Abstract:Aim To clone a novel rabbit’s gene involved in hyperlipidemia. Methods The hypercholesterolemic rabbits were obtained after feeding atherogenic diet for 150 days. The differentially expressed gene was obtained by suppression subtractive hybridization and rapid amplification of cDNA end. After identified by cDNA microarray and fluorescent semi-quantitative PCR, its tissue distributing was analyzed. Results A novel rabbits gene involved in hypercholesterolemia had been cloned. GenBank Accession No. is AY248719. The full length of cDNA is 1 386 bp and encodes 321 amino acids, which has a 92% identity with that of human GANS1 gene. Conclusions These results will contribute to reveal the molecular mechanism of the hyperlipidemia

Abstract:Aim To study the large amount of differentially displayed genes and expressed sequence-tags (EST) obtained by gene clone is a difficult problem. It is important to exploit new methods for further study. Methods EST were acquired from human umbilical vein endothelial cells induced by cholesterol by suppression subtractive hybridization (SSH). Bioinformatics analysis of atherosclerosis related genes was done by the bioinformatical databases and bio-softwares such as BLAST, ExPasy analyse soft box, DNAMAN soft, BioEdit soft and RasMol soft. Results We obtained a 684 bp complete cDNA sequence by electronic clone, which was homogeneous to cytochrome C oxidase subunit Ⅱ gene (COX2). The complete genome of COX2 sited in homo sapiens mitochondrion, and included a complete open reading frame (ORF) coding 227 amino acids. The analysis of its protein sequence indicated that COX2 gene encode a 25.6 kDa protein, which was a weak acid signal anchor. The three-dimensional structure of COX2 was a representative chair-like stucture, containing a hydrophobicity region, a transmembrane domain and a periplasmic domain. The periplasmic domain of the COX2 protein sequence was high conservative in the process of evolutions by the analysis of the protein evolutions. Conclusion We obtained the information of COX2 about nucleic acid sequence and protein sequence by bioinformatics analysis

Abstract:Aim To explore the preparation of the polyclonal antibody of a novel Homo sapiens synapse associated protein (FRG4). Methods FRG4 full-length sequence was obtained by PCR from human fetal liver library, by bioinformatics to detect the second structure of amino acids encoded by FRG4 and its epitope and motifs, according to the hydrophility, second structure, coupling and experiment difficulty etc, the peptide with 13 amino acid (PKLVKEEVFWRNY) was selected and synthesized by Solid-phase Peptide Synthesis (SSPS) method. After coupling,the peptide was immunized to rabbits through the basic and 6 fortified-immunities. 10 to 14 days later just after the fourth fortified-immunity,serum,separated from blood exsanguinated from carotid,was lyophilized and then stored at -20℃. Results Analyzed by ELISA and Western blot,the antibody dilution was 1∶16 000 and the blot was showed at the location of 40 kDa,the FRG4 protein was detected in cytoplasm of HepG2 cells by immunohistory. All these proved that the antibody has a good reaction and speciality. Experiments showed that the SSPS method was convenient to control the synthesis of peptides, spare time and labours,also obtain a good dilution. Conclusion A novel Homo sapiens synapse associated protein (FRG4) antibody was synthesized successfully,and worked as a foundation to the further study of FRG4 protein function playing in cardiovascular disease.

Abstract:Aim To elucidate the role of the motifs in the cytoplasmic domain of scavenger receptor A (SR-A). Methods A SR-A DNA mutant (SR-AΔ_ 1-27) was constructed by truncating the 1 to 27 amino acid residues of the N-terminal cytoplasmic domain. SR-A and SR-A_ Δ1-27 were transfected into CHO cells by Lipofectamine 2000. Western blot, laser confocal microscopy, flow cytometry and cell adhesion assay were used additionally. Results Compared with SR-A, expression of the SR-A_ Δ1-27 was increased and amount of cell-associated Dil-labeled ac-LDL in SR-A_ Δ1-27 expressing cells were almost reduced 80%. SR-A_ Δ1-27 was able to assemble in the plasma membrane and cytoplasm. SR-A_ Δ1-27 could bind its lipoprotein ligands but decline to internalize the bound ligands greatly. Incited by Ac-LDL, SR-A_ Δ1-27 located not only in membrane and plasma, but also in the nuclear. The SR-A_ Δ1-27 display decreased adhesion. Conclusion The 1 to 27 amino acid residues of the N-terminal cytoplasmic domain mediate SR-A expresssion and location, lipid uptake and cell adhesion

