Abstract:Aim To explore the expression and significance of cyclooxygenase(COX-2)during the focal ischemic reperfusion injury of middle cerebral artery in rats.Methods The model of focal ischemic reperfusion injury of middle cerebral artery was built in rats by placing an intraluminal suture.80rats were divided into8groups,which were control group,ischemia for2hours,ischemic reperfusion for3hours,6hours,12hours,24hours,48hours,72hours.The nerve function was evaluated and the change of brain tissue injury was observed by HE stain.Northern blot,Western blot and immunohistoche-mistry were used to dectect the expression of COX-2.Results Manifestation of neurologic impairment gradually aggravatedwith the extension of reperfusion time.The mRNA and protein expression of COX-2increased significantly after ischemia for2hours,and gradually reinforced after reperfusion,which reached the peak from12hours to24hours after reperfusion(p<0.01).Conculsion The expression of COX-2gene in focal cerebral iscliemia reperfusion injury was dynamic changing process.The expression of COX-2protein may be involved in the delayed neuronal death after ischemic reperfusion injury.

Abstract:Aim To study on the effect of over-expressing acyl coenzyme A cholesteryl acyltransferase-1 (ACAT-1) gene in the formation of foam cells in three kinds of cells. Methods ACAT-1 gene were transfected into cultured human THP-1 monocytic cells, human embryonic kidney 293 (HEK293) cells and murine macrophages RAW264.7 cells. The over-expressing ACAT-1 gene cells were incubated with acetyled low density lipoprotein (ac-LDL) and stained with oil red O to detect the formation of foam cells. Results The formation of foam cells became easier from over-expressing ACAT-1 gene THP-1 monocytic cells and RAW264.7 cells than control. But no apparent changes were observed in over-expressing ACAT-1 gene HEK293 cells. Conclusion Over-expressing ACAT-1 gene accelarated the formation of foam cells in monocytic and macrophagic cell lines.

Abstract:Aim To investigate the effect and mechanism of retinoid X receptor agonist 9-cis retinoid acid on the differentiation of macrophage into dendritic cell induced by LPS. Methods LPS-treated RAW264.7 murine macrophage cell line differentiated into dendritic like cells after 48h. The effect of 9-cisRA on the differentiation induced by LPS was studied. Cell morphology was observed by phase contrast microscope and cell surface markers involved in dendritic cell immune maturation and activation (CD40, CD86, CD83) was analyzed by FACS. Cellular reactive oxygen species production was detected by CM-H2DCFDA fluorescent probe. Results 9-cisRA partly prevented dendritic like morphology induced by LPS. Upregulated cell surface markers CD40、CD86 and CD83 by LPS were decreased about 50%、40% and 38% respectively by 9-cisRA. And the effect of 9-cisRA was dose dependent. LPS-treated RAW264.7 acquired significantly increasing cellular reactive oxygen species (MFI 98.9±9.6 vs 12.3±1.8,p<0.05), which was significantly reduced by 9-cisRA at 10-8M and 10-7M to 69.4±5.8 and 37.0±4.2 respectively. Conclusion RXR agonist 9-cisRA partly inhibits the differentiation of macrophage into dendritic cell induced by LPS, which may be related to reducing oxidative stress injury.

Abstract:Aim To observe the effects of dehydroepiandrosterone on expression of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in vascular smooth muscle cells, and investigate whether its mechanism has something to do with the catalysis of the cytochrome P450 aromatase (CYP19). Methods Transient transfected the plasmid with or without CYP19 into cultured vascular SMC respectively by lipidosome transfection. 24 h later, the cells were stimulated with ox-LDL and DHEA. Using the method of RT-PCR, Realtime PCR, ELISA, the gene and protein of MCP-1 expression levels of each group were detected. Results Compared with the group stimulated by ox-LDL, the secretion level of MCP-1 was obviously reduced after given ox-LDL and DHEA (p<0.05). There wasn’t obvious difference of MCP-1 expression in the groups of transfected plasmid with or without CYP19 (p>0.05). Conclusion DHEA shows inhibiting effects on ox-LDL-induced MCP-1 expression in vascular SMC, which may be one of the mechanisms of its antiatherosclerosis. And this effect is not mediated by CYP19.

