植物过氧化物酶研究进展

过氧化物酶 [peroxidase,POD,EC1 .1 1 .1 .7(X) ]是广泛存在于各种动物、植物和微生物体内的一类氧化酶。催化由过氧化氢参与的各种还原剂的氧化反应 :RH2 H2 O2 →2 H2 O R。植物过氧化物酶的研究可追溯到 1 80 9年用愈创树脂为底物进行的颜色反应。但直到一个世纪之后才开展此酶的分离和命名。已知的催化反应底物超过 2 0 0种 ,以及多种过氧化物和辅助因子。迄今被研究最深入的应首推辣根过氧化物酶 (horseradish pero-xidase,HRP)。早在 1 94 0年 ,Thorell即用电