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Objective To evaluate the relationship between the platelet reactivity detected by two methods and CYP2C19 gene polymorphism in patients with ACS after taking clopidogrel, and analyze the correlation between different platelet detection methods. Methods Forty-three patients with ACS, admitted in the General Hospital of Northern Theater Command from July to October 2019, met the diagnostic criteria and received dual antiplatelet therapy, were enrolled in present study. The gene chip was used for CYP2C19 genotype detection, vasodilatory stimulated phosphorylated protein (VASP) method and two revulsiveinduced phototurbidimetric methods [arachidonic acid-induced phototurbidimetry (AA-LTA), diphosphate adenosine-induced phototurbidimetry (ADP-LTA)] were used to evaluate the antiplatelet reactivity, and the correlation between different evaluation methods was compared. Results Of the 43 patients, 17(39.5%) were EMs, 19(44.2%) were IMs, and 7(16.3%) were PMs. The results of VASP were higher in PMs and IMs patients than those in EMs, and the results of AA-LTA were higher in PMs than those in IMs and EMs. The differences were significant (P

Objective To investigate the effect of exosome in cultured in vitro H9C2 myocardial cells injury of diabetic mice and its mechanism. Methods The mouse model of diabetic myocardial injury was established by using db/db mice (n=10) and their mate mice db/+ (n=5). Serum exosomes were isolated and quantitated using the exosome isolation reagent and EXOCET Quantitation kit. The serum exosomes were labeled with PKH26 (red fluorescent cell linker) to detect the endocytosis in H9C2 cells. The expressions of exocrine associated protein and inflammatory cytokines in H9C2 cells with or without exosome stimulation were detected by Western blotting. TUNEL was used to detect apoptosis. A neutralizing antibody of Rab1a was used for blocking experiment. Results Db/db mice produced more exosomes than db/+ mice (30.95×109/ml vs. 10.45×109/ml, P

Objective To construct the promoter enhanced green fluorescent protein (pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1A-stimulated genes (hCREG), and compare the transcriptional activity of each promoter to determine the hCREG core promoter region. Methods The promoter fragment with length of 2003 (–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information (NCBI) database and combining with the characteristics of the promoter. Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003, pEGFP1_hCREG_945, pEGF P1_hCREG_586, pEGFP1_ hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid. The 293T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours. The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope, and the mRNA expression of each promoter was detected by real-time quantitative PCR, and the core promoter region was determined. Bioinformatics was used to predict the transcription factors that might bind to the core promoter region. Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing. The results showed that the transcription activity of pEGFP1_ hCREG_586 was the highest (P0.05), implying that –867/ –509 bp is a negative regulatory region, and there existed enhancer sequences in –400/–281 bp and –508/–401 bp, so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp. Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53, C/EBPβ, GR-β, GATA-1, GR-α, c-Jun, PRB/ PRA, YY1, RXR-α, AP-2, FOXP3, GR, TFIID, STAT4 and c-Ets-1. Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed, the core promoter of which is located in –508/–281 bp, where several transcription factors might be bound. DOI: 10.11855/j.issn.0577-7402.2019.07.09

The low spatial frequency error (LSFE) was an important component of the measurement error of star trackers. In order to improve the star tracker measurement accuracy better than 1 arcsecond, the calibration method of LSFE was studied. Firstly, the error composition, source and importance of ground calibration were introduced. Then, the traditional calibration and evaluation methods were described, and the problems in the application of the method to the star trackers which accuracy better than 1 arcsecond were analyzed. Then, according to the consistency of LSFE in a small range of field and the random characteristics of high spatial frequency errors (HSFE), an error separation and calibration method was proposed. Finally, the new calibration method was simulated and verified by experiments based on a sub-arcsecond precision star tracker. The Experimental results show that the calibration residual of the new method is 1.96/100 pixels, which is 38% lower than the traditional calibration method. This method is accurate, stable and reliable, and can be used for ground calibration of various space starlight measuring instrument such as star tracker and star camera.

Objective To investigate the effect of C-C motif chemokine receptor 2 (CCR2) on phenotypic transformation of cardiac fibroblasts after hypoxia. Methods The mouse myocardium primary fibroblasts were extracted by collagenase digestion. Cells were divided into control, hypoxia-24h and hypoxia-48h, the mRNA and protein expression of CCR2 was examined by real-time PCR and Western blotting. CCR2 low expression cell (si-CCR2) was established using by small interfering RNA. Cells were divided into four groups including si-control, si-CCR2, si-control+hypoxia, si-CCR2+hypoxia. The mRNA and protein expressions of CCR2, α smooth muscle actin (α-SMA) and Collagen 1A (Col 1A) were detected by real-time PCR and Western blotting, cell proliferation was detected by Cell Counting Kit-8 (CCK8) and Bromodeoxyuridine (BrdU). Results Compared with control, the mRNA and protein levels of CCR2 significantly increased in hypoxia-24h and hypoxia-48h group (P