Abstract:Aim To observe the mRNA expression of endothelial nitric oxide sythase (eNOS) at different time intervals after acute myocardial infarction in rat hearts and examine the effect of acute myocardial ischemic stimulation on the protein production of inducible nitric oxide sythase (iNOS) in rat hearts. Methods Forty-eight healthy Sprague-Dawley rats (200～250 g) were randomly divided into two groups: control group and ischemia group. Myocardial ischemia was induced by the left anterior descending coronary artery (LAD) ligation. Animals with LAD occlusion (ischemia group) were sacrificed at 1 h, 2 h, 8 h, and 24 h after operation. The eNOS gene expression in ischemic myocardium was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) standardized with β-actin; the iNOS protein in spare cardiomyocytes in the infarcted region after operational 8h was detected using immunohistochemedical analysis. Results Ligating of LAD for 2 h decreased significantly the eNOS mRNA levels in ischemic myocardial tissue compared with that in controls (p<0.05). The decent of eNOS mRNA level sustained for post-operational 24 h. There is no significant difference in expression level between ligating for 2 h group and ligating for 24 h group. iNOS protein was abundant in the 8 h infarcted heart in spared cardiomyocytes in the infarcted region. Staining of sham-operated rat hearts indicated the absence of iNOS protein. Significant difference in expression levels were observed between ischemia experiment groups and sham-operated control groups (p<0.01). Conclusion The eNOS gene expressed in normal rat hearts, whereas there was no iNOS protein expression in normal rat hearts. The expression of eNOS mRNA in ischemic myocardium after myocardial infarction in rats decreased in the early time. Acute myocardial infarction induced iNOS protein levels expressing abuntantly in the ischemic myocardium tissue in rats.

Abstract:Aim To investigate the dynamic expression of transforming growth factor-β1(TGF-β1), matrix metalloproteinase-1(MMP-1), and matrix metalloprotinase tissue inhibitor-1 (TIMP-1), and their relations with intimal hyperplasia in autogenous vein grafts in rats. Methods Animal models were constructed. The vein grafts were collected on day 3, day 7, day 14, and day 28 after operations. HE and Verhoffe staining and computer image analysis system were used to explore the thickness changes of intima and wall at different time points. Western blot and reverse transcription polymerase chain reaction(RT-PCR) were used to detect the protein and mRNA level of TGF-β1, MMP-1, and TIMP-1 in vein grafts and controls. Results Histomorphological analysis showed that the intimal thickness increased remarkably on day 7 after operation compared with control(p<0.05), and reached a peak on day 14. Western blot revealed that protein level of TGF-β1 increased on day 3, peaked on day 7 and returned to the baseline on day 28. MMP-1 protein was not significantly different with control on day 3 and 7 respectively, but decreased significantly on day 14 and 28. TIMP-1 protein increased significantly on day 14 and 28. Their mRNA level had similar tendency with the results of Western blot. Conclusions TGF-β1 decreased MMP-1 activity by increasing TIMP-1 expression, and facilitate the excessive accumulation of ECM so as to cause intimal hyperplasia and stenosis.

Abstract:Aim To analyze the specificity of the on/off switch mediated by high fidelity proofreading DNA polymerases and 3'-phosphorothioate-modified allele-specific primers for screening certain sites of single nucleotide polymorphism (SNP) in genomic DNA. Methods The genomic DNA is used as the template. The 3'-phosphorothioate-modified allele-specific primers were evaluated in their effects on primer-extension with genomic DNA harboring SNP sites allele. Results The products from Taq had many mixed bands, and some of them clearly showed false positives. However,at most of the situations, high fidelity DNA polymerase of Pfu yielded single products. Conclusion These data suggest that the “on /off-switch" mediated by exo+ polymerase is more reliable as compared to exo-polymerase in SNP assay and the novel “on/off” switch has enormous application in the diagnosis of monogenic diseases.

Abstract:Aim To investigate the effect of advanced glycation end products (AGE) modified protein on morphological changes of actin cytoskeleton in endothelial cell and the role of receptor for AGE (RAGE) and oxidant stress in this pathological procedure. Methods Human umbilical vein endothelial cells (hUVEC)-derived cell line (ECV304) were incubated with AGE modified human serum albumin (AGE-HSA) of concentrations of 12.5, 25, 50, and 100 mg/L respectively, for 2, 4, 8, 12 and 24 h. As control, HSA of the same concentration was administrated to ECV304 cells. To visualize the morphological changes of actin cytoskeleton,the treated cells were incubated with rhodamine-phalloidin or Oregon Green-Dnase I to stain F-actin or G-actin. ECV304 were treated with anti-RAGE IgG before AGE-HSA applications or both together. Apocynin, a specific inhibitor of NADPH oxidase, was pre-administrated to the endothelial cells in concentrations of 10, 100, 250, and 500 μmol/L respectively, for 30 min, then the cells were rinsed with DMEM for three times and exposed to 50 mg/L AGE-HSA for 8 h. The morphological changes of actin cytoskeleton were observed after the above-mentioned procedure. Results Morphology of F-actin and G-actin in endothelial cells were changed greatly under the stimulation of AGE-HSA in a concentration and time-dependent manner. Exposure of ECs to AGE-HSA caused a shift in F-actin distribution from web-like structure to polymerized stress fiber. Cells subjected to higher-concentration and longer-time AGE-HSA exposure showed more and more stress fiber accumulation.Also the edge of G-actin became coarse and illegible and merged with each other, even formed tufts of flock extending all around nuclear area. And the changes can be inhibited by not only pretreatment with anti-RAGE IgG or NADPH oxidase inhibitor Apocynin, but also co-administrated with anti-RAGE IgG and AGE-HSA. The unmodified HSA,anti-RAGE IgG and Apocynin did not affect morphology of actin cytoskeleton. Conclusion AGE induced morphological changes of actin cytoskeleton in endothelial cells and it required receptor occupancy on cells surface and intracellular activation of NADPH oxidase.

