Abstract:Aim To investigate the inhibitory effect of antisense monocyte chemotactic protein-1 (MCP-1) expression mediated by recombinant gene transfer on the migration of monocytes into arteries in vivo, as a first step, a recombinant retroviral vector expressing antisense MCP-1 gene was constructed and its expression was observed in vitro. Methods An antisense MCP-1 recombinant vector was constructed by inserting rabbit MCP-1 cDNA into LNCX plasmid in a reversed orientation. Recombinant viral DNA was transfected into φ-2 packaging cells by lipofectamine transfecting procedure. The transiently produced viruses were used to transfer PA317 amphotropic packaging cells. G418 resistant positive PA317 clones were isolated and expanded. Virus surpernatant from G418 resistant PA317 cells was used to determine virus titer and to infect aortic smooth muscle cells. After the selection with G418 medium, the integration and expression of recombinant gene in cells were measured. Results The highest retroviruses' titer was 5.6×10 7 CFU/L. PCR result showed that recombinant viruses were integrated into genome of the infected NIH3T3 cells and cultured smooth muscle cells. RNA slot blot analysis showed that antisense MCP-1 mRNA expressing was detected in the infected smooth muscle cells. There was obvious inhibition of MCP-1 mRNA expression obtained in the infected smooth muscle cells in comparing to that the uninfected cells. Conclusions The recombinant retroviral vector containing antisense MCP-1 could inhibit target gene expression in cultured smooth muscle cells and may be used for further experimental studies in vivo.

Abstract:Aim To well understand the role of oxidized low density lipoprotein (ox-LDL) in the pathogenesis of thrombotic complications in atherogenesis. Methods LDL was isolated from normal heparinized blood by density gradient ultracentrifugation and oxidized by CuCl 2. Total RNA was extracted from human umbilical vein endothelial cells (hUVECs) exposed to LDL or ox LDL by guanidinium isothiocyanate method. The quantification of tissue factor pathway inhibitor (TFPI) mRNA in hUVECs was carried out by reverse transcriptase PCR (RT PCR). Results hUVECs were able to express TFPI mRNA constitutively. The expression was not affected by LDL but effectively inhibited by ox LDL in a time and dose dependent manner. Conclusions Oxidized LDL may play an important role in inducing coagulation in atherosclerotic lesion by inhibition of expression of TFPI in vascular endothelial cells.

Abstract:自由基生物学与自由基医学是70 年代初发展起来的一门新兴交叉学科。到90 年代初它以迅猛的速度向基础医学和临床医学渗透,为某些疾病的发生机理的阐明和防治提供理论基础。在这种形势下,于1980 年第一军医大学生物化学教研室成立了脂质过氧化损伤研究组。开始围绕组织的脂质过氧化损伤和抗氧化系统中的抗氧化剂维生素E 和抗氧化酶SeGPx 进行了基础研究和临床研究。首先从细胞水平说明维生素E和SeGPx 协同地终止脂质过氧化作用?...

Abstract:Aim This study was conducted to understand whether malondialdehyde (MDA) had a chemotactic activity for human peripheral blood monocytes. Methods The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber. MDA was prepared by the method described by Kikugawa. Results The mean of migration distances (85.37 ± 10.44 μm, 109.03 ± 7.88 μm, and 122.67 ± 6.25 μm) induced by MDA at low (0.05 mmol/L), middle (0.5 mmol/L), and high (1 mmol/L) concentrations, respectively, were significantly longer than that of the random migration group (69.88 ± 8.19 μm). The analysis of variance showed that there was significant difference between the low concentration group and the random migration group (P<0.05), whereas the mean of migration distances in the middle and high concentration groups and in the random migration group was highly statistically different (P<0.001). Furthermore, the monocyte migration in the chemotaxis group was enhanced by MDA in a dose-dependent manner. The mean of migration distances in the chemokinesis group (110.72 + 7.32 μm) was significantly longer than that of the random migration group as well. Conclusions Malondialdehyde is significantly chemotactic and chemokinetic for peripheral blood monocytes. It suggests that malondialdehyde might play certain role in the recruitment of the monocytes/macrophages in the arterial intima during early stage of atherogenesis.