Objective To establish a new, simple and efficient method for culturing pulmonary microvascular endothelial cells (PMVECs) of primary neonatal mouse. Methods The C57BL/6 mice aged less than one week were sacrificed. The primary PMVECs were isolated and cultured with agarose microsphere antibody screening method. Inverted microscope was used to observe the cell morphology, immunofluorescence staining and matrigel angiogenesis test with CD31 related antigen and vascular Willebrand factor (vWF) related antigen were performed to identify the cells, and CCK-8 method was employed to draw cell growth curve. Results Under the inverted microscope, the primary PMVECs showed short spindle and round in shape, and integrated in monolayer as paved stone like. CD31 related antigen and vWF immunofluorescence staining were observed in endothelial cells, the positive rates were 91.35%±3.06% and 92.99%±2.67%, respectively. The vascular lumen was formed 8h after inoculation of PMVECs on the matrigel. The growth curve drawn by CCK-8 method showed that PMVECs extracted with agarose microsphere antibody screening method were in the logarithmic growth period 2-5 days after cultivation. Conclusion Agarose microsphere antibody screening method is a simple and efficient method to isolate and cultivate the primary PMVECs of neonatal mouse, and is a worthwhile approach. DOI: 10.11855/j.issn.0577-7402.2018.05.12

Objective To investigate whether chemokine CC motif 2 (CCL2) is involved in the high residual platelet response, and the mechanism of CCL2 being involved in the regulation of platelets. Methods Forty patients with ST elevation myocardial infarction (STEMI) were admitted. P2Y12 reaction unit (PRU) was detected by VerifyNow. Forty patients were divided into high platelet reactivity group (high reactivity group, n=24) and normal platelet reactivity group (normal reactivity group, n=16) according to the results of PRU detection. Plasma CCL2 concentration of the STEMI patients was examined by ELISA. The expressions of CCL2 and CCR2 in the platelets were detected by Western blotting. After CCL2 stimulation, the kinases of which phosphorylation was changed in the platelets were screened by ARY003B protein chips. The phosphorylation of p38MAPK and HSP27 in the platelets was tested by Western blotting after CCL2 stimulation in the presence or absence of CCR2 antagonist (RS 102895) or p38MAPK signal pathway inhibitor (SB 203580). Results The plasma CCL2 concentration of high reactivity group was markedly higher than that of normal reactivity group. Moreover, compared with normal reactivity group, the expressions of CCL2 and CCR2 in the platelets of high reactivity group significantly increased. After the platelets were stimulated by CCL2, the phosphorylation of p38α and HSP27 enhanced in the platelets by protein chips screening. When RS 102895 or SB 203580 was treated before CCL2 stimulation, the phosphorylation of p38MAPK and HSP27 decreased. Conclusions CCL2 participates in high residual platelet response in an autocrine/paracrine way. CCL2/CCR2 might affect the function of platelets through p38MAPK- HSP27 signal pathway. DOI: 10.11855/j.issn.0577-7402.2017.05.09

Objective To observe the effects of miR-124 on the proliferation and apoptosis of endothelial cells of umbilical cord vein in order to explore the role of miR-124 in the pathogenesis of atherosclerosis. Methods The expression levels of miR124 in plasma of 40 patients with coronary heart disease and 40 healthy control participants were examined using quantitative RTPCR. CCK8 assay and BrdU incorporation assay were used to analyze the effects of miR-124 on the proliferation of human umbilical vein endothelial cells (HUVECs). The effect of miR-124 on HUVEC apoptosis was examined by TUNNEL staining. The effects of miR-124 on the expression of proapoptotic protein cleaved caspase 3 and anti-apoptotic protein Bcl-2 in HUVECs were examined by Western blotting. Results Compared with healthy controls, plasma miR-124 was down-regulated in patients with coronary artery disease. In vitro experiment, miR-124 significantly promoted HUVEC proliferation, and it also inhibited HUVECs apoptosis by affecting the expressions of cleaved caspase 3 and Bcl-2. Conclusions MiR-124 could promote the proliferation and inhibit the apoptosis of HUVECs. This may provide a theoretical basis for clarifying the mechanisms of atherosclerosis. DOI: 10.11855/j.issn.0577-7402.2016.01.02

Objective To investigate the effects of chemokine ligand 16 (CXCL16) in high salt induced myocardial remodeling in salt sensitive hypertensive rats. Methods Sixty-four Dahl salt sensitive (Dahl-SS) rats were randomly divided into normal salt (NS, 0.3% NaCl) and high salt (HS, 8.0% NaCl) group, 32 rats for each group. Blood pressure was measured by tail-cuff method every week. Hearts were harvested and the expression of CXCL16 in heart tissues was detected by Western blotting at the 2nd, 4th, 6th and 8th week after NS or HS feeding. Immunofluorescence double staining was used to detect the colocalization of CXCL16 with both cardiomyocytes and cardiac fibroblasts. Immunofluorescence and immunohistochemical staining were used to detect the expression of CXCL16 and the biomarker of macrophage in the myocardial tissue after 6 week and 8 week of NS or HS feeding. Myocardial fibrosis was evaluated by HE and Masson trichrome staining. Results Compared with that in NS group, the systolic pressure and diastolic pressure in HS group increased significantly after 1 week of HS feeding (147.10±3.67mmHg vs128.57±6.32mmHg, P