Abstract:Aim To investigate the physiological role of Battenin interactive protein (BIP) and screen the proteins interacting with BIP in human fetal cDNA library. Methods The cDNA coding BIP and binding domain of plexA were recombined as a “bait", proteins interacting with BIP were screened from human fetal cDNA library by yeast two-hybrid system. Results Seven positive clones were isolated through plexA-HERPUD1 screening the human fetal brain cDNA library by yeast hybrid technique. One of them has 99% homology with the TEGE gene of human, which codes Bax inhibitor-1 (BI-1)(testis enhanced gene transcript). Bioinformatic analysis shows that: Bax inhibitor-1 which interacts with BCL-2 and BCL-X is a gene transcript enhanced factor and a regulatory factor of apoptosis and probably associated with gene expression regulation for cell's growth and development. The gene that codes Bax inhibitor-1 is TEGT located in chromosome 12q12-q13, containing 8 extrons and 7 introns. Conclusions The interactive proteins of BIP were screened through yeast two-hybrid system successfully, and one of the proteins is Bax inhibitor-1. Bax inhibitor-1 is a gene transcript enhanced factor and a regulatory factor of apoptosis and probably associated with gene expression regulation for cell's growth and development

Abstract:Aim So far, research on the relationship of mast cells, atherosclerosis and angiogenesis is mostly pathological observation and cytology experiments. Based on the status, compound 48/80 and apolipoprotein E-knockout mice were used to investigate the effect of mast cell degranulation on plaque. Methods 40 apoE-/- mice were fed a Western-type diet from 10 weeks old. At 16 weeks old, mice were divided into 2 groups and treated for 8 days as follows: group 1, mice were intraperitoneal injected with compound 48/80, 0.5 mg/kg, every other day; and group 2, control mice were intraperitoneal injected with buffer. After half an hour of the fouth injection, euthanasia, blood was collected from the orbit for measurement of plasma lipids and tryptase, mice were in situ perfusion fixed with 10% neutral buffered formalin and aortas were removed. Then aortas were fixed in neutral buffered formalin, and embedded in paraffin. Sections were routinely stained with hematoxylin and eosin. Corresponding sections on separate slides were stained immunohistochemically with antibodies against a macrophage-specific antigen (Mac3), α-smooth muscle actin, VE-cadherin. The images were analyzed with a Olympus BX51 microscope and HMIAS2000 software to distinguish the maximum cross section area of aorta plaque and expression of Mac3, α-smooth muscle actin, VE-cadherin between the two groups. Results Tryptase levels were 0.54±0.29 versus 0.36±0.13 mmol/L in the Compound 48/80 and control groups, respectively(p<0.05). There was no difference on serum lipids content. Compound48/80 group were compared with control group,the maximum cross section area of aorta plaque was significantly increased [(1.25±0.36)×10 4 μm 2 VS (0.79±0.24)×10 4 μm 2 in control group, p<0.05],the percent of Mac3+ cell was increased (58.6%±17.3% vs 28.5%±16.4%, p<0.05), the percent of α-smooth muscle actin cell was reduced (36.2%±14.9% vs 69.7%±31.3%, p<0.05) and the MOD of VE-cadherin of aorta endothelium was reduced (48±8 vs 65±10, p<0.05). Conclusions Compound 48/80 promotes mast cell degranulation and increase the maximum cross section area and the percent of Mac3+ cell, reduce the the percent of α-smooth muscle actin cell and the MOD of VE-cadherin of aorta endothelium of aorta plaque.