Abstract:Aim To investigate the effects of dimethyl sulfoxide (DMSO), an antioxidant, on neutrophils adhesion to tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC), and the expression of adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) on TNF-α-stimulated endothelial cells. Methods The expression of adhesion molecules on HUVEC was measured by a cellular enzyme-linked immunoabsorbent assay(ELISA)method,and the adherence of neutrophils to activated HUVEC was detected by Rose Bengal assay. Results After stimulation of HUVEC with TNF-α for 6 h, the adherence of neutrophils to endothelial cells was increased compared with control group (P<0.01). Exposure HUVEC to TNF-α for 4 h or 18 h induced E-selectin expression and increased constitutive expression of ICAM-1 (P<0.01). Pretreatment of HUVEC with 2.5% DMSO for 1 h before stimulation with TNF-α produced a decrease in the neutrophil adhesion and inhibition in the expression of E-selectin and ICAM-1 (P<0.01). Conclusion DMSO decrease neutrophil adhesion to TNF-α-activated endothelial cell by inhibiting the expression of E-selectin and ICAM-1. This result may contribute to the anti-inflammatory and antiatherosclerotic potential of DMSO.

Abstract:Aim To observe the effects of cholesterol-rich lipoproteins [β-very low density lipoprotein (β-VLDL), normal low density lipoprotein (n-LDL)and oxidized low density lipoprotein(ox-LDL)] on lipid accumulation in smooth muscle cells (SMCs) and to explore its mechanism . Methods β-VLDL, n-LDL or ox-LDL was incubated with rabbit aortic SMCs for 48 hours, total cholesterol(TC)and triglyceride(TG)of the cells were extracted and determined. The increasing concentrations of lactoferrin (specific ligand of LDL receptor-related protein ,LRP) were added withβ-VLDL, n-LDL or ox-LDL, respectively, in the culture and TC and TG of the SMCs were determined as above. SMCs were incubated at 4℃ in M199 containing different concentrations of unlabeled lactoferrin and FITC-β-VLDL, FITC-n-LDL or FITC-ox-LDL, respectively, for 4 hours. The cell surface bound lipoproteins were determined by fluorospectrometry. Results β-VLDL, n-LDL and ox-LDL can all increase TC and TG accumulation in SMCs. More TC and TG were accumulated withβ-VLDL as compared with n-LDL or ox-LDL. The effects can be inhibited by lactoferrin. Moreover, lactoferrin competed with FITC-β-VLDL, FITC-n-LDL or FITC-ox-LDL on binding to SMCs. Conclusion β-VLDL, n-LDL and ox-LDL may contribute to lipid accumulation in SMCs by LRP mediated pathway.

Abstract:Aim To study the function of apolipoprotein E gene polymorphism on coronary heart disease (CHD) and to evaluate the effect of apolipoprotein E gene polymorphism on plasma lipid levels. Methods By using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), we determined apolipoprotein E genotype of 220 CHD patients and 180 healthy controls. Other plasma lipid levels are measured by routine way. Results 5 genotypes of apolipoprotein E , E3/3, E3/2, E4/3, E4/2 and E4/4 are detected in our study. There are statistically significant differences in apolipoprotein E4/3 and ε4 frequencies between CHD patients and controls (P<0.01). We also find statistically difference in apolipoprotein E3/3 frequencies between the two groups (P<0.05). CHD patients have significantly higher serum total cholesterol (TC) and low density lipoprotein cholesterol (LDLC) than controls (P<0.01). Among different apolipoprotein E genotypes of CHD patients, there are statistically difference in TC and LDLC levels. Conclusion These results suggest that apolipoprotein E gene polymorphism is involved in the occurrence and the development of CHD and affects plasma lipid levels. apolipoprotein Eε4 might be an important genetic factor of CHD.