Objective To investigate the effect of cellular repressor of E1A stimulated genes (CREG1) on cardiac function in mouse with myocardial fibrosis. Methods CREG1 knockout mice (CREG1+/–) and CREG1 wild-type mice (CREG1+/+) were used to reproduce the model of myocardial fibrosis by subcutaneous pump burying of angiotensin Ⅱ (AngⅡ). After being stimulated with AngⅡ for 14 days, myocardial fibrosis was verified by HE staining and Masson trichrome staining. Western blotting and immunohistochemistry were used to detect the expression of CREG1 in myocardium before stimulation and 3, 7, 14 days after the AngⅡ stimulation. The cardiac function was evaluated by echocardiography after AngⅡ stimulation for 14 days. The CREG+/+ mice were given AngⅡ for 14 days, and at the same time recombinant CREG1 protein [respectively 15, 30, 60 and 300μg/(kg.d), intraperitoneal (IP) injections] (treatment group) and NaCl (control group) were administered for treatment, and then cardiac function and myocardiac apoptosis were examined. Results Western blotting and immunohistochemistry showed that the expression of CREG1 in heart tissue was significantly lower in CREG+/– mice than in CREG+/+ mice (P

Objective To investigate the correlation of the single nucleotide polymorphism (SNP) of PON1 (Paraoxonase-1) gene rs854572 to the occurrence of clopidogrel resistance (CR). Methods A case-control method was employed in present study. A total of 850 hospitalized patients with coronary artery diseases (CAD) in General Hospital of Shenyang Command were enrolled. The residual platelet aggregation rate (RPA) induced by 20μmol/L of adenosine diphosphate (ADP) was detected by optical nephelometry, and RPA≥70% was defined as CR. Accordingly, all the enrolled 850 patients were then divided into CR group (n=215) and non-CR (NCR) group (n=635). Polymerase chain reaction (PCR) and pyrophosphate sequencing were executed to determine the genotypes and the allele frequencies of PON1 gene rs854572. Results The genotype frequencies in rs854572 of PON1 gene conformed well to the Hardy-Weinberg equilibrium in both CR group and NCR group. Three frequencies of genotype CC, CG and GG were 23.7%, 49.3%, 27.0% in CR group, and 24.1%, 50.2%, 25.7% in NCR groups, respectively. No significant difference in genotype and allele frequency existed between CR group and NCR group (P=0.93 and 0.76, respectively). Logistic regression analysis revealed that no correlation between rs854572 SNP of PON1 gene and the formation of CR in patients with CAD after adjustment of correspondent factors including age, gender, body mess index, smoking, hypertension, diabetes mellitus and hyperlipidemia. Conclusions It is considered that no correlation exists between PON1 gene rs854572 polymorphism and clopidogrel resistance in patients with coronary heart disease.

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca2+ -dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation.

AIMS The current study sought to evaluate the clinical impact of newly reported genetic variations and their association with clopidogrel high on-treatment platelet reactivity (HTPR) in acute coronary syndrome (ACS) patients after drug-eluting stent (DES) implantation. METHODS AND RESULTS The study enrolled 1,016 consecutive patients with ACS undergoing DES implantation. A total of 19 tag single nucleotide polymorphisms (SNPs) were selected from CYP3A4/5, CYP2C19, P2Y12 and ABCB1 genes. ADP-induced light transmittance aggregometry (LTA) was performed to test the post-procedure maximum platelet agglutination (MPA). The primary endpoint was a composite of cardiovascular death, non-fatal myocardial infarction (MI), stent thrombosis, and ischaemic stroke at one-year follow-up after DES placement. The secondary endpoint was the incidence of bleeding events. The post-procedure MPA was calculated and the cut-off point was determined for the HTPR. Using multivariate logistic regression analysis, the carriage of two CYP2C19 LOF alleles was an independent predictor of the post-procedure HTPR (OR: 2.8, 95% CI: 1.70-7.23, p

To explore the effects of astragali radix extract on the expressions of matrix metalloproteinase 9 (MMP-9) and the formation of atherosclerotic plaque in aortic atherosclerotic plaques of apolipoprotein E-deficient mice (ApoE-/-).Male 8-week-old ApoE-/- mice fed with high fat diet were randomly divided into four groups (n=12 each): control group (saline 0.2 ml/d), atorvastatin group (atorvastatin 10 mg×kg(-1)×d(-1)), low-dose astragali radix extract group (1.25 g×kg(-1)×d(-1)) and high-dose astragali radix extract group (5 g×kg(-1)×d(-1)). After 12 weeks, serum oxLDL was measured by the method of ELISA. The formation of atherosclerotic plaque was determined in HE and oil red O stained aortic slice. The expressions of macrophage and MMP-9 in the aortic plaque were detected by immune fluorescence and immunohistochemistry staining method.Similarly as atorvastatin, astragali radix extract significantly decreased the level of serum oxLDL in ApoE-/-1 mice in a dose-dependent manner. The level of oxLDL in the high-dose astragali radix extract group [(5.2±6.1) µg/ml] was significantly lower than that in the control group [(15.8±5.4) µg/ml, P0.01]. The area of atherosclerosis plaques was smaller (17.24%±4.22% vs. 49.87%±9.37%, P0.01) and the penetration degree of plaques in the arterial wall was relieved in the high-dose astragali radix extract group compared to those in the control group (P0.01). The expressions of Mac3 in atherosclerosis plaques of the high-dose astragali radix extract group was also significantly lower than in the control group (P0.01). The mean absorbance value of the expression of MMP-9 in the high-dose astragali radix extract group (0.0154±0.0014)was significantly lower than that in the control group (0.0263±0.0065) (P0.01).Similar as atorvastatin, astragali radix extract can dose-dependently inhibit the expression of MMP-9 and the formation of the atherosclerotic plaque in ApoE-/- mouse, probably by reducing the serum oxLDL, inhibiting macrophage infiltration, migration and secretion of MMP-9.