Abstract:Aim To construct and identified the eukaryotic expression recombinant plasmids containing caveolin-1 gene and mutants for the potential functional analysis. Methods Using a novel high fidelity gene expression profiling strategy mediated by Pfu proofreading polymerases combining with phosphorothioate modified primers, PCR-amplified ORFs of caveolin-1 and its mutants were inserted into a N-GFP tagging expression vector with the help of topoisomerase I-mediated ligation. After transformation into chemically competent One Shot TOP10 E.coli,the respective destination clones were selected for by plating on ampicillin selective agar plates. The correct orientation and reading frames of the constructs were identified through the PCR approach with the respective sense primer and the BGH antisense primer. The corrected reconstructs would add about 100 bp more than the RT-PCR fragements. The integrity of ORFs was verified by sequencing to exclude errors introduced in the amplification step. MTT and western blot approaches were carried out to determine whether the overexpressed tagged proteins present the same protein activities as endogenous proteins. Results We obtained 3 recombined caveolin-1 mutant plasmids. In addition,we demonstrated that overexpression of two caveolin-1 mutants in HepG2 cells could result in apoptosis of HepG2. However the other caveolin-1 mutant could promote the growth of HepG2 cells. The overexpressed tagged proteins present the same protein activities as endogenous caveolin-1 protein. Conclusion The eukaryotic expression recombinant plasmids containing caveolin-1 gene and mutants were successfully constructed.

Abstract:Aim To investigate whether caveolae/caveolin-1 participates in the progression of atherosclerosis. Methods C57BL/6J mice were fed with normal chow plus 1.5% cholesterol and 15% lord for 24 weeks. The area of atherosclerotic plaque, thickness of intima and media of aorta were determined by graphic analysis computer system; Electron microscopy was used to investigate the expression of caveolae in vascular smooth muscle cell (VSMC); the expression of caveolin-1 in vascular wall was detected by western blot. Results The serum levels of total cholesterol, triglyceride, and low density lipoprotein cholesterol in the atherosclerotic mice were elevated significantly by comparing with those in the normal mice. And the atherosclerotic lesion was widely distributed in the aorta of the atherosclerotic mice as shown by the lesion area of 3 744±340 μm 2 in the atherosclerotic mice. At the same time, the results of transmission electronic microscope showed that the caveolae in the VSMC from the atherosclerotic mice was fewer than that from normal mice. And no caveolae was observed in the foam cell derived from VSMC. Western blot demonstrated that expression of caveolin-1 decreased obviously in the aortic wall of the atherosclerotic mice. Conclusions It indicates that caveolae vary negatively with formation of VSMC and decreased expression of caveolin-1 may be the cause of the blocking of caveolae formation in the atheromatous area.

Abstract:Aim To study the therapeutic action of extract of maritine shellfish (EMS) on experimental atherosclerosis in quails. Methods The experimental atherosclerosis model in quails was induced exogenously by hyperlipidic feed for 8 weeks. Then EMS was orally given. The influence of EMS on lipid level in serum was detected with enzyme at the second and fourth weekend after administration. The effects of EMS on the atherogenic plaque in artery and fatty liver were observed by histopathological methods. Results At the second weekend after administration EMS 5, 10 and 20 g/kg significantly reduced the level of triglyceride (TG) in serum (p<0.05, p<0.01). EMS 20 g/kg evidently lowered the serum total cholesterol (TC) and low density lipoprotein cholesterol (LDLC). The high density lipoprotein cholesterol (HDLC) concentration was raised (p<0.05). In therapeutic oral administration for 4 weeks EMS 5, 10 and 20 g/(kg·d) markedly reduced the volume of TG, TC, LDLC in serum. At the same time, TC and TG in the myocardial and aortic wall were reduced (p<0.05, p<0.01). The serum HDLC was increased (p<0.05, p<0.01). Compared with model group, the degree of atherosclerotic lesion in aorta and coronary artery and fatty degeneration of liver in EMS group was reduced (p<0.05). Conclusion EMS shows action of the regression for atheroscleritic lesion and the regulation of hyperlipidemia in therapeutic administration.

Abstract:Aim To study the effect of monocyte chemotactic protein-1 (MCP-1) on the proliferation of human umbilical vascular smooth muscle cells (hUVSMC). Methods hUVSMC were cultured in vitro. Growth-arrested hUVSMC were stimulated with different concentrations of MCP-1 (0.1, 1.0, 10 and 100 μg/L). The response of hUVSMC to these treatments was observed in comparison with that of platelete derived growth factor (PDGF). The proliferation of hUVSMC was evaluated by cell counting, MTT assay and flow cytometry. The expression of c-fos mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Results MCP-1 stimulated the proliferation of hUVSMC in a dose-dependent-manner. Compared with PDGF, the effect of MCP-1 at high concentration was significant (p<0.01). Meanwhile, MCP-1 induced the expression of c-fos mRNA. Conclusions MCP-1 could promote the proliferation of hUVSMC and the expression of c-fos mRNA. The effect of MCP-1 at high concentration was more significant than that of PDGF. MCP-1 may exert its effects on cells through c-fos.