Abstract:Aim To investigate the relationship between advanced glycosylation end products (AGEs) and the changes of extracellular matrix (ECM) of aorta in diabetic rats in the development of atherosclerosis. Methods Wistar rats were divided into three groups: induced diabetic rats (DM group), aminoguanidine (AG) treated diabetic rats (AG group) and the control rats (Control group). After 1, 2, 3 or 4 months, the levels of Hb-AGEs and procollagen Ⅲ, collagen Ⅳ, collagen Ⅳ-AGEs and laminin were determined. Results Hemoglobin-AGEs (Hb-AGEs) in DM group was elevated significantly as compared with AG group and Control group; it increased with the prolongation of the time course of diabetes (after 1, 2, 3, and 4 months the Hb-AGEs was 6.88±1.23, 10.26±0.63, 15.3±1.49 and 18.57±2.90 ku/g, respectively), and was closely related to FBG. Procollagen Ⅲ of group DM increased monthly (after 1, 2, 3 and 4 months, it was 15.20±3.03, 21.44±1.79, 27.19±3.28, 33.99±4.96 μg/L, respectively) which was related to Hb-AGEs and collagen Ⅳ-AGEs, and was significantly higher than that of group AG and C ontrol group. Collagen Ⅳ acted in the same way as procollagen Ⅲ (it was 23.67±1.49, 30.37±2.86, 36.65±1.98 and 45.46±5.77 μg/L, respectively) with the correlation to Hb-AGEs and collagen Ⅳ-AGEs, so did the changes of collagen Ⅳ-AGEs (0.79±0.15, 1.25±O.22, 1.54±0.06 and 1.80±0.14 AGEs Mu/g in 1～4 months) with the correlation to Hb-AGEs and collagen Ⅳ. There was no difference of laminin among three groups. Conclusion It could be concluded that AGEs may be the cause of the increase of procollagen Ⅲ, collagen Ⅳ and collagen Ⅳ -AGEs in the aorta of diabetic rats, thus suggesting a role of AGEs in the development of atherosclerosis in diabetes mellitus.

Abstract:Aim To reduce the level of apolipoprotein B (Apo B) via the inhibitory effect of antisense oligodeoxynucleotides (AODN) on expression of Apo B gene in cultured liver cells and search its mechanism. Methods The cultured liver cells were treated with synthesized Apo B gene antisense, sense ODN (SODN) and 0.9% salt solution respectively. Apo B100 concentration was measured by Auto-biochemical Instruction. The mRNA level was observed by using of reverse transciption-polymerase chain reaction (RT-PCR). Results Apo B gene AODN 5, 10, 15, and 20 μmol/L inhibited Apo B concentration 26.6%, 34.2%, 34.2%, and 45.8% respectively. The inhibitory effect appeared in a concentration-dependent manner. RT-PCR showed that Apo B gene AODN downregulated Apo B mRNA expression obviously. Conclusions he ApoB gene AODN inhibited Apo B gene expression obviously and reduced Apo B concentration. The possible mechanisms are to downregulate Apo B mRNA level and inhibit translation of Apo B gene.

Abstract:Aim In order to see the effects of continued and stable expressive antisense c-myc on the apoptosis of vascular smooth muscle cell(VSMC) . Methods Three recombinant retroviral expressive vectors,aM1,aM2 and aM3,which respectively loading reverse fragments of exon 1,2 and 3,were transferred into rat artery SMC and assayed 1 month after their stable expression. Results Apoptosis ratioes of aM3 group were increased to 360%,while those of aM1 and aM3 groups were significantly decreased. In addition,SMC transfected with aM1 and aM3 (especially aM3),emerged morphological features of apoptosis and autophagy,while aM2 commonly appeared bunches of myofilament in electron microscopy. Conclusion Stable expression of antisense c-myc can induce SMC into apoptosis in vitro, and aM3 has the most powerful ability to induce apoptosis and autophagy.