Tumor necrosis factor-α (TNF-α) is known to induce changes in endothelial cell morphology and permeability. The aim of this study is to determine the underlying signaling mechanisms involved in these responses.Cultured human umbilical vein endothelial cells (HUVECs) were exposed to TNF-α, and HUVEC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of horseradish peroxidase (HRP)-albumin across the EC monolayers. To explore the signaling pathways behind TNF-α-induced changes in HUVEC morphology and permeability, HUVECs were treated with either the Rho GTPase inhibitor Y27632 or the mitogen-activated protein kinases (MAPK) inhibitors PD98059 and SB203580 before TNF-α administration. To further elucidate possible involvement of the RhoA and ERK pathways in TNF-α-induced HUVEC changes, retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA, namely T19NRhoA and Q63LRhoA, were pre-infected into HUVECs prior to TNF-α exposure.TNF-α induced F-actin cytoskeleton rearrangement and increased HUVEC permeability in a dose and time-dependent manner. The maximal increase in the HRP-BSA flux (40 ng/ml) was seen in cells exposed to TNF-α at 100 ng/ml after 2 h. Preconditioning of HUVEC monolayer with Y27632 or PD98059 significantly reduced TNF-α induced permeability increase (HRP concentration from 40 ng/ml decreased to 12.5 ng/ml, P0.05) and F-actin cytoskeleton rearrangement, HUVEC pre-infection with activated forms of Q63LRhoA increased HUVEC permeability and upregulated pERK compared to GFP infection, while HUVEC pre-infection with inhibited forms of T19NRhoA attenuated the effects of TNF-α on HUVEC permeability.These results indicate that TNF-α-induced EC barrier dysfunction and morphological changes of the F-actin via activating RhoA-ERK/MAPK signal pathway.

To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-beta1 for further research on the effects of TGF-beta1 on vasculogenesis and angiogenesis.Three pairs of siRNA target sequences coding from the mRNA of TGF-beta1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, the three resulting TGF-beta1 shRNA expression vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-beta1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression.RT-PCR and Western blot showed that one of the TGF-beta1 shRNA expression vectors pDS_Tc downregulated TGF-pl mRNA and protein expression markedly in NIH/3T3 cells.ShRNA eukaryotic expression vectors targeting TGF-beta1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-beta1 regulates vasculogenesis and angiogenesis.

To observe the dynamic changes of plasma matrix metalloproteinases (MMPs) and investigate the effect of early or delayed percutaneous coronary intervention (PCI) in the presence or absence cilostazol on left ventricle (LV) remodeling in patients with non-ST elevation myocardial infarction (NSTEMI).One hundred and sixty-four patients undergoing PCI with NSTEMI were randomized to early PCI (PCI within 24 h) group or delayed PCI group (PCI after 36 h), and patients in both group were further assigned to cilostazol or no cilostazol group. Plasma MMP-2 and MMP-9 concentrations were measured at 2, 4 days and 2 and 4 weeks after PCI. Left ventricular end-diastolic volume (LVEDV), left ventricle ejection fraction (LVEF), left ventricle posterior wall (LVPW) and interventricular septum (IVS) were measured by echocardiography at baseline and 1 year after PCI.MMP-2 concentration at 2 weeks after PCI is higher than that at 2, 4 days and 4 weeks after PCI. MMP-9 concentration at 4 days is higher than that at 2 days, 2 weeks and 4 weeks after PCI. MMP-2 and MMP-9 were significantly lower in cilostazol group compared with that in non-cilostazol group at 4 days, 2 weeks and 4 weeks after NSTEMI (all P0.05). Changes of LVEDV and LVEF were significantly less in cilostazol group and early PCI group than that in no cilostazol group and delay PCI group (P0.05 or P0.01) at 1 year after NSTEMI.Early PCI and Cilostazol use are associated with less LV remodeling in patients with NSTEMI. Cilostazol attenuated LV remodeling possibly by reducing concentration of MMP-2 and MMP-9 after PCI.