Abstract:Aim To clone human vascular endothelial growth factor-165 (VEGF-165) gene and human fibroblast growth factor-2 (FGF-2), and clone core hypoxia-response enhancer RTP801 (RTP801HRE) of RTP801 promoter. Using plasmid pIRES as backbone to construct eukaryotic expression vector, then transfecting it into human embryo kidney 293 cell and observing the expression of VEGF-165 and FGF-2 under normal condition and anoxic condition. Methods The 337 bp～511 bp of core hypoxia-response enhancer (core HRE) in RTP801 promoter was obtained by polymerase chain reaction (PCR) from mouse genomic DNA, to replace the CMV enhancer (150 bp～390 bp) in pIRES. The fragment containing VEGF-165 and FGF-2 was acquired by PCR, and then inserted into the recombinant plasmid pIRES/Igκ-VEGF-165/IRES/Igκ-FGF-2, and transfected recombinant plasmid pIRES/RTP801HRE/Igκ-VEGF165/IRES/Igκ-FGF-2 into the 293 cells with biodegradable hyperbranched polyethylenimine (PEI) in vitro. The infected cells were cultivated for 36 hours under normal and anoxic condition respectively. The expression of VEGF-165 and FGF-2 were detected by Western blot and ELISA. Results Eukaryotic expression vector pIRES/RTP801HRE/Igκ-VEGF-165/IRES/Igκ-FGF-2 has been confirmed to be successfully constructed by PCR, enzyme digestion and sequencing. Highly efficient co-expression of VEGF-165 and FGF-2 by the recombinant plasmid under the regulation of the RTP801 core enhancer in anoxic 293 cells has been shown by both Westernblot and ELISA. Conclusion The RTP801 core hypoxia-response enhancer can enhance the expression of downstream genes, so the constructed eukaryotic vector which contains this enhancer can high-efficiently express VEGF-165 and FGF-2 under anoxic condition

Abstract:Aim To investigate the effect of selective retrograde coronary venous bypass (SRCVB) on nitric oxide (NO) and endothelin (ET) of myocardial ischemia model in dogs. Methods 16 dogs were divided into ischemia group (n=8) and SRCVB group (n=8). The levels of plasma NO and ET were measured in each group. In SRCVB group, 10 mL ink were utilized to perfuse the heart through SVG graft in vivo. Results The differences of NO and ET were not statistically significant before ligation of PDA in both groups (p>0.05), but changed significantly in SRCVB group after the ligation (p<0.01). The ink perfused retrogradely from graft distributed the myocardium tissue uniformly. Conclusions SRCVB could guarantee effective perfusion of myocardium and ameliorate the abnormal concentration of NO and ET in ischemic myocardium, decrease injury to endothelial cell and protect the heart.

Abstract:Aim To design and construct the expression vector that can express the short interfering RNA(siRNA) against apolipoprotein M (ApoM)gene, screen the effective target sequence of siRNA and explore the function of ApoM. Methods The four siRNA against ApoM gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilencerTM 2.1-U6, and the ApoM gene was inserted into the reporter gene in order to construct the recombinant plasmid vector plucF-ApoM. The recombinant plasmid and siRNA producing plasmid were cotransfected into 293T cells and screened out the effective siRNA that inhibiting the expression of luciferase, siRNA was transfected into L-02, the expression of ApoM mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) to further identify the inhibiting function of siRNA on ApoM expression, enzyme linked immunosorbent assay (ELISA) method was used to detect the effect of effective siRNA on synthesis and secretion of apolipoprotein A_Ⅰ (ApoA_Ⅰ), apolipoprotein B (ApoB), apolipoprotein E (ApoE). Results Two siRNA of the four synthesized siRNA displayed inhibitory effects on the lucifermase expression with the inhibitory rate being 86% and 91% respectively, the expression of ApoM mRNA was specially inhibited, effective siRNA could inhibit synthesis and secretion of ApoA_Ⅰ effectively (p<0.05), while there was no change in ApoB and ApoE secretion (p>0.05). Conclusion Effective siRNA plasmids against ApoM gene were constructed and screened out successfully which created a favourable condition for exploring the mechanisms of coronary heart disease.

