<p>OBJECTIVETo determine the change of P-selectin in avulsion-injured vessels.p><p>METHODSDifferent stretch forces of 60, 70, 80 and 90 g were applied to a vascular injury model. The expression changes of P-selectin were evaluated by RT-PCR.p><p>RESULTSThe expression of P-selection mRNA in the injured vessels increased with the stretch force.p><p>CONCLUSIONThe result associated with our previous study indicated that P-selectin may be involved in thrombosis.p>
Animals ; Endothelium, Vascular ; P-Selectin ; metabolism ; RNA, Messenger ; metabolism ; Vascular System Injuries ; genetics ; metabolism

<p>OBJECTIVETo construct a luciferase reporter gene vector of p-selectin gene promoter and determine its transcriptional activity for screening the effect of drugs on the transcriptional activity of p-selectin promoter.p><p>METHODSPrimers were designed based on human p-selectin promoter sequence from UCSC software. The p-selectin promoter from human genome DNA was then amplified. After digestion of pGL3-Basic vector and p-selectin promoter with Kpn I and Xho I, p-selectin promoter was inserted into pGL3-basic vector. The recombinant plasmid, namely pGL3-p-selectin-promoter, was transiently cotransfected into 293F cells with pRL-SV40 as the control vector, and the activity of the dual luciferase was detected. The transcription activity of serially truncated segments of the p-selectin promoter reporter gene was quantified by luciferase expression. 293F cells transfected with pGL3-p-selectin-promoter reporter gene and dual luciferase were stimulated with LPS, TNF-α and As2O3, and the transcriptional activity of p-selectin promoter were assessed.p><p>RESULTSpGL3-p-selectin-promoter was constructed successfully as verified by restriction digestion and sequence analysis. The luciferase activity was higher in pGL3-p-selectin-promoter/pRL-SV40 group than in pGL3-basic/pRL-SV40 group (0.8573±0.4703 vs 0.03955±0.05894). pGL3- 1826 bp was actively transcribed compared with pGL3-1092 bp and pGL3-3738 bp. LPS, TNF-α and As2O3 significantly enhanced the transcriptional activity of p-selectin promoter.p><p>CONCLUSIONpGL3-p-selectin-promoter can be transcribed and activated in 293F cells. This study provided an important basis for acquiring transcriptional factors and screening inflammatory factors and drugs.p>
Genes, Reporter ; Genetic Vectors ; HEK293 Cells ; Humans ; Luciferases ; P-Selectin ; genetics ; Promoter Regions, Genetic ; Transcriptional Activation ; Transfection

<p>OBJECTIVETo investigate whether P-selectin gene -2123 polymorphism is associated with the pathogenesis of Henoch-Sch-nlein purpura (HSP) in children.p><p>METHODSPolymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) is used to identify the distribution of allele and genotype frequencies of P-selectin gene promoter -2123 polymorphism in 86 children with HSP (including 40 cases of purpura nephritis) and 70 healthy controls.p><p>RESULTSCompared with the healthy controls, the frequencies of GG genotype and G allele of P-selectin promoter -2123 in children with HSP increased significantly (P<0.05). There were no significant differences in P-selectin promoter -2123 genotype and allele frequencies between the patients with and without nephritis.p><p>CONCLUSIONSP-selectin gene promoter -2123 polymorphism appears to be associated with the pathogenesis of HSP in children.p>
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Male ; P-Selectin ; genetics ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Purpura, Schoenlein-Henoch ; etiology ; genetics

Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection (TCUMGT) via ultrasound-targeted microbubble destruction (UTMD) to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles (MBs) with pcDNA_3.1-human vascular endothelial growth factor 165 (pcDNA_3.1-hVEGF₁₆₅) plasmids targeted to P-selectin (MB+P+VEGFp) were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups: microbubble only (MB), microbubble+P-selectin (MB+P), microbubble+pcDNA_3.1-hVEGF₁₆₅ plasmid (MB+VEGFp), and microbubble+ P-selectin+pcDNA_3.1-hVEGF₁₆₅ plasmid (MB+P+VEGFp). The reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB+P+VEGFp delivery showed greater improvement than MB+VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA_3.1-hVEGF₁₆₅ recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.
Animals ; Genetic Therapy/methods* ; Male ; Microbubbles ; Myocardial Ischemia/therapy* ; P-Selectin/genetics* ; Rats ; Rats, Sprague-Dawley ; Transfection/methods* ; Ultrasonics ; Vascular Endothelial Growth Factor A/genetics*