Abstract:Aim To investigate the role of angiotensinⅡ(AngⅡ) and aldosterone (Ald) in myocardial hypertrophy and fibrosis in hypertensive rats by blocking renin angiotensin system at receptor level. Methods Thirteen 2 kidney 1 clip (2K1C) hypertensive rats began to be administered valsartan in the drinking water (10 μg/g·d) at the sixteenth week after operation. Nineteen 2K1C rats were used as untreated controls. Nineteen sham operated rats were also used as controls. Systolic blood pressure (SBP), left ventricular weight index (LVWI), plasma and myocardial AngⅡ /Ald concentration, myocardial collagen concentration (MCC) and collagen volume fraction (CVF) were measured when three groups rats were killed at the sixteenth week, the twentieth week and the twenty eighth week after operation. Results All measured facts in 2K1C hypertensive group were significantly higher than in sham operated group (P<0.05 or 0.005). Compared with age matched 2K1C group, plasma AngⅡ concentration was higher significantly(P<0.05), but SBP, LVWI, MCC, CVF and plasma Ald concentration in treated group decreased significantly (P<0.05). Conclusions Left ventricular myocardial remodeling in renovascular hypertensive rats was closely related to myocardial AngⅡ and Ald concentration. Ang Ⅱ 1 (AT 1) receptor antagonist valsartan can block the pathophysiological effects of AngⅡ, inhibit Ald secretion and reveres left ventricular remodeling (mainly decrease deposition of type Ⅰ collagen). It suggests that AT 1 receptors may mediate the biological effects of AngⅡ in local tissue.

Abstract:Aim To investigate the atherogenic role of very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) fraction isolated from plasma of LDL receptor (LDLR)-knockout mice. Methods The lipoprotein fraction containing both VLDL and IDL fraction (LDLR ko-VLDL/IDL) was isolates from plasma of LDLR-knockout mice by ultracentrifugation. The lipid content of the fraction was determined on a Hitachi 7450 automatic analyzer, and its electrophoresis mobility and apolipoprotein components were respectively determined with agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, its interaction with J774 macrophages was studied. Results After LDLR ko-VLDL/IDL was incubated with J774 macrophages , the cholesterol ester (CE) content [(73±0.5) μmol/(g. cell protein)] in macrophages increased markedly, which was an 9-fold increase over the corresponding lever induced by native LDL [(8±7) μmol/(g. cell protein)] and significantly higher than non-loaded group (P<0.005). Conclusions Unmodified LDLR ko-VLDL/IDL fraction could be actively taken up by J774 macrophages and transferred the macrophages to foam cells .

Abstract:Aim The vasorelaxation responses of some peripheral vessels to fenoldopam, a selective dopamine-1 receptor agonist, were observed in N G-nitro-L-arginine (L-NNA), a selective NO synthase antagonist, induced pulmonary artery hypertension (PAH) rats. Methods Rings of rat pulmonary, renal, mesenteric artery were to dopamine-1 (DA 1) receptor agonist responsiveness. All experiments were performed in the presence of indomethacin (10 μmol/L) and propranolol (3 μmol/L) to prevent the production of vasoactive prostanoids and the binding of norepinephrinewith beta receptor. Results It was shown that, during the development of PAH, the vasorelaxation responses to fenoldopam were significantly reduced in peripheral vessels, especially in the pulmonary vessel. After 28days, the Emax (%) in the pulmonary artery was 45.5%±4.1%, being lower than the control group of 97.3%±10.6% and the K A was 2042±221, being lower than the control group of 4272±512 (P<0.01), while in the mesenteric and renal vessels, the vasorelaxation responses were only moderately decreased after 28 days. Conclusion There results show that the detereorated peripheral vessels DA 1 receptor-mediated vasorelaxation might be one of the mechanisms underlying development of hypertension.

Abstract:高血压病患者27 例,男性15 例,女性12 例,平均年龄66.8±6.5 岁,伴高TG18 例,伴低HDLC9 例,其中合并冠心病11 例。低脂饮食、停用一切降脂药物4 周以上,收缩压控制在160 mmHg 以下,舒张压控制在95 mmHg 以下。排除继发性高血压、急性心肌梗死( 半年内) 、脑血管意外、肝肾功能不全及糖尿病等影响血脂代谢的疾病。洛伐他汀( 罗华宁) 片,每天晚饭后服20 mg,连服8 周,同时低脂饮食及坚持降压治疗。治疗前和治疗后8 周分别取早晨空?...