Abstract:Aim To study the relation of myocardial apoptosis and interstitial fibrosis in non-infarct zone during the subsequent stages after infarction and its mechanisms by setting up an infarcted model of rat. Methods 10-12 femal rats underwent sham operation or left coronary artery ligation, were killed after 24 h, 1 week, 2 weeks and 4 weeks of operation respectively. Left ventricular structure was observed by HE staining, Messon staining was used for the determination of collagen volume fraction (CVF), TUNEL was used for detection of apoptotic cells, radioimmunoassay was used for cardiac AngⅡ and Ald contents and reverse transcriptase-polymerase chain reaction (RT-PCR) for genes expression of collagen type 1 and collagen type 3. Results The infarction sizes were 24%-33%. From 7 days to 28 days after operation, the apoptotic cells and the apoptotic index increased, the Ang Ⅱ level increased gradually (p<0.05～0.001), collagen type 3 mRNA grey level was significantly higher in MI group (p<0.05). From 14 days to 28 days after operation, the CVF increased gradually along with the course of infarction (p<0.05～0.01), ALD level increased gradually and collagen type 1 mRNA grey level was markedly higher in MI group (p<0.05～0.01). Conclusions ①Apoptosis happened in non-infarct zone 14 days after MI in rats. ②There was interstitial fibrosis in non-infarct zone 14 days after MI in rats. ③The interstitial fibrosis might be associated with activation of cardiac RAAS. ④The apoptosis of cardiocytes in non-infarct zone might be related with the excess deposition of interstitial collagen.

Abstract:Aim To study how Hcy leaded to NF-κB activation and induced endothelial inflammation and how simvastatin antagonized the inflammatory response. Methods Human umbilical vein endothelial cells (hUVEC) were treated with Hcy, with or without simvastatin, for different times. Dynamic changes of NF-κB activation were measured by electrophoretic mobility shift assay (EMSA), TransAMTM NF-κB P65 assay system. Western blotting was performed to detect inflammatory proteins expression respectively. Results Treatment with both low (0.5 mmol/L) and high (3.0 mmol/L) concentrations of Hcy induced hUVEC NF-κB activation was accompanied by an increased level of MMP-2, MMP-9 and IL-1β expression. The NF-κB activation reached its maximum at 30 min and 6 h induced by high (3.0 mmol/L) concentrations of Hcy. EMSA and Western blotting showed Hcy induced NF-κB activation due to the increased phosphorylation of the inhibitory protein (IκBα) as well as the degradation of IκBα, while simvastatin almost completely blocked the NF-κB activation as well as the phosphorylation and degradation of IκBα. Conclusion Hcy may induce hUVEC inflammation via a pathway involving IκBα and NF-κB and simvastatin can inhibit homocyteine-induced endothelial dysfunction and inflammatory response.

Abstract:Aim To elucidate effects of aspirin on the proliferation of vascular smooth muscle cells (VSMC) and the mechanism involved. Methods The rat aortic smooth muscle cells were treated with various concentrations of aspirin for 24 hours, then MTT assay was performed to determine the number of the cells. The expression of proliferating cell nuclear antigen (PCNA) was detected by immunocytochemistry. In addition, reverse transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression of PCNA. Results High concentration (5×10-3 mol/L) of aspirin inhibited the proliferation of VSMC and the relative inhibition ratio was 31.5%. Compared with the control group, the expression of PCNA protein and mRNA in the 5×10-3 mol/L aspirin group were significantly decreased (312±48 and 0.663±0.035, p<0.05). Conclusion Aspirin may arrest the proliferation of VSMC by inhibiting the expression of PCNA.

Abstract:Aim To investigate the effect of Angiotensin(1-7) (Ang(1-7) on cell proliferation and collagen synthesis of cardiac fibroblasts(CF) induced by Angiotensin Ⅱ (Ang Ⅱ). Methods CF were isolated and cultured from Neonatal SD rats. Cell number and cell cycle were evaluated by MTT assay and flowcytometry technique. Type and Ⅲ collagen mRNA expressions in CF were analyzed by RT-PCR. Results ①1 μmol/L Ang Ⅱ significantly increased absorption value of CF(0.24±0.01)compared with control group (0.14±0.01, p<0.01), 0.001～1.0 μmol/L Ang(1-7) attenuated the absorption value of Ang Ⅱ group in a concentration dependent manner (0.22±0.01, 0.21±0.01, 0.18±0.01 and 0.16±0.01, p<0.01). ②Compared to the control (5.4%±0.7%, 10.8±2.4), cells in S stage (14.0%±0.9%) and proliferatiion index (23.4±1.8), they were markedly increased in 1 μmol/L Ang Ⅱ group (p<0.01). In the presence of 0.1 μmol/L Ang(1-7), cells in S stage (8.5%±0.7%) and the proliferatiion index (16.2±2.0) were significantly lower than that of Ang Ⅱ group (p<0.01). ③mRNA expressions of typeⅠand Ⅲ collagen in Ang Ⅱ group were significantly higher than those of the control (p<0.01), and in the presence of 0.1 μmol/L Ang(1-7), mRNA expressions were reduced in a concentration dependent manner (p<0.05). Conclusion The results indicate that Ang(1-7) could inhibi the proliferation of CF and the synthesis of type Ⅰand Ⅲ collagen.