Abstract:Aim To elucidate the effect of adrenocoticoids on viral myocarditis. Methods Two hundreds male 4-week-old Balb/c mice were divided into five groups randomly, including normal control group, infected control group, group of infected mice with adrenocorticoids treatment at early stage, group of infected mice with adrenocorticoids treatment at middle stage, group of infected mice with adrenocorticoids treatment at late stage. Normal control group mice were inoculated intraperitoneally with Eagle’s medium 0.1 mL. Infected group were inoculated intraperitoneally of 10 7.5 TCID₅₀ Coxsackievirus B₃ at a dose of 0.1 mL per mouse. Five mice of each group were sacrificed on 3, 7, 10, 14, 21, 30, 40 and 50 days post-inoculation. Pathologic examination and ultrastructure of heart, virus titer in heart by plaque-forming assay, serum cardiac troponin I with chemiluminescence immunoassay were examined. Results The myocardial histopathological score of mice in each infected group on day 7 to 10 after infection were significantly higher than that in normal control group, but no significant difference in each infected group. On day 14 after infection, the myocardial histopathological score of mice with adrenocorticoids treatment at middle stage were significantly lower than that in other infected groups, and no myocardial lesions were noted 30 days later post-infection while cardiac lesions could be noted in other infected groups. Virus titer in group of infected mice with adrenocorticoids treatment at early stage was significantly higher than that of in other infected group. Cardiac troponin I values were significantly greater in infected group than that in normal control group. There was a positive correlation between the level of cardiac troponin I and the change of myocardial histopathological score. Cardiac troponin I values was much lower after adrenocorticoids therapy, especially in group of infected mice with adrenocorticoids treatment at middle stage. Conclusions Adrenocorticoids therapy ameliorated the severity of myocardial lesions in viral myocarditis, especially used in middle stage of the disease. Adrenocorticoids therapy reduced the inflammatory infiltration in myocardium, inhibited autoimmune response. Adrenocorticoids therapy reduced the level of serum cardiac troponin I in viral myocarditis.

Abstract:Aim To test the effects of Xanthone on the serum lipids and oxidative state of hypercholesterolemic rats. Methods In the preventive experiment, the rats were treated with Xanthone (10, 30 and 90 mg/kg) for 2 days and then treated with cholesterol for 10 days in the presence of Xanthone; In the therapeutic experiment, after the rats were fed with food containing cholesterol for 4 weeks to induce hypercholesterolemia, the animals were treated with Xanthone for 3 weeks. Serum lipid and malondialdohyde were measured. Results Xanthone, given preventively or therapeutically, markedly decreased total cholesterol and low density lipoprotein cholesterol level. Moreover, in the therapeutic experiment, xanthone also decreased malondialdohyde content. Conclusion Xanthone could decrease serum lipid and malondialdohyde level of hypercholesterolemic rats.

Abstract:Aim To investigate the effects of quercetin (QUE), isorhamnetin (ISOR) and norepinephrine (NE) on collagen synthesis of cultured human aortic vascular smooth muscle cell (VSMC). Methods Human aorta VSMC was cultured with QUE, ISOR, NE, phentolamine(Ph). Collagen synthesis was evaluated by measuring 3 H-proline incorporation. Results NE 10 μmol/L could stimulate collagen synthesis of VSMC, and the most effects of the stimulation occurred at 72 h. The effects can be inhibited by Ph. QUE and ISOR could inhibited collagen synthesis of VSMC. When VSMC were treated by 1～200 μmol/L of QUE or ISOR, the best inhibitory effects occurred at 200 μmol/L. There were dose-dependent inhibitory effects in different concentration of QUE and ISOR and the best effect of QUE occurred at 72 h. QUE and ISOR markedly inhibit collagen synthesis of VSMC induced by NE in a dose-dependent manner and the best effects occurred in 72 h after treated with QUE or ISOR. At the same time QUE and ISOR had cooperative effects on inhibition of the stimulation of NE. QUE and ISOR had more powerful inhibitory effect on collagen synthesis of VSMC co-incubation with NE than without NE. QUE and ISOR also had more powerful inhibitory effect on collagen synthesis of VSMC co-incubation with NE than Ph. Conclusions QUE and ISOR could effectively inhibit the collagen synthesis of VSMC, especially inhibit the collagen synthesis of VSMC stimulated by NE.