To observe expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC during early embryonic vascular development, and to initially investigate the differentiation effect of platelet derived growth factor-BB (PDGF-BB) on vascular smooth muscle cells (VSMCs) during that period.Murine embryonic stem cell line expressing the enhanced green fluorescent protein (GFP) under the transcriptional control of the smooth-muscle-specific SM22alpha promoter was used to make embryoid bodies,and to analyze the expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC by immunofluorescence stainings, RT-PCR and Western blot. Then AG1296 (PDGF receptor inhibitor) 0 micro-mol/L(control group), 10 micromol/L and 50 micromol/L were used to treat EBs respectively in order to analyze the differences of SMa-actin, SM22alpha, myocardin and SMMHC at gene and protein levels among the three groups.SMalpha-actin, myocardin, SM22alpha and SMMHC expression in EBs were found to begin at day 0 (ESCs), 8, 11, 13 respectively during early embryonic vascular development. There were no clear differences in SMa-actin, SM22alpha, myocardin and SMMHC protein expression and SM22alpha, myocardin and SMMHC mRNA level among the three groups of different concentrations of AG1296.A spontaneous VSMCs differentiation occurs during EBs development, SMalpha-actin is the first to be detected,the following are myocardin, SM22a and SMMHC. PDGF-BB may not be indispensable for the regulation of expression of VSMCs markers during early EBs differentiation.

To purify the recombinant human cellular repressor of EIA stimulated gene (hCREG)/myc-His glycoprotein and confirm the biological function of hCREG/myc-His which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro.The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The recombinant hCREG/myc-His protein was desalted by HiTrap Desalting Column. The effect of recombinant hCREG/myc-His glycoprotein of different concentration (0.5 microg/ml, 1 microg/ml and 2 microg/ml) on proliferation of HITASY cells was studied by flow cytometric analysis and the effect of recombinant protein on proliferation of HITASY cells was confirmed by BrdU incorporation method.The recombinant hCREG protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The concentration of recombinant hCREG protein which has been concentrated and desalted was determined to be 1.6 mg/ml and the purity of recombinant protein reached 92%. The protein was identified to be glycosylated. The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro and the inhibition effect was stronger in low-dosage group than that in high-dosage group by flow cytometric analysis. The proliferation of HITASY cells cultured in vitro with 2 microg/ml recombinant hCREG protein was inhibited significantly compared with that in control group according to the BrdU incorporation result. There was statistical difference among the groups (P0.05).The purification of recombinant hCREG/myc-His glycoprotein with biological activity provides an experiment platform for function study and engineering production of hCREG protein.

In designing the routing protocols for Wireless Sensor Networks, clustering is one of the methods used to manage network energy consumption efficiently.LEACH is one of the most famous clustering mechanisms. It elects a cluster head based on probability model. This approach may reduce the network lifetime because LEACH does not consider the energy remains of each node. In this paper, we present an unequal cluster-head selection mechanism algorithm. Simulation results demonstrate that the proposed algorithm has higher efficiency and can achieve better network lifetime and energy consumption.

To investigate the relationship between matrix metalloproteinase (MMP) 1 gene -519A/G polymorphism and the risk of coronary heart disease (CHD) in Northern Chinese Han population.A total of 517 patients with CHD and 380 healthy adults diagnosed by coronary angiography were genotyped by polymerase chain reaction-restriction fragment length polymorphism and DNA sequence technology for the -519A/G polymorphism in MMP1 gene.(1) The frequency of AA genotype was significantly higher in patients with CHD than that in controls [67.70% (350/517) vs. 40.26% (153/380), OR = 1.64, P0.001, 95%CI: 1.44 - 1.86]. People carrying A allele had increased risk for CHD (OR = 1.49, P0.001, 95%CI: 1.33 - 1.69). (2) The frequency of AA genotype was higher in patients with acute coronary syndrome (ACS) than patients with stable angina pectoris [68.81% (278/404) vs. 51.76% (44/85), P0.01, 95%CI: 1.04 - 1.27]. The A allele carriers were more likely to develop ACS (OR = 1.11, 95%CI: 1.01 - 1.21, P0.05).Our data shows MMP1 gene -519A/G polymorphism is associated with the risk of CHD, and A allele carriers are more susceptible for CHD in Northern Chinese Han population.

To investigate the changes of high-sensitive C-reactive protein (hs-CRP) and monocyte chemotactic factor-1 (MCP-1) following percutaneous coronary interventional procedures (PCI) in patients with coronary artery disease (CAD), and evaluate the impact of PCI on the inflammatory indices and postoperative vascular restenosis.This study involved 80 patients undergoing PCI procedures for CAD compromising a single coronary artery. Forty healthy individuals with normal findings by coronary angiography were selected as the control group. Before and after PCI or coronary angiography, plasma hs-CRP and MCP-1 were measured in all the subjects by immunonephelometry and enzyme-linked immunosorbant assay (ELISA), respectively.In the CAD patients, the plasma hs-CRP level was significantly elevated after PCI as compared with the preoperative level (2.37-/+0.56 microg/L vs 1.59-/+0.41 microg/L, P0.01), whereas in the control group, the hs-CRP level underwent no significant changes after coronary angiography (1.18-/+0.37 microg/L vs the preoperative level of 1.13-/+0.32 microg/L, P0.05). PCI procedures also resulted in significant elevation of plasma MCP-1 level in the CAD patients (26.04-/+5.43 pg/L vs the preoperative level of 18.07-/+4.30 pg/L, P0.01), but in the control group, MCP-1 showed no significant variation after coronary angiography (9.80-/+2.64 pg/L vs the preoperative level of 9.63-/+2.52 pg/L, P0.05).Plasma hs-CRP and MCP-1 are elevated in CAD patients following PCI procedures, but their roles in the vascular restenosis following the procedures need further investigation.