The study was purposed to develop a novel cryopreserved agent (CPA) for platelets, to investigate the morphology of cryopreserved platelets in different CPA and the CD62P expression on membrane of platelets after stimulating by thrombin, as well as to compare the effect of adding UDP-Gal on preserved efficiency of preservation solutions. A novel cryopreserved agent consisting of 2% DMSO, thrombosol and UDP-Gal was developed on basis of using higher concentration of DMSO. The morphology of chilled platelets was observed by transmission electron microscope and compared with fresh platelets. The expression of CD62P on the membrane of platelets was detected at 0, l, 3 months. The results indicated that the significant effect of cryopreservation on morphology of platelets was found according to percentages of round, dendritic and irregular shapes of cryopreserved platelets. The protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal were better than that of 5% DMSO. Compared with fresh platelets, the expression of CD62P on platelet membrane decreased obviously after cryopreservation, but not observed difference at preservation for 1 month and 3 months, as well as among 3 kinds of different CPA. It is concluded that the protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal on morphology of platelets are similar, but better than that 5% DMSO. The reaction of cryopreserved platelets to thrombin decreases, while the significant difference is not found among these 3 kinds of CPA. The addition of UDP-Gal to cryopreserved agents not show the protective effect on platelets.
Blood Platelets ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Humans ; P-Selectin ; biosynthesis ; genetics ; Uridine Diphosphate Galactose ; pharmacology

The study was aimed to investigate the expression of Toll-like receptor 4 (TLR4) on platelets and to determine whether platelet TLR4 involves in its activation induced by lipopolysaccharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy individuals pretreated with a concentration of 0.2 microg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4, CD62P (P-select) and CD40L on platelets were detected by flow cytometry, and platelet TLR4 expression was further determined by Western blot analysis. The results indicated that the percentage of TLR4-positive platelets induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, p < 0.05). TLR4 expression on platelets treated with LPS was remarkably elevated in the presence or absence of thrombin. However, the expression level of the former was much higher than that of the latter and thrombin stimulation alone (p < 0.05). Moreover, the similar results were found in Western blot analysis. Synchronously, expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin (42.68% and 14.8%) and LPS respectively, and the increases of expression of CD62P and CD40L were more significant when stimulated with both LPS and thrombin (63.03% and 13.94%). Although anti-TLR4 antibody inhibited significantly the increase of TLR4, CD62P and CD40L on platelets induced by LPS, which did not affect their increase induced by thrombin. In conclusion, the evidence has been shown that functional TLR4 can be expressed on human platelets. It may involve in platelet activation as an important mediator of LPS-induced CD62P and CD40L expressions on platelets.
Adult ; Blood Platelets ; metabolism ; CD40 Ligand ; genetics ; Humans ; Lipopolysaccharides ; pharmacology ; Middle Aged ; P-Selectin ; metabolism ; Platelet Activation ; Toll-Like Receptor 4 ; metabolism

Cell adhesive molecular P-selectin was cloned, expressed and anchored on CHO cell membrane through GPI for selection specific antibodies. Total human platelet RNA was extracted and the functional segment of P-selectin gene was cloned by RT-PCR. The P-selectin functional segment gene was cloned into a eukaryotic expression vector pMCEw2-GPI containing an attenuated neo gene together with a downstream GPI, which was synthesized by overlapping PCR. The recombinant plasmid pMCEw2-GPI-P-selectin was then transfected to CHO(dhfr-) cells and screened with G418. ELISA, western-blot and immunofluorescence were carried out to detect the stability of P-selection expression on cell membrane. These results provided a necessary basis for the following study of selection the antibodies targeting P-selectin.
Animals ; Antibodies ; metabolism ; CHO Cells ; Cell Membrane ; metabolism ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Epidermal Growth Factor ; metabolism ; Glycosylphosphatidylinositols ; metabolism ; Lectins ; metabolism ; P-Selectin ; biosynthesis ; genetics ; immunology ; RNA ; metabolism ; Transfection

<p>OBJECTIVETo comparatively study the expressive conditions of platelet activation related factors (GP I b, GP II b- III a and GMP-140) in healthy subjects and patients with coronary heart disease (CHD) of blood-stasis (BS) or non-blood-stasis (non-BS) syndrome, and to analyze the relationship between the activities of various glycoproteins and the polymorphism of genes.p><p>METHODSWith case control design adopted, patients with the CHD (40 of BS, 37 of non-BS) and 39 healthy subjects for control, all fitting to the inclusion criteria, were selected in this study. The number of affected coronary branches was recorded by the contrast examination. The mean fluorescence intensity (MFI) of GP I b, GP II b- III a, and GMP-140 (CD42b, CD61, CD62p) in patients and healthy persons was measured with flow cytometry, the polymorphism of HPA-3 gene was detected by Taqman probe technique and that of HPA-2 gene was determined by gene sequencing.p><p>RESULTSMFI of CD61 and CD62p was higher in the CHD patients than in the healthy control, which was also higher in patients of BS syndrome than in patients of non-BS syndrome (P<0.05); MFI of CD42b was lower in the CHD patients than in the healthy control (P<0.05), but showing insignificant difference between BS and non-BS syndrome (P>0.05); at the same time, no significant difference of all the above-mentioned three MFI could be found in patients with various numbers of affected coronary branches, neither in patients with different genotypes at GP II b HPA-3 and GP I b HPA-2 polymorphism loci (P>0.05).p><p>CONCLUSION(1) The activities of GP II b- III a and GMP-140 were obviously increased in the genesis and developing process of CHD and CHD of BS syndrome, and so they could be taken as one of the objective indexes for microscopic diagnosis of BS syndrome. (2) The level of GP I b was lower in CHD patients than in healthy persons, but it was not a sensitive indicator for BS syndrome of CHD. (3) Levels of GP II b- III a, GP I b and GMP-140 were not related with the number of affected coronary branches in CHD patients. (4) The changes in amino-acids expression induced by the two loci brought no significant influence on GP I b and GP II b- III a activities.p>
Coronary Disease ; blood ; genetics ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; P-Selectin ; blood ; genetics ; Platelet Activating Factor ; analysis ; genetics ; Platelet Glycoprotein GPIIb-IIIa Complex ; analysis ; genetics ; Platelet Glycoprotein GPIb-IX Complex ; analysis ; genetics