Abstract:Aim To investigate the effects of C-reactive protein (CRP) on expression of matrix metalloproteinase-2 (MMP-2) in human umbilical vein endothelial cells (hUVEC)and the influence of CRP on matrix remodeling in atherogenesis and plaque rapture. Methods hUVEC were cultured in vitro and intervented by different concentrations of recombination human CRP and provastatin. The MMP-2 mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MMP-2 protein was measured by Western blot. Results In comparison with the control, the expression of mRNA and protein of MMP-2 was significantly increased in 20 mg/L and 100 mg/L CRP groups (p<0.05), and this up-regulation of the expression of MMP-2 could be inhibited in provastatin group. Conclusions CRP can enhance the expression of MMP-2 in hUVEC and may cause advanced inflammation in athelosclerosis plaques. It may provide an explanation for the phenomenon that patients who have high concentration of CRP are prone to have atherosclerotic lesions and plaque rapture.

Abstract:Aim To probe into the effect of proteasome inhibitor MG-132 on myocytic apoptosis in rat with myocardial ischemia-reperfusion injury. Methods The ischemia-reperfusion model was established after 30 min ligation of left anterior descending (LAD) coronary artery. MG-132 (0.75 mg/kg in 2 mL DMSO) was injected 5 min prior to reperfusion. Electron microscopy, histology, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) method, and reverse transcription-polymerase chain reaction was applied to observe myocardial cell apoptosis. Results Functional effects of MG-132 on polymorphonnuclear neutrophillic leukocytes(PMN) accumulation, activation of nuclear factor kappa B (NF-κB) p65 mRNA and protein levels, and apoptosis were characterized in rat myocardial tissue. MG-132 time-dependently inhibited myocardial p65 mRNA expression and reduced myocardial apoptotic index (AI) after reperfusion for 2 h, 6 h and 24 h (p<0.01, respectively). Moreover, MG-132 time-dependently decreased Bax protein levels, while increased Bcl-2 protein levels in ischemic and reperfused myocardium (p<0.05), these effects peaked 24 h after reperfusion. Conclusion MG-132 reduced myocardial reperfusion injury by inhibiting myosytic apoptotic cell death and blocking the activation of NF-κB, down-regulating Bax expression and up-regulating Bcl-2 expression as well as elevating Bcl-2/Bax ratio.

Abstract:Aim To observe the serum ferritin (SF) expression in cerebral infarction and carotid atherosclerosis. Methods Carotid atherosclerosis of all enrolled patients were evaluated by ultrasonography and the level of SF were detected in blood plasma. The SF content in cerebral infarction group was compared with control group; the relationship between SF and carotid interia-media thickness (IMT)/carotid plaque scores was analysed. Results 77 (70.6%)patients were detected to have carotid atherosclerosis in cerebral infarction group, and 30 (39.5%) patients were detected to have carotid atherosclerosis in control group. The level of SF in cerebral infarction group was higher than that in control group (248.5±107.4 μg/L vs 197.6±94.8 μg/L, p<0.05). Partial correlation analysis of SF and carotid IMT/ plaque scores showed positive correlation, and the partial coefficient were 0.56 and 0.48 respectively (p<0.05). The level of SF was positively correlated with low density lipoprotein in plasma, and the pearson coefficient was 0.51 (p<0.05). Conclusions The SF level increases in cerebral infarction patients; the level of SF is positively correlated with the severity of carotid atherosclerosis.