Abstract:Aim To investigate the process of neointimal formation, level of platelet activation and thrombin receptor mRNA by means of a rat aortic balloon injury model in order to study the mechanism of acute and late restenosis after percutaneous transluminal coronary angioplasty(PTCA). Methods Forty eight male Wistar rats were divided into two groups randomly. Group 1 (n=24) served as controls, group 2 (n=24) were given aortic balloon injury by self-made 2F balloon catheters. The process of intimal thickening, number of platelet GMP-140 and level of thrombin receptor mRNA were investigated at day 3, 7, 14 and 28 after balloon injury by histological method, radioimmunological method and reverse transcription polymerase chain reaction (RT-PCR) technique, respectively. Six rats were examined at each group. Results The expression of thrombin receptor mRNA was at a low level in endothelium and smooth muscle cell of normal arteries, but increased significantly at day 3 after balloon injury, reached its peak at day 14 and decreased at day 28. The number of platelet GMP-140 was higher at day 3 and began to decrease at day 7 after injury. The migration and proliferation of vascular smooth muscle cell had existed at day 3 after balloon injury. The intimal thickening began at day 7 after injury, and it was more significant at day 14. The proliferation of VSMC decreased at day 28, but extracellular matrix increased and the intimal thickening continued. Conclusions The expression of TR mRNA and the number of platelet GMP-140 increase in the process of intimal thickening after balloon injury.

Abstract:Aim To investigate the mechanism of activation of nuclear factor-κB(NF-κB) by advanced glycation end products (AGE) in endothelial cell. Methods Human umbilical vein endothelial cell(ECV304) were stimulated with AGE-human serum albumin(AGE-HAS), the level of IκBα was mesured by Western blot, and the activation of NF-κB was detected by electrophoretic mobility shift assay(EMSA). Results The degradation of IκBα and the activation of NF-κB could be induced by AGE-HSA in time and dose dependent way in ECV304. The activation of NF-κB could be blocked by inhibiting the degradation of IκBα. Conclusions AGE-HSA might activate NF-κB in IκBα degradation dependent in ECV304. This pathobiological effect of advanced glycation end products might contribute to the development of atherosclerosis.

Abstract:Aim Restenosis after percutaneous transluminal coronary angioplasty was one of the main clinic problems at present. A successful restenosis animal model was essential for its solution. So the rat and rabbit carotid restenosis models were built and compared to select more practical ones. Methods Balloon catheter was used to injure one of rats’ and rabbits’carotids. One week later the treated carotids were cutted, sliced and HE stained. Rat carotid well cells were cultured in vitro and analysed with α-actin immunohistochemistry. Results The main histopathologic changes of rabbit carotids after balloon injury were smooth muscle transfer and blood cells adheration. While the main change of rat’s was cell proliferation,and α-actin immunohistochemistry analysis confirmed the cell was smooth muscle cell. Conclusions Rat restenosis model was more practical for study of smooth muscle cell proliferation associated diseases,such as restenosis after percutaneous transluminal coronary angioplasty.

Abstract:Aim To investigate the relationship between the polymorphism of apolipoprotein C3 gene -482C>T and the levels of serum lipids as well as lipoproteins. Methods The genotypes of the subjects were determined by using polymerase chain reaction-restriction fragments length polymorphisms (PCR-RFLP), and the serum lipids and apolipoproteins were determined. Results The carrier with minor allele of -482C>T have higher levels of serum triglycerides. At the same time the carrier with minor allele of -482C>T have higher levels of serum apolipoprotein C3 and higher apolipoprotein C2. Conclusions The results strongly support that the single nucleotide polymorphisms, -482C>T in apolipoprotein C3 gene, is related to the levels of serum triglyceride (TG) and high density lipoprotein cholesterol (HDLC) and those of other several lipoproteins in the Chinese population.

Abstract:Aim To investigate the relationship between angiotensin converting enzyme (ACE), apoE gene polymorphisms and the risk of subclinical atherosclerosis (As) in newly diagnosed type 2 diabetes after multifactorial intensified intervention. Methods The DNA polymorphisms in ACE and apoE were determined by PCR-RFLP between 157 newly diagnosed diabetic patients and 93 controls in Han's population of Hunan. Data of body weight, HbA1c, blood pressure, blood lipid and insulin resistance were compared among the groups with different genotypes of ACE and apoE. Characteristics of ACE and apoE gene polymorphisms were compared between patients with and without subclinical As. The association of the risk of subclinical As and apoE, ACE genotypes and their synergistic effects were determined by Logistic regression analysis. Results Frequencies of apoE genotypes in 157 individuals were not different between patients and controls (p>0.05), whereas the frequencies of ACE-I alleles were significantly higher than that in the controls (0.707 vs 0.581, p<0.05). No significant relationship was identified between ACE polymorphisms and blood pressure in this population. ApoE4/X carriers had higher LDLC than that in 2/X carriers (3.19±0.84 mmol/L vs 2.42±0.37 mmol/L, p<0.01). No synergistic effect was found in ACE and apoE geno-type on sBP or LDLC level. There was no association between targeted value of metabolic parameters, arterial intima-medial thickness and the two gene polymorphisms. Meanwhile, no correlation was found between apoE genotypes and the incidence of subclinical As. None of the patients with ACE-DD homozygote suffered from subclinical As, approaching significant difference compared to patients with ACE -II and ID genotype(P=0.059). Conclusions ApoE gene polymorphism may benotapre-dictor, but ACE-DD genotype might be a negative one for the development of subclinical As in patients with newly diagnosed type 2 diabetes after one-year multifactorial intervention.