Abstract:Aim To determine the influence of exogenous testosterone on early atherosclerosis in cholesterol-fed male Wistar rats and its possible mechanisms. Methods Thirty-six male Wistar rats were randomly assigned to the three groups: normal group, cholesterol group, treated group (testosterone cypionate,2.5 mg/kg, im.); plasma lipids and the lipids content of aorta and liver were analyzed with enzymatic methods after twelve weeks, aortic intima were examined with light microscope. Results Plasma lipids and lipoproteins were not significantly different between cholesterol group and treated group, the treated rats had significantly higher plasma lipid oxidative peroxidation products value, lower hepatic lipids contents, but higher aortic lipids contents than the cholesterol group, the number of monocytes adhering to endothelial cells and oil red O positive foam cells was more in the treated group than the cholesterol group. Conclusion Exogenous testosterone accelerated early atherosclerosis in male hypercholesterolemic rats.

Abstract:Aim To study the effects of cucurmin on the metabolism of low density lipoprotein and lipoprotein (a). Methods Cucurmin was injected into the body of hedgehogs via the armpit vein 2 minutes before the 125 iodine low density lipoprotein ( 125 ?I-LDL), 125 iodine lipoprotein(a) ( 125 I-Lp(a)), 125 iodine desialylated low density lipoprotein ( 125 I-ds-LDL) or 125 iodine desialylated lipoprotein(a) ( 125 I-ds Lp(a)) were injected. The animals were put to death in 1 hour. The radioactivity of 125 I-LDL, 125 I-Lp(a), 125 I-ds-LDL and 125 I-dsLp(a) in the blood, liver, kidney, spleen, gall and adrenal were measured . Results Experiments showed that the absorption of LDL in liver, adrenal and gall increased 228.1%, 86.7% and 70.3% respectively, but decreased 54.4% in spleen and 25.8% in blood. The effects of cucurmin on Lp(a) were similar to that on LDL. The absorption of Lp(a) in liver and adrenal increased 34.7% and 43.1% respectively, but decreased 34.8% in kidney and 19.2% in blood. The absorption of ds-LDL in liver, kidney, adrenal and blood decreased 15.1%, 43.6%, 68.2% and 31.8% respectively, but increased 31.7% in gall. Cucurmin has slightly effects on the metabolism of ds Lp(a). Conclusion Experiments proved that cucurmin may promote the metabolism of LDL and Lp(a) in liver and adrenal, increase excretion of LDL by gall and inhibit the absorption of LDL in spleen. It make the concentration of LDL and Lp(a) in blood decrease and achieve the effects of lowering lipids and antiatherosclerosis.

Abstract:Aim To investigate the effect of antioxidant Vitamine E and Selenium on the expression of cellular adhesion molecules(CAMs) induced by lipid peroxidation(LPx) in cultured endothelial cells and monocytes(MC) adherence to EC. Methods LPx was initiated withdiamide(DM).Effect of Vitamine E and Selenium on the expression of vascular adhesion molecule-1(VCAM-1) and endothelial leucocyte adhesion molecule-1(ELAM-1) was studied under laser confocal microscope,MC adherence to EC was measured. Results Vitamine E and Selenium could decrese the content of lipid peroxid,reduce VCAM-1 and ELAM-1 expression andMC adherence to EC incubated with DM. Conclusion Antioxidant could inhibite VCAM-1 and ELAM-1expression induced by LPx to reduce Mc adherence to EC and may possiboly prevent the development of athrosclerosis (As) lesion.

Abstract:Aim To compare the lipid-lowering efficacy and security of zocor with homemade-simvastatin. Methods 40 patients with Hypercholesterolemia were randomized into two groups, 20 patients in simvastatin group, the others in zocor group as controls. Serum lipids levels were determined prior to lipid lowering treatment and at the end of 4 and 8 week treatment respectively. Results At the end of 4 and 8 week treatment, levels of total cholesterol (TC), low density lipoprotein chdesterol (LDLC) and TC/HDLC (high density lipoprotein chdesterol) in both groups significantly reduced (P<0.01), while HDLC significantly raised (P<0.01). At the end of 8 week treatment, levels of triglyceride (TG) in zocor group significantly reduced (P<0.01), while not in simvastatin group. There was no obvious side effects during the period of homemade-simvastatin and zocor treatment and the patient's tolerance was good. Conclusion Homemade-simvastatin is a effective medicine for the treatment of hypercholesterolemia.