To evaluate the interaction of atorvastatin or pravastatin with clopidogrel on platelet activation and aggregation function in patients with acute coronary syndromes (ACS) undergoing coronary stenting.Between April and December 2006, a total of 150 hospitalized ACS patients undergoing coronary stenting were randomized to receive atorvastatin (n = 50), pravastatin (n = 50) or no statin (n = 50) one day post procedure. All patients received standard antiplatelet treatment including aspirin 300 mg/d and loading dose 300 mg of clopidogrel followed by maintenance dose 75 mg/d. The expressions of CD62P and PAC-1 and the maximal platelet aggregation rate (MPAR) induced by 20 micromol/L ADP were measured at day 1 before statin therapy (baseline) and day 3 after procedure.Baseline clinical characteristics and levels of CD62P, PAC-1 and MPAR at the baseline were comparable among three groups. After 3-day statin treatment, the changes of CD62P [(4.69 +/- 16.78)% vs. (1.35 +/- 10.86)% vs. (2.97 +/- 10.21)%], PAC-1 [(12.78 +/- 22.07)% vs. (8.01 +/- 21.23)% vs. (10.65 +/- 21.39)%] and MPAR [(5.44 +/- 18.68)% vs. (7.15 +/- 19.59)% vs. (3.76 +/- 23.42)%] among three groups were not significantly different (all P0.05). Subgroup analysis showed that DeltaCD62P [(7.50 +/- 19.35)% vs. (3.24 +/- 11.18)% vs. (2.53 +/- 8.87)%], DeltaPAC-1 [(13.40 +/- 24.62)% vs. (11.28 +/- 19.90)% vs. (10.11 +/- 21.29)%] and DeltaMPAR [(7.56 +/- 19.11)% vs. (7.87 +/- 23.60)% vs. (6.75 +/- 23.30)%] in ACS patients were also similar among three groups (all P0.05).Neither atorvastatin nor pravastatin attenuates the antiplatelet function of clopidogrel in ACS patients early post coronary stenting.

To investigate the association between the connexin 37 (CX37) C1019T polymorphism and the susceptibility to coronary artery disease (CAD) in northern Han population of China.A total of 514 CAD patients and 400 healthy controls diagnosed by angiography were genotyped by using polymerase chain reaction-restriction fragment length polymorphism and polyacrylamide gel electrophoresis.The genotype frequencies of CC, TC and TT in the CX37 C1019T polymorphism was 22.37%, 53.31% and 24.32% in CAD patients, 17.75%, 46.50% and 35.75% in the controls respectively (P = 0.0007). The frequency of the CX37 C allele in CAD patients was significantly higher than that of the control group (49.03% vs 41.00%, OR = 1.38, 95% CI = 1.15 - 1.66, P = 0.0006). The frequency of the C allele carriers (CC + TC) was 75.68% in the CAD group and 64.25% in the control group (P = 0.0002). Compared with the TT homozygote, the CAD risk was significantly increased in the carriers of C allele (CC + TC) (OR = 1.73, 95% CI = 1.30 - 2.30). Subsequent stratified analysis revealed that the frequency of C allele was significantly higher in the male CAD patients than in the male controls (49.37% vs 39.60%, OR = 1.49, 95% CI = 1.18 - 1.89, P = 0.0009). The CAD risk was nearly two-fold increased in the carriers of C allele (CC + TC) than in the TT homozygote (95% CI = 1.38 - 2.78). However in the female population, there was no difference in the CAD risk between the carriers of (CC + TC) type and the TT homozygote (P = 0.24).The C allele in the CX37 gene might be associated with the susceptibility to CAD and potentially plays an important role in the manifestation of coronary atherosclerosis among Chinese.