Abstract:Aim To observe the changes of serum level of the CD62p, CD63, glucose protein (GP)_b/Ⅲ_a which indicated the platelet activity and serum levels of von willebrand factor (vWF), flow-mediated dilatation (FMD), endothelium 1 (ET-1), nitrogen oxide (NO) which indicated function of vascular endthelium after percutaneous coronary intervention (PCI) in patients with acute coronary syndrome (ACS). Methods The expression levels of CD62p, CD63, GP_b/Ⅲ_a and vWF, ET-1, NO and FMD were examined in brachial artery at the same time using ultrasonography from before treatment, immediate and 24 hours after treatment of PCI in 60 patients with ACS and compared with control. Results The ACS patients' blood CD62p, CD63, GP_b/Ⅲ_a levels increased significantly compared with healthy control group and stable angina pectoris (SAP) (p<0.05 or p<0.01), and those of post PCI immediate group increased significantly than pre-PCI group ( p<0.01). There were no significant difference (p>0.05) between post-PCI 24 hour group and pre-PCI group. The blood vWF and ET-1 levels of ACS patients increased significantly (p<0.01), FMD and NO decreased significantly (p<0.05 or p<0.01) compared with healthy control group and SAP group. The vWF and ET-1 levels of post-PCI immediate group increased significantly (p<0.05 or p<0.01), FMD and NO decreased significantly (p<0.05) compared with pre-PCI group, and vWF and FMD had significant difference (p<0.05) between post PCI 24 hour group and pre-PCI group. While ET-1 and NO had no significant difference (p>0.05) between post-PCI 24 hour group and pre-PCI group. Conclusion Platelet activation and vascular endothelial cell impairment played an important role in the occurrence and development of ACS. The vascular endothelium function was impaired and platelet were obviously activated to some extent in patients with ACS after PCI immediate.

Abstract:Aim To evaluate the diagnosis value of 64-row spiral CT in follow-up investigation of patients after coronary artery revascularization. Methods 28 patients underwent coronary artery bypass graft (CABG) and 114 patients underwent percutaneous coronary intervention (PCI) were performed with 64-row spiral CT coronary angiography and left ventricular function analysis. The restenosis of the grafts or stents and the variation of left ventricular function were evaluated, in the purpose of comparison of CABG and PCI. Results Patients underwent CABG and PCI got an evident increase of left ventricular ejection fraction (LVEF) and left ventricular stroke volume (LVSV). Follow-up investigation showed patients underwent CABG got a lower rate of restenosis and a better long-term curative effect. Conclusion 64-row spiral CT is of great clinic value in follow-up investigation of patient underwent revascularization.

Abstract:Aim To observe the clinic and the coronary artery’s aspect of coronary heart disease. Methods 967 patients of coronary heart disease diagnosed by coronary artery angiography were collected, and divided into three groups according to the level of blood glucose: the DM, the IGT and the NBG, and their clinic aspect and the results of coronary artery angiography were compared. Results The stroke and the coronary artery of left main and left anterior descending branch is higher, the age is younger in DM than in NBG (p<0.05), the multi-artery diseases and the grave artery diseases are higher in DM and in IGT than in NBG(p<0.05). Conclusions The coroanry heart disease with DM or IGT of ten have extensive and grave artery diseases, so we should control the level of blood gloucose from many aspects as early as possible.

Abstract:Aim To investigate the relationship of the concentration of serum resistin and insulin resistance in the elderly hypertension patients. Methods The patients (the young 41,the old 43) were divided into hypertension group and normal group. The concentration of fasting serum resistin was detected by enzyme linked immunosorbent assay (ELISA), fasting blood sugar and insulin, serum lipid, and insulin sensitive index (ISI) were determined too, at the same time, waist circumstance,hip circumstance and waist haunch ratio (WHR) of the people involved were measured. Results The concentration of fasting serum resistin was significantly higher in the elder group than that in control group (p<0.05). There was no difference of serum resistin between the hypertension group and normal blood pressure group in the elder patients, and it was little higher in hypertension group than that of normal blood pressure in the control group but the difference was not significant (p>0.05). Plasma resistin was positively correlated with age (r=0.31,p<0.01;r=0.28, p<0.05). The concentration of fasting serum resistin was positively correlated with body mass index (BMI) (r=0.23,p<0.05), negatively correlated with ISI (r=-0.31,p<0.05). But there were no significance between the concentration of resistin and BMI among the test groups. BMI, WHR, blood pressure, serum lipid, fasting serum sugar and insulin was not found to be related to the concentration of serum resistin. Conclusion Fasting plasma resistin was positively associated with age and BMI, but negatively associated with ISI.