Abstract:Aim To establish a method of cystathionine β-synthase activity assay and detect cystathionine β-synthase activity in various tissues of rat. Methods We first extracted cystathionine β-synthase from various tissues of rat. And then the final concentration of the assay components in 0.4 mL of incubation mixture were: 100 mmol/L Tris-HCl (pH 8.6), 0.25 mmol/L pyridoxal 5'-phosphate, enzyme protein (2.5～40 mg), 20 mmol/L serine, and 20 mmol/L L-homocysteine. After incubation for 60 min at 37℃, the reaction was stopped by addition of 0.05 mL 50% trichloroacetic acid. Following 5 min incubation on ice, the precipitated protein was removed by centrifugation. To a 0.2 mL aliquot of a supernatant, 3.3 mL of ninhydrin reagent was added and the color was developed. The absorbance at 465 nm was read against an substrate and enzyme-free blank was subtracted. Results Colorimetry to detect the cystathionine β-synthase activity was established; and cystathionine β-synthase activity in various tissues of rat was different. Conclusion The method established can detect cystathionine β-synthase activity successfully. It had the merit of high precision and good repetition and was easy to operate.

Abstract:在动脉粥样硬化病变发展过程中,斑块表面破裂并发血栓形成是极常见的。斑块破裂的危险性取决于斑块的组成成分,往往是巨噬细胞丰富、纤维帽薄、脂质池大的斑块容易破裂。血液中的单核细胞与病变好发区内皮表面粘附分子结合,在趋化因子作用下,迁入内膜,然后转化为巨噬细胞,进而经特异受体介导的胞吞作用蓄积脂质,转变成泡沫细胞,形成动脉粥样硬化的早期病变,即脂纹。此时的巨噬细胞不仅形态上变为泡沫细胞,而且新增许多功能,如合成组织因子、载脂蛋白E及一系列介导免疫反应与信息传递的细胞因子,还可以分泌基质金属蛋白酶,降解纤维帽内的胶原纤维,使纤维帽弱化。巨噬细胞死亡,可促进脂质池形成,即细胞外脂质大量聚积。因此认为,巨噬细胞对斑块破裂及血栓形成有重要影响。

Abstract:冠状动脉粥样硬化斑块破裂是导致血栓形成引起急性缺血冠状动脉综合征的主要原因,斑块自身组成成分与斑块破裂的关系非常密切。本文概述了斑块组成成分在斑块破裂中所起的作用,并对易破斑块的识别和斑块破裂的预防作一简要叙述。

Abstract:研究糖皮质激素在动脉粥样硬化中的作用和地位可以从一个崭新的角度去探讨动脉粥样硬化的发病机理。本文综述了近15 年来的国外相关研究文献,以其讨论糖皮质激素与核受体的作用原理及其与脂蛋白代谢和动脉血管壁细胞生物学行为的关系,并从基因水平阐述了糖皮质激素对某些动脉粥样硬化相关基因转录和表达的调节作用。

Abstract:肝脂酶是一种糖蛋白,由肝实质细胞合成,在内质网中糖化后转移至高尔基体,形成具有催化活性的成熟酶并转运到窦状隙内皮细胞表面发挥作用。肝素可将结合在肝窦状隙内皮细胞表面的肝脂酶释放入血,选择性测定肝素化血浆脂肪酶活性可了解体内肝脂酶的活性。基因及蛋白质结构分析均表明肝脂酶是脂肪酶家族的成员之一,能水解乳糜微粒、中间密度脂蛋白及高密度脂蛋白中的甘油三酯和磷脂,在乳糜微粒残骸的清除、低密度脂蛋白的形成及胆固醇逆向转运过程中起着十分重要的作用。肝脂酶活性的改变会引起脂蛋白代谢变化、特别是血浆高密度脂蛋白胆固醇浓度的改变;肝脂酶基因变异是血浆高密度脂蛋白胆固醇水平的重要遗传决定因素之一,与动脉粥样硬化及冠心病关系密切。