To explore the effects of triple antiplatelet therapy on platelet aggregation and activation in patients who underwent coronary stenting.120 in-hospital coronary heart disease patients with coronary stenting were randomized to two groups receiving either triple antiplatelet drugs of aspirin and clopidogrel combined with cilostazol or dual antiplatelet drugs of aspirin and clopidogrel. On the first day after stenting cilostazol was added to the triple group patients who were previously administered aspirin and clopidogrel. Expressions of PAC-1 and CD62p which indicate platelet activation were assessed with flow cytometry and the maximal platelet aggregation rate (MPAR) induced by 5 and 20 micromol/L ADP was measured at the day before receiving cilostazol and the fifth day after stenting, respectively.The baseline clinical characteristics did not differ significantly between the two groups. There were no significant differences in the baseline level of MPAR CD62p and PAC-1 at the first day after stenting between the two groups. The margins between the two measurements were [(6.44 +/- 14.44)% vs (5.41 +/- 13.77)%, P0.05] for DeltaMPAR induced by 5 micromol/L ADP, [(8.50 +/- 15.50)% vs (7.84 +/- 14.21)%, P0.05] for DeltaMPAR induced by 20 micromol/L ADP, [(5.12 +/- 11.25)% vs (1.08 +/- 4.97)%, P0.05] for DeltaCD(62)p and [(12.12 +/- 12.30)% vs (2.22 +/- 15.15)%, P0.01] for DeltaPAC-1 in the triple and dual group patients, respectively. Among the above measurements, DeltaCD62p and DeltaPAC-1 in the triple group patients were significantly higher than those in the dual group patients although DeltaMPAR did not significantly differ between the two groups at the fifth day after stenting. Subgroup analysis for patients with acute coronary syndrome (ACS) showed that DeltaMPAR induced by 5 micromol/L ADP [(8.68 +/- 10.35)% vs (2.92 +/- 13.06)%, P = 0.018], DeltaMPAR induced by 20 micromol/L [(11.05 +/- 11.14)% vs (5.16 +/- 13.27)%, P = 0.019], DeltaCD62p [(5.57 +/- 12.08)% vs (1.35 +/- 4.42)%, P = 0.028] and DeltaPAC-1 [(11.62 +/- 12.73)% vs (1.29 +/- 15.73)%, P = 0.001] in triple group were significantly higher than that in dual group. At 3-month clinical follow-up, the rate of major adverse cardiac and cerebral events was 0 in the triple group and 3.3% (2/60) in the dual group, and the rate of hemorrhage was 5% (3/60) in the triple group and 3.3% (2/60) in the dual group, the differences were not statistically significant.Compared with dual antiplatelet regimen with aspirin plus clopidogrel, triple antiplatelet therapy with aspirin and clopidogrel combined with cilostazol is more efficient in inhibiting platelet activation and aggregation after coronary stent implantation. Large scale clinical trials are needed to confirm efficacy and safety of the triple antiplatelet regimen.

To investigate the effects and molecular mechanisms of the cellular repressor of E1A-stimulated genes (CREG) on the apoptosis of vascular smooth muscle cells (VSMCs), the human internal thoracic artery-Shenyang (HITASY) cells were infected with sense-CREG [pLNCX(2)(+)/CREG] and antisense-CREG [pLXSN(-)/CREG] retrovirus respectively. The stably infected cells were obtained by screening the G418-resistant clones. DAPI nuclei staining and Annexin V/PI FASC assay indicated that over-expression of CREG in HITASY cells infected with pLNCX(2) (+)/CREG inhibited VSMC apoptosis induced by serum deprivation, accompanied with decreased expression of caspase-9 mRNA detected by RT-PCR. Furthermore, Western blot analysis showed that p38 mitogen activated protein kinase (p38 MAPK) expression and activation were significantly enhanced in HITASY cells infected with pLNCX(2) (+)/CREG. The inhibition of CREG protein expression in cells infected with pLXSN(-)/CREG promoted the VSMC spontaneous apoptosis, as well as down-regulated p38 MAPK expression and activation, when cells were cultured with 10% fetal bovine serum (FBS) mediums. These results implicate that the CREG protein has the ability to regulate VSMC apoptosis in which the activation of p38 MAPK is possibly involved. To further identify the role of p38 MAPK in VSMC apoptosis, SB203580, a specific inhibitor of p38 MAPK, was used to inhibit p38 MAPK activity. When p38 MAPK signaling pathway was blocked, the effects that over-expression of CREG protein inhibited VSMC apoptosis disappeared. Taken together, the present work indicates that over-expression of CREG protein inhibits VSMC apoptosis, and this inhibitory effect is partly mediated by p38 MAPK signaling pathway.

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cell (HUVEC) senescence, we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-beta-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-beta-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96 h. Accompanied with these changes, the expression of activated Rac1 increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVEC senescence, HUVECs were transiently infected with the constitutively active form of Rac1 (V12Rac1) or dominant negative form of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs, V12Rac1-HUVECs) after hypoxia for 48 h and 96 h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48 h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-beta-Gal activation, PAI-1 expression increase, G(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96-hour hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96 h. All the results identified that the activation of Rac1 might accelerate HUVEC senescence induced by hypoxia and that inactivation of Rac1 could partly block the cell senescence. To further investigate the mechanism of HUVEC senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and Western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12Rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggest that activation of Rac1 accelerates endothelial cell senescence and inhibition of Rac1 activity prevents HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.

To identify the relationship between p53-dependent apoptosis associated genes and tumor metastasis.mRNA differential display (mRNA DD) was adopted for gene cloning after the different metastatic potential lung cancer cell lines were infected by Adv-p53 (a reconstructed adenovirus encoding wild type p53 gene). RT-PCR, Northern blot and Western blot assays were used to confirm the result from mRNA DD.After induction by p53 gene, the ANNEXIN A2 gene had differential expression in the cell lines; its level was down regulated in all the cells infected by Adv-p53 gene, especially in the Anip973 cell lines with high metastatic potential. RT-PCR, Northern blot and Western blot assays confirmed the consequence.The experimental data suggest that the ANNEXIN A2 gene may relate to cellular apoptosis induced by p53 gene. The affirmative relationship between ANNEXIN A2 gene and p53 needs further investigation.