Abstract:Aim To explore the effects of remedy-given insulin with a different dosage on the onset of coronary events in middle aged type-2 diabetes mellitus. Methods 53 middle-aged type-2 diabetes mellitus cases who were received insulin treatment with coronary heart disease were processed with insulin and peptic C measure and angiography. The coronary lesions and involved vessel score were repectively analysed using linea regression study. And another 32 type-2 diabetes mellitus without insulin regimen were placed in control group. Samples were analyzed with immunoreactiveinsulin and peptides C. Correlation between these variety scores for coronary lesions and 3 insulin sensitivity parameters was respectively obtained using linear regression analysis, and the stepwise regression analysis was used to assess dependence of associations. Results Serum insulin levels in coronary events group was significantly higher than those in control group. Most patients in coronary group consumed insulin more than 30 IU each day. Insulin and peptide C in coronary group was correlated with coronary lesion score or diseased vessel score. And the correlation was still significant with the insulin consumed consistance. Stepwise regression analysis showed that insulin and peptide C were independently correlated with the severity of coronary lesions. Conclusions The severity of coronary lesions was positively correlated with insulin and peptide C. Remedy-given hyperinsulinimia is only independently correlated with coronary events trigger in those type-2 diabetes mellitus. There might be certain association in the acute pathophysiology process of coronary lesion events.

Abstract:Aim To observe the effect of Pravastatin on plasma endothelin (ET), nitric oxide (NO)﹚ and C-reaction protein (CRP) in the early treatment of acute coronary syndromes (ACS). Methods 60 patients with ACS were randomly divided into the Pravastatin group (n=30) and the conventional medication group (n=30). The course of therapy was 2 weeks. The plasma concentration of ET, NO and CRP were measured before and after treatment. Results The levels of plasma CRP (32.7±10.8 μg/L), ET (50.3±17.2 μg/L) were lower and the level of NO (50.3±10.2 μmol/L) wes higher in the Pravastatin group than the levels of plasma CRP (44.3±9.7 μg/L), ET (72.4±16.4 μg/L), NO (42.8±8.7 μmol/L)in the conventional medication group (p<0.01). Conclusion Pravastatin can inhibit the inflammatory reaction so as to stabilize atherosclerotic plaque and improve endothelial function which relieve coronary artery spasm.

Abstract:Aim To understand the association of fibrinogen and its gene β148, β854 polymorphism with coronary heart disease. Methods 1 254 subjects were divided into coronary heart disease group (coronary artery narrow≥50%, n=836) and control group (coronary artery narrow <50%, n=418) according to coronary angiographic results. TaqMan MGB probes (FAMTM and VICR dye-labeled) polymerase chain reaction (PCR) was performed to screen the fibrinogen gene β148, β854 polymorphism in the appropriate Sequence Detection System Instruments (7900HT). Plasma fibrinogen levels and blood lipids were simultaneously measured in the patients with coronary heart disease and controls. Plasma fibrinogen concentrations were assayed by thrombin method. Total cholesterol (TC), triglyceride (TG) and high density lipoprotein cholesterol (HDLC) levels were assayed by standard enzyme method. Results Age, sex, smoking, hypertension, diabete mellitus, blood lipids and fibrinogen levels in coronary heart disease group were very different from that of control group (p<0.01). No significant difference was found in the frequencies of fibrinogen β148, β854 alleles gene and genotype between the controls and the patients with coronary heart disease (p>0.05). There was no significant association between fibrinogen β148, β854 genotypes and fibrinogen and the patients with coronary heart disease (p>0.05), but age, sex, smoking, hypertension, diabete mellitus, TG, and HDLC, lipoprotein (a) and fibrinogen levels were closely associated with coronary heart disease (p<0.01). Conclusions Fibrinogen levels were closely associated with coronary heart disease, but not with fibrinogen β148, β854 genotypes.

Abstract:Aim To investigate the relationships between apolipoprotein E gene polymorphism and the occurrence of coronary heart disease (CHD). Methods The distributions of the Hha Ⅰ polymorphisms of the apolipoprotein E gene and blood lipids levels were determined among 132 Chinese subjects in relation to circulating lipids and coronary angiography. Results The control group had higher apolipoprotein E ε2 frequencies than the CHD group (p<0.001) and the apolipoprotein E gene alleles were associated with the plasma levels of lipids and lipoproteins (all p<0.05). ε2 was significantly correlated with CHD (p<0.001). Conclusion The results suggest that the apolipoprotein E gene polymorphism do have influence on circulating levels of lipids and lipoproteins and individuals with apolipoprotein E ε2 are likely to have a reduced risk of developing CHD.