<p>OBJECTIVETo investigate the effect of ischemic reperfusion(I-R) on the expression of P-selectin(Ps) of platelets, CD11b of leukocytes, monocyte chemotactic protein-1(MCP-1) mRNA of myocardial tissue and the protective role of MgSO(4).p><p>METHODSIschemic reperfusion rat model was established in the experiments. All animals were randomly divided into 3 groups sham group, I-R group and Mg(2+) group. The expression of Ps of platelets and CD11b of leukocytes was determined by flowcytometry and the expression of MCP-1 mRNA of myocardial tissue was measured by RT-PCR admission and at 5, 30, 60, 180, 360 min of reperfusion (R5, R30, R60, R180, R360).p><p>RESULTSThe expression of Ps of platelets in I-R group began to rise at R5 and peaked at R60 P<0.01); the expression of CD11b of leukocytes was significantly higher than that of the control groups P<0.01 and P<0.05) the expression of MCP-1 mRNA of myocardial tissue began to rise at R60,reaching the highest at R360 P<0.01) the concentration of Ca(2+) of the serum was significantly higher than that of the control groups P<0.01 and P<0.05). MgSO(4) inhibited the expression of Ps of platelets and MCP-1 mRNA of myocardial tissue and decreased serum Ca(2+)concentration, but did not affect the expression of CD11b of leukocytes.p><p>CONCLUSIONI-R can increase the expression of cell adhesion molecules and cell chemotactic factor. MgSO(4) may protect myocardial tissue from ischemic reperfusion injury by inhibiting the expression of Ps of platelets and MCP-1 mRNA of myocardial tissue and decreasing the concentration of Ca(2+) of the serum.p>
Animals ; CD11b Antigen ; genetics ; Calcium ; blood ; Chemokine CCL2 ; genetics ; Gene Expression ; Magnesium Sulfate ; therapeutic use ; Male ; Myocardial Reperfusion Injury ; metabolism ; prevention & control ; P-Selectin ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

<p>OBJECTIVETo investigate the therapeutic effects of propyl gallate (PrG) in combination with standard medication on patients with non-ST-elevation acute coronary syndrome (NST-ACS), including unstable angina and acute non-ST-elevation myocardial infarction, and its influences on serum inflammatory marker and platelet activation.p><p>METHODSFifty-five patients with NST-ACS were randomly assigned to two groups. Accessory to the standard Western medicine, the 27 patients in the tested group treated with PrG and the 28 in the control group with salvia composite (SC), all being medicated for 14 days. Effects on angina pectoris and electrocardiogram were observed. The positive rate and mean fluorescence density (MFI) of GP IIb-IIIa and CD62p expression on platelet surface were detected using flow cytometer; the serum concentration of high sensitive C-reactive protein (Hs-CRP) was determined using ELISA before and after treatment respectively.p><p>RESULTSThe therapeutic effects on angina and electrocardiogram between the two groups showed no significant difference. Serum level of Hs-CRP, GP IIb-IIIa MFI and CD62p positive rate were significantly lowered after treatment in both groups (P < 0.05), no significant difference was found between groups, though the lowering of Hs-CRP and GP IIb-IIIa MFI in the tested group displayed a further decreasing trend.p><p>CONCLUSIONIn combination with standard medication of Western medicine, PrG and SC showed no obvious difference in the therapeutic effect and influences on angina pectoris and electrocardiogram in patients with non-ST-elevation acute coronary syndrome.p>
Acute Coronary Syndrome ; drug therapy ; genetics ; metabolism ; Adult ; Aged ; C-Reactive Protein ; metabolism ; Female ; Gene Expression ; drug effects ; Humans ; Male ; Middle Aged ; P-Selectin ; genetics ; metabolism ; Propyl Gallate ; therapeutic use