To obtain the human cellular repressor of E1A-stimulated gene (hCREG) protein and the polyclonal antibody against the hCREG, and to further observe the expression and localization of hCREG protein in human internal thoracic artery cells (HITASY).The hCREG cDNA was amplified by PCR and cloned into the pGEX-4T-1. Glutathione-S-transferase (GST)-hCREG fusion protein was expressed in E.coli BL21 and was used to immunize rabbits to obtain anti-hCREG serum, which was purified by protein A and GST immobilized on glutathione-Sepharose beads. The titer and specificity of polyclonal antibodies were determined by ELISA and Western blot analysis. The expression and localization of hCREG protein were detected with immunofluorescence staining in HITASY cells after serum removal. The proliferation of HITASY cells affected by hCREG protein was examined by means of BrdU stain.It was confirmed that hCREG cDNA was correctly inserted into the vector by endonuclease digesting and DNA sequencing. The expressed GST-hCREG protein was purified by the gel-filtration and its purity was up to about 90%. The anti-hCREG polyclonal antibody was specific with high titer (1:10(5)) and the hCREG protein was expressed in a perinuclear pattern in HITASY cells after serum deprivation. It was also observed that the proliferation of HITASY cells was obviously inhibited by hCREG protein.The hCREG protein was highly expressed in HITASY cells and inhibited HITASY cell proliferation, suggesting that it may be involved in the process of proliferation and differentiation of vascular smooth muscle cells.

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.

The phenotypic modulation of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesis of arteriosclerosis. The purpose of this study was to investigate the expression of the cellular repressor of E1A-activated genes (CREG) at the transcriptional and protein level in human internal thoracic artery smooth muscle cells (HITASY), which express different patterns of differentiation markers after serum withdrawal.After cloning and recombining the CREG vector, the antiserum against the CREG protein was produced from the rabbits immunized by the purification CREG protein. The specificity of purified polyclonal antibody was detected by Western blot assay. The DNA synthesis of HITASY cultured in serum-free and serum-supplemented medium was measured by the [(3)H]-thymidine incorporation. Western blot analysis detected the expression of smooth muscle-specific markers (smooth muscle alpha-actin, calponin). The localization of CREG in cells was examined with immunohistochemistry staining and expression of CREG mRNA and protein were analyzed by RT-PCR and Western blot in HITASY after serum withdrawal.The high specificity of polyclonal antibody against CREG obtained from rabbits was confirmed by Western blot assay. In response to serum withdrawal, cultured HITASY cells exhibited phenotypic conversion from synthetic into contractile one as evidenced by the data of [(3)H]-thymidine incorporation and Western blot. The DNA synthesis of HITASY precipitously dropped to background levels after serum withdrawal and nearly restored after reintroduction of serum to culture medium 2 days later. Western blot revealed a reversible upregulation of smooth muscle alpha-actin and calponin in HITASY after serum deprivation. Moreover, serum withdrawal also induced a prominent increase of CREG mRNA and protein expression which reached a peak on 3 days and decreased gradually on 5 approximately 7 days after serum withdrawal. Immunohistochemistry stain indicated the CREG protein mainly localizes in a perinuclear pattern in HITASY cells.Those data provide evidence that the coordinated changes in CREG gene and protein expression as well as smooth muscle-specific markers may take place in connection with the process of phenotypic modulation of VSMC in vitro.

Smad proteins transduce signals from transforming growth factor beta superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. TGF-beta/Smads signal pathway not only has transforming potential but can also drive tumourigenesis, malignant progression, invasion and metastasis of human cancers. Using the immuno-histochemistry, we investigate the expression and location of TGF-beta R II, Smad2, Smad4 and Smad7 in 20 lung cancer specimens and 8 lung cancer cell lines. The results suggest that aberrant smads protein expression is significantly related to lung cancer tumoruigenesis and progression. Interestingly, TGF-beta R II and Smad7 strongly express in high metastasis cell lines. High expression of TGF-beta R II and smad7 in the cell lines with high-metastatic potential showed a conceivable TGF-beta signal pathway independent Smads in the lung cancer, and that might mediate invasion and metastasis of lung cancer.

To study the growth inhibitory effects of p21WAF1 and p53 overexpression in human lung adenocarcinoma cell line.The p21WAF1 and p53 gene were transfected respectively into a human lung adenocarcinoma cell line, GLC-82. Flow cytometry (FLC), transmission electron microscopy (EM) and TUNEL technique were used to evaluate cell growth and identify apoptosis.The GLC-82 transfected by p21 plasmid showed increased cell number in G1 phase of cell cycle, decreased proliferation potential and decreased cloning efficiency. Apoptosis have not been detected neither on EM nor by TUNEL technique, whereas the GLC-82 infected by Ad-p53 showed significantly decreased proliferation potential and some of them even died, in addition apoptosis was confirmed by TUNEL technique.The results indicate that p21WAF1 and p53 can inhibit proliferation; p53 also can induce apoptosis of lung adenocarcinoma cell. Therefore, these two genes should have a wide application in gene therapy of tumors in future.