Abstract:Aim To explore the correlation of high-sensitivity C-reative protein (hs-CRP), fibrinogen(Fg) and the ultrasound indexes of carotid atherosclerosis in acute cerebral infarction. Methods The blood concentration of hs-CRP and Fg were tested while the carotid arteries were examined by color Dopplle and B-ultrasound in 103 subjects which were divided into the following groups: acute cerebral infarction group (ACI, n=33), old cerebral infarction group (OCI, n=34), normal controls (n=36). Results The results showed that the carotid intimal-media thickness (IMT) were significantly increased in all of the diseased groups than those in controls(P all<0.01). The total plaque score(TPS) was thicker in the group of OCI(8.65±5.76 mm)than in the group of ACI (4.68±2.98 mm)(p<0.01) and also in control(3.71±1.35 mm)(p<0.05). The incidence rate of atherosclerous plaque of carotid artery was higher in group of ACI (72.72%) than those in group of OCI (70.59 %)(p<0.05) and control (16.67%) (p<0.05). IMT, TPS and the incidence rate of atherosclerous plaque were different between ACI group and OCI group. The serum concentration of hs-CRP in the groups of ACI (19.54±37.96 mg/L) was higher than that in the group of OCI (4.01±7.22 mg/L)and also in control (1.66±1.17 mg/L) (all p<0.01); there was no difference in the group OCI and control(P>0.05). The 95% confident interval of plasma hs-CRP was 1.24～2.08 mg/L;4.52～34.56 mg/L; 1.42～ 6.62 mg/L respectively in the ACI, OCI and control. Plasma concentration of Fg was not different in the three groups (p>0.05). Multiple regression stepwise analysis revealed that the serum concentration of hs-CRP was positively correlated with the plasma concentration of Fg and TPS; and it was negatively correlated with resistant index. There was negative correlation between the plasma concentration of Fg and high-density lipoprotein cholesterol. Conclusions In the acute phase of cerebral infarction, the change in the plasma of Fg was not sensitive. The concentration of hs-CRP elevated was not correlated with IMT thickening, while closely correlated with the plasma Fg and the total plaque score.

Abstract:Aim To investigate the clinical relevance between the matrix metalloproteinase-2 (MMP-2) and acute coronary syndrome (ACS). Methods There are 61 coronary heart disease (CHD) patients diagnosised by coronary angiography (CAG) including 46 ACS patients, 15 stable angina pectoris (SAP) patients, and 24 non-CHD patients are excluded by CAG as the control group, and 46 ACS patients are separated into 16 acute myocardial infarction (AMI) and 30 unstable angina pectoris (UAP) patients. Every sample’s serum MMP-2 levels are detected by enzyme linked immunosorbent assay (ELISA). Results Before CAG, the concentration of MMP-2 in ACS, SAP, AMI, UAP and control group was 56.9±11.9 μg/L, 48.2± 10.8 μg/L, 58.6±14.8 μg/L, 53.4±9.3 μg/L and 46.6±10.5 μg/L respectively. There are remarkable statistic difference among AMI, UAP and SAP group, and so between SAP group and control group. There was obvious correlation among the level of MMP-2 in serum and the C-reactive protein (CRP) level, white blood cell (WBC) number, pathologic coronaries’ score and pathologic stenosis’s degree, but there was no obvious correlation between the level of MMP-2 and the pathologic coronaries’ number,the level of troponin I (Tn I), MB isoenzyme of creatine kinase (CK-MB), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDLC), low density lipoprotein cholesterol (LDLC) and the number of platelet. After percutaneous transluminal coronary angioplasty (PTCA), the level of MMP-2 is higher than that before PTCA. Conclusions The concentration of MMP-2 in ACS patients increased obviously, which was related to the stability of coronary plaque; MMP-2 contributes to type the CHD and could be the important monitering markers for CHD in clinic practices.

Abstract:Aim To study the effects of atorvastatin cholesterol-lowering therapy on vascular endothelial function in unstable angina pectoris (UAP). Methoed 62 patients with unstable angina were randomly divided into atorvastatin therapeutic group (n=30) and conventional therapeutic group (n=32). Vascular endothelial function in form of flow-mediated dilatation was assessed in the brachium artery by high-resolution ultrasound technique and the levels of NO were assayed before and after 12 weeks treatment in two groups. Results After 12 weeks atorvastatin treatment, vascular endothelial function were obviously improved and the levels of NO were markedly increased compared with that before treatment (p<0.01). It had no significant difference in control group(p>0.05). Conclusion The cholesterol-lowering therapy with atorvastatin could improve vascular endothelial function of patients with unstable angina pectoris.