AIM: To observe the changes of plasma content of Glu-plasminogen (plasmin, Pln) in patients during blood coagulation and/or fibrinolytic system activation. METHODS: Using specific McAb to antigenic determinant in NH 2 terminal (1-65 aa) of Pln and specific McAb to antigenic determinant in heavy chain of plasminogen, the sandwiched ELISA method was established to detect Glu-plasminogen and total plasminogen in human plasma collected from 220 normal controls and 40 patients after heart surgery. RESULTS: The average total plasminogen was (231.8?62.1) mg/L and average Glu-plasminogen was (231.9?45.8) mg/L in 220 normal controls, the ratio of Glu-plasminogen to total plasminogen (G/P) was 0.91. The ratio of Glu-plasminogen [(152.4?68.1) mg/L] to total plasminogen [(268.9?73.3) mg/L] in 40 patients after heart surgery was significantly lower than that in normal controls ( P

Objective To produce anti-thrombomodulin antibodies.Methods Using genetic immunization: Eukaryotic expression plasmid pcDNA3.1/TM(LEO),encoding all the extracellular domain of human thrombomodulin and signal peptides but lacking the transmembrance and cytoplasmic domains was constructed, which recombinant thrombomodulin was secreted soluble product. The plasmid was isolated from large-scale bacterial cultures by treatment with alkali and SDS, purified by precipitation with polyethylene Glycol (PEG). Recombinant plasmid was injected into tibial muscle of BALB/c mice. The productions of TM and anti-TM have been detected. Results The positive of RT-PCR and expressed TM identified the function of the recombinant plasmid. The pcDNA3.1/TM(LEO) induced higher titer of anti-TM. The antibody titer peaked between the 5th and 7th injection with a titer of 1∶8 000 detected by cell-ELISA coated with EVC-304. Specificity has been identified by western blot and immunohistochemistry.Conclusion The production of antibody through genetic immunization was a feasible method due to the difficulties in obtaining and purification of natural thrombomodulin.

This study investigated the protective effect of ATP on skeletal muscle satellite cells damaged by H2O2 in neonatal rats and the possible mechanism. The skeletal muscle satellite cells were randomly divided into four groups: normal group, model group (cells treated with 0.1 mmol/L H2O2 for 50 s), protection group (cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h, and then with 0.1 mmol/L H2O2 for 50 s), proliferation group (cells treated with 16, 8, 4, 2, 1, 0.5, or 0.25 mmol/L ATP for 24 h). MTT assay, FITC+PI+DAPI fluorescent staining, Giemsa staining and immunofluorescence were performed to examine cell viability and apoptosis, and apoptosis-related proteins. The results showed that the survival rate of skeletal muscle satellite cells was decreased and the apoptosis rate was increased after H2O2 treatment (P<0.01). Different doses of ATP had different effects on skeletal muscle satellite cells damaged by H2O2: the survival rate of muscle satellite cells treated with ATP at 4, 2, or 1 mmol/L was increased. The protective effect was most profound on cells treated with 2 mmol/L ATP. Immunofluorescence showed that ATP could increase the number of Bcl-2-positive cells (P<0.01) and decrease the number of the Bax-positive cells (P<0.01). It was concluded that ATP could protect skeletal muscle satellite cells against H2O2 damage in neonatal rats, which may be attributed to the up-regulation of the expression of Bcl-2 and down-regulation of Bax, resulting in the suppression of apoptosis.

Objectives To investigate and evaluate the preventive and therapeutic effects of celecox ib,a selective cyclooxygenase-2(COX-2)inhibitor,on multiple colorectal adenorna and compare it with aspirin.Methods Ninty-six patients with colorectal multiple adenoma were randomly divided into A,B and C groups.Adenomas in all patients were removed with high-frequency eleetrocoagulation,electroexci- sion or argon plasma coagulation(APC)under colonoscopy.Then,group A were administered celecoxib 200 mg twice daily,group B aspirin 50 mg twice daily,group C served as control.Colonoscopy was per formed every 6 months in the first year,and every year in order to observe and evaluate the recurrence rate of adenoma and the side effects after the treatment.Results Twenty-seven patients in group A,26 pa- tients in group B and 27 patients in group C had completed the treatment.At the end of the treatment, on PP/ITT analysis,the cure rate of the eolorectal adenoma were 84.4%/100% ,78.1%/96.2% and 75.0%/88.9% in group A,B and C,respectively.During the first year of follow-up,there were 1 ,1 and 6 cases which were found recurrences of the adenomas in group A,B and C,respectively.The recurrence rates of coloreetal adenomas in group A(3.7%)and group B(4.0%)were significantly low er than that in group C(24.0%) (P＜0.05 and＜0.05,respectively).At the end of follow-up,the total recurrence rate of colorectal adenomas in group A(14.80%)and group B(19.2%)were significant- ly lower than that in group C(46.2%)(P＜0.05 and＜0.05).While the side-effective rate regroup A (3.3%)was significantly lower than that in group B(22.5%)(P＜0.05).Conclusions After re- section of the multiple colorectal adenomas,both the selective inhibitor of COX-2,celeeoxib and the non- selective inhibitor of COX-2,aspirin,may reduce its recurrence rate,but the former has a good tolerance and lower side-effects.

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress (ER stress) in endothelial cells stimulated with cigarette smoke extract (CSE).Human umbilical vein endothelial cells (HUVECs) were cultured and divided into 3 groups:CSE-stimulated group,CSE-stimulated with 4-PBA group,and negative control group.HUVECs were cultured and stimulated with CSE at concentrations of 5％,10％ and 20％,respectively,mRNA of CXCL-8 and GRP78 was detected by real-time PCR.ELISA was performed to test the expression of CXCL-8 protein,and neutrophils migration was detected by Transwell board test.The NF-κB,ERK,p38MAPK and transforming growth factor beta (TGF-β) were detected by flow cytometry.The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs (P＜0.05).Furthermore,it was concentration-dependent.4-PBA significantly reduced the expression of CXCL-8 protein (P＜0.05) and neutrophil migration (P＜0.05).The TGF-β,rather than the NF-κB,ERK and P38MAPK pathway might be involved in ER stress stimulated by CSE.CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells.ER stress might play a role in the effect of neutrophils migration stimulated with CSE,and TGF-β pathway may contribute to the ER stress in HUVECs.

Seashore landfill aquifers are environments of special physicochemical conditions (high organic load and high salinity), and microbes in leachate-polluted aquifers play a significant role for intrinsic bioremediation. In order to characterize microbial diversity and look for clues on the relationship between microbial community structure and hydrochemistry, a culture-independent examination of a typical groundwater sample obtained from a seashore landfill was conducted by sequence analysis of 16S rDNA clone library. Two sets of universal 16S rDNA primers were used to amplify DNA extracted from the groundwater so that problems arising from primer efficiency and specificity could be reduced. Of 74 clones randomly selected from the libraries, 30 contained unique sequences whose analysis showed that the majority of them belonged to bacteria (95.9%), with Proteobacteria (63.5%) being the dominant division. One archaeal sequence and one eukaryotic sequence were found as well. Bacterial sequences belonging to the following phylogenic groups were identified: Bacteroidetes (20.3%), β, γ, δ and ε-subdivisions of Proteobacteria (47.3%, 9.5%, 5.4% and 1.3%, respectively), Firmicutes (1.4%), Actinobacteria (2.7%), Cyanobacteria (2.7%). The percentages of Proteobacteria and Bacteroides in seawater were greater than those in the groundwater from a non-seashore landfill, indicating a possible influence of seawater. Quite a few sequences had close relatives in marine or hypersaline environments. Many sequences showed affiliations with microbes involved in anaerobic fermentation. The remarkable abundance of sequences related to (per)chlorate-reducing bacteria (C1RB) in the groundwater was significant and worthy of further study.

OBJECTIVE: To observe the expression pattern of caspase-3 and HCLS1-associated protein X-1 (HAX-1) at different time after cerebral contusion in rat, and explore the new method for estimating the injury interval. METHODS: The cerebral contusion model was established using adult SD male rats. Then the rats were randomly allocated into 8 groups: 2 h, 6 h, 12 h, 1 d, 3 d, and 7 d after cerebral contusion, sham-operation and normal control. Expression of caspase-3 and HAX-1 protein after cerebral contusion in rat was detected by Western blotting. Laser scanning confocal microscope was used to observe the number of HAX-1 positive cells and TUNEL-stained cells after cerebral contusion. RESULTS: The expression of caspase-3 increased parallelly with the time after cerebral contusion and reached the peak value on 3 d. The expression of caspase-3 decreased gradually and still maintained a high level expression on 7 d (P < 0.05). The expression of HAX-1 positive cell went up after injury, and reached the peak value at 6 h (P < 0.05), then turned down gradually after 12 h and went out of detection after 3 d. The number of TUNEL-stained cells increased obviously at 2 h and reached the peak value on 3 d. The number of TUNEL-stained apoptotic cells decreased gradually and still maintained a high level expression on 7 d (P < 0.05). CONCLUSION The expression of caspase-3 and HAX-1 after cerebral contusion has time sequential regularity, which may provide new evidence for forensic diagnosis of cerebral contusion interval.
Animals ; Blotting, Western ; Brain Injuries/pathology* ; Carrier Proteins/metabolism* ; Caspase 3/metabolism* ; Cerebellum/pathology* ; In Situ Nick-End Labeling ; Intracellular Signaling Peptides and Proteins ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of serum containing Gumibao (Chinese character: see text) on the proliferation and differentiation of osteoblast induced by dexamethasone.

METHODSOsteoblasts were extracted from skulls in newly born (within 24 hours) SD rats, and digested with collagenase. The first passage of cells were used for experiments. Cells were cultured in the medium containing different concentrations of dexamethasone (0, 10(-8), 10(-7), 10(-6), 10(-5) ,10(-4) mol/L). Alkaline phosphatase staining were carried out after 1 week and numbers of mineralized nodes with alizarin red staining were observed after 3 weeks. Accordingly, following the treatment of 10(-5) mol/L dexamethasone for 1 week, cells were cultured in the medium with serum containing Gumibao (Chinese character: see text). One week after Cumibao (Chinese character: see text) treatment, cells were stained with Alkaline phosphatase and collagen I and PCNA were examined by Western-blot. However, the observation of numbers of mineralized nodes with alizarin red stain required one more week.

RESULTSHigh concentration of dexamethasone could inhibit the expression of PCNA, collagen I, alkaline phosphatase and reduce the number of mineralized nodes of osteoblast, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

CONCLUSIONHigh concentration of dexamethasone could inhibit the proliferation and differentiation of osteoblastic cells, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; analysis ; Dexamethasone ; pharmacology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Female ; Osteoblasts ; cytology ; drug effects ; physiology ; Proliferating Cell Nuclear Antigen ; analysis ; Rats ; Rats, Sprague-Dawley

Three sesquiterpenoids and nine iridoids were isolated from the roots and rhizomes of Valeriana jatamansi by various chromatographic methods. Their structures were identified by physicochemical properties, NMR and MS data. Among them, valeriananoid G (1) was a new patchoulol-type sesquiterpenoid, and compound 3 was isolated from the genus Valeriana for the first time. Compounds 3 and 10 exhibited significant inhibitory effects on nitric oxide production induced by lipopolysaccharide in RAW 264.7 macrophages, with IC₅₀ values of 19.00 and 3.66 μmol·L^-1, respectively. In addition, compounds 4, 6 and 12 showed anti-influenza virus activity with IC₅₀ values of 51.75, 51.40 and 102.08 μmol·L^-1, respectively.

Background The sex hormones plays an important role in the incidence of dry eye,especially for the regulation of function.However,the effects of sex hormones on lacrimal gland epithelial cells are below understand.Objective This study was to investgate the effects of estradiol and testosterone on the apoptosis of lacrimal gland cells induced by H2O2.Methods The lacrimal gland tissue was obtained from 2- or 3-month-old clean male New Zealand rabbits and the lacrimal gland epithelial cells were cultured in vitro using esplant culture method.The cells were identified by pan cytokeratin antibodies with immunocytochemistry.lacrimal gland epithelial cells were incubated in the 96 well plate at the density of 5 × l04 cells/ml for 44 hours.Estradiol or testosterone with the concentrations of 1 × 10-5,1 × 10-6,1 × 10-7,1 × 10-8 mol/L were added into the medium for 24 hours respectively and 1× 10-4 mol/L H2O2 treated the cells for 1 hour to induce the apoptosis in experimental groups.The cells treated by only 1 × 10-4 mol/L H2O2 were used as apoptotic control group,and the cells cultured by regular method were used as blank control group.The cell viability in different groups was detected using MTT at 570 nm ( A570 ),and the apoptotic rates of the cells were assayed using Annexin V/PI double staining.This use and maintain of experimental animals followed the Regulation for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The cultured cells showed the irregular polygon in shape,and about 80％ cells was positive response for cytokeratin.MTT assay showed that the lower A570 values were detected in the H2O2-induced group,various concentrations of estradiol or testosterone groups compared with blank control group (P＜0.01 ).The A570 values in 1 × 10-5,1 × 10-6,1 × 10-7 mol/L estradiol groups or 1 × 10-6 mol/L testosterone group were significantly higher than ones of H2 O2-induced group (P＜0.01 ).Compared with corresponding concentrations of testosterone groups,the A570values in various concentrations of estradiol groups were elevated( P＜0.01 ).The apoptosis rates at the early and later phase were significantly declined in both estradiol group and testosterone group in comparison with H2 O2-induced group (P ＜ 0.01,P＜ 0.05 ),and those in estradiol group were lower than the testosterone group( P＜0.01,P＜0.05 ).Conclusions Estradiol and testosterone suppress the apoptosis of lacrimal gland cells induced by H2O2,and the stronger effect is found in estrogen.The inhibition of estrogen on lacrimal gland cell apoptosis show a dose-dependent manner to some extent.