acid and citric acid in pulp, seeds were 0.0123%, 0.0090%, and 22.8810%, 3.8990%, respectively.Conclusion The protocatechuic acid was increased significantly after being processed, but citric acid showed a small change and had a slight downward trend. The two organic acids in pulp were higher than the two in seeds.

BACKGROUND:Recent studies have shown that icari n is a good bone-inducing factor that can promote the osteogenic differentiation of mesenchymal stem cel s, providing a new hope for the treatment of bone defects. OBJECTIVE:To review research achievements in pharmacological effects of icari n effects on bone tissue metabolism as wel as its effect to promote osteogenic differentiation of mesenchymal stem cel s. METHODS:The first author retrieved CNKI and PubMed databases for relevant Chinese and English literatures using keywords of“icariin, stem cell, osteogenesis”, respectively. Articles regarding icariin, stem cells, osteogenesis were included, and repetitive studies were excluded. Totally 754 articles were retrieved initially. In accordance with inclusion and exclusion criteria, 41 articles were included in result analysis. RESULTS AND CONCLUSION:As the main ingredient of Herba epimedi , icari n functions as a good osteogenetic growth factor to promote the osteogenic differentiation of bone marrow mesenchymal stem cel s. In recent years, icari n has been shown to promote adipose-, umbilical cord-, and periodontal ligament tissue-derived mesenchymal stem cel s to differentiate into osteoblasts. But such studies are less reported. Until now, mesenchymal stem cel s stil exhibit unsatisfactory osteogenic ability in in vivo experiments. Given this, osteogenetic growth factors contribute to the osteogenic differentiation of mesenchymal stem cel s. Therefore, the use of icari n is expected to provide a good strategy for bone defect repair.

Abstract Objective:FSM-2117 and FS-5416 are two bivalent hybrid strains of S.flexneri and S.sonnei, constructed by this laboratory-The FS-5416 expresses four invassive plasmid antigens(IpaA、IpaB、IpaC and IpaD) and has a good contact heamolysis activity(CHA+ ), butFSM-2117 hasn' t. To observe the immunogenicity of Shigelk vaccines through three different mucosal administration rout, the changes of ASCin the spleen and Peyer's patch(PP) , mesenteric lymph nodes(MLN) lymphocytes are detected.Methods:BALB/c mice were divided intothree groups, 20 mice per group. Mice were immunized respectively with the Ipa+ or Ipa- vaccines(4 x 10~7 CFU) three times with an intervalof two weeks by intranasal、intragastric or intraintestinal administration routs. The spleen , PP , MLN lymphocytes were isolated of seventh dayat random after immunization. An BA-EIISASPOT was done to account the numbers of ASC.Results:The numbers of SIgA and SIgG ASC ofspleen , PP , MLN lymphocytes of intranasal and intraintestinal group were significantly increased . Significant difference were only observed inthe number of spleen lymphocytes SIgA and SIgG ASC of intranasal group between Ipa+ and Ipa- . Conclusion:Two bivalent Shigelk vaccinescan induce spleen and GALT immunity reaction by nasal or small intestinal mucosal in a low dosage compared with intragastric rout (about 1/20). Ipa can significantly increased the number of spleen lymphocytes SIgA and SIgG ASC of intranasal group.

Objective To construct the red fluorescent protein reporter gene vector containing high mobility group box 1 protein(HMGB1) promoter sequence and study the regulation mechanism of the expression of HMGB1gene under mechanical stretch.Methods HMGB1 promoter was subcloned into a red fluorescent protein vector,pDsRed1-1.After identified by PCR,enzyme digestion and DNA sequencing,the recombinant vector pDsRed1-1-HMGB1P was then transfected into HEK293 cells.Blank vector or pDsRed-1 was transfected into 293 cells and served as controls.The expression of red fluorescent protein and its reaction to mechanical stretch were observed under a fluorescent microscope.HEK293 cells transfected with pDsRed1-1 vector served as control.Results PCR,double restriction enzyme digestion and DNA sequence analysis showed that the recombinant vector,pDsRed1-1-HMGB1P,was constructed correctly.This vector was lowly expressed in HEK293 cells of resting state.But after stimulated by mechanical stretch,strong red fluorescence was observed.No red fluorescence was observed in the control cells.Conclusion A red fluorescent protein reporter gene vector containing HMGB1 promoter sequence has been constructed successfully and expressed highly in mammalian cells.Since it responds to mechanical stretch effectively,it can thus provide a convenient tool to study the regulation mechanism of the expression of HMGB1 gene by mechanical stress.

Objective In order to investigate the roles of hIGF\|1 in treatment of diabetes mellitus and diabetic syndromes,the gene of human insulin like growth factor type Ⅰ(IGF\|1) was cloned and constructed into eukaryotic expression vector,then the expression and activity were determined. Methods Total cellular RNA of human fetal liver was abstracted and the RT\|PCR amplification of the cDNA fragment was performed.The fragment was cloned into pUCM\|T vector and sequenced.The eukaryiotic expression vector was recombined and transfected into fibroblast cell line,COS\|7.The expression of hIGF\|1 was examined by in situ hybridization and immunohistochemistry.The effect of hIGF\|1 on cultured islet cells was observed by glucose\|stimulated insulin release assay. Results The cDNA fragment of 710bp with additional Kozak sequence was amplified by RT\|PCR.Eukaryiotic expression vector pCI\|neo/hIGF\|1 was constructed and IGF\|1 gene expressed in COS\|7.The biological activity of hIGF\|1 was proved by increasing inslin secretion from islet cells.Conclusion\ The newly constructed vector,pCI\|neo/hIGF\|1 could be transfected into COS7 cells and its expressed product showed to have the biology activity of hIGF\|1.\;[

AIM: To screen the binding proteins to HMGB1 promoter by phage display technique. METHODS: HMGB1 promoter was incubated with phage display library. Unbound phages were eluted and phages bound to HMGB1 promoter were amplified. Twenty individual clones were randomly selected and identified by enzyme-linked immunosorbent assay (ELISA). Positive clones were characterized by DNA sequencing and the sequences were subjected for computer analysis. RESULTS: Positive phages binding to HMGB1 promoter were enriched after 4 rounds of biopanning. Twenty phage clones were selected and eleven clones of which were identified to bind specifically to HMGB1 promoter. The sequences in full length were obtained and searched for homologous sequences from GenBank. Altogether eight coding sequences were obtained, six of which were known proteins including activator protein-1(AP-1) and two of which were uncharacterized ones. CONCLUSION: Several proteins were obtained that bind specifically with HMGB1 promoter. The results will be useful for further studying the expression and regulation mechanism of HMGB1.

Objective To investigate the role of extracellular regulated protein kinase (ERK) signal pathway in mechanical stretch induced high mobility group box 1 protein (HMGB1) expression on alveolar epithelial cells (A549). MethodsA549 cells were cultured and seeded at 1×10~5 cells/ml in 6-well Bioflex cell culture plates. Subsequently, the cells were exposed to cyclic mechanical stretch at 14% (group B) elongation for 4 hours using Flexercell 4000T cell stretching unit. In group C, cells were pretreated with PD98059 for 2 hours before mechanical stretch. Cells in group A without stretch were served as control. The expression of HMGB1 protein and mRNA in A549 cells were detected by immunocytochemisty staining and RT-PCR, respectively. ERK activity was measured by Western blotting method. Results Immunocytochemisty staining indicated that the expression of HMGB1 protein in A549 cells was increased obviously in group B (P<0.05) and decreased in group C (P<0.05). Polymerase chain reaction (RT-PCR) showed that the expression of HMGB1 mRNA was also significantly increased in group B (P<0.05) and decreased in group C (P<0.05). Western blotting analysis confirmed the activation of ERK in A549 cells by mechanical stretch (P<0.05). PD98059, an inhibitor of ERK, might significantly inhibit mechanical stretch induced HMGB1 protein and mRNA expression in A549 cells (P<0.05). Conclusion Mechanical stretch could regulate the expression of HMGB1 gene and protein in A549 cells through ERK signal pathway.

This study is to investigate the therapeutic effect and mechanism of indole-3-carbinol (I3C) on pig serum-induced liver fibrosis of rats. The liver fibrotic model of rats was induced by pig serum. After models were successfully established, rats in the treatment groups were administered with I3C through intraperitoneal injection or curcumin by intragastric administration, daily for 17 days. Hepatic hydroxyproline (Hyp) content was measured. The liver histology and immunohistochemistry with a-smooth muscle actin (alpha-SMA) were assayed. Hepatic stellate cells line, HSC-T6 was incubated with different concentrations of I3C (25, 50, and 100 micromol x L(-1)) for 24 h. The effect of I3C on cell apoptosis was identified by FITC-Annexin V/PI double labeled assay. And the mRNA expressions of Bax and Bcl-2 were measured by real time RT-PCR. The results showed that hepatic content of Hyp decreased by I3C treatment, as compared with the fibrotic model control. Histopathological changes, such as steatosis, necrosis, deposition of collagenous fiber reduced remarkably and the expression of alpha-SMA was significantly down-regulated in the I3C-treated groups (P < 0.01). Apoptosis analysis showed that I3C significantly increased HSC-T6 apoptosis rate and the expressional ratio of Bax to Bcl-2. The results indicated that I3C could effectively cure pig serum-induced liver fibrosis in vivo by inducing HSC apoptosis and promoting ECM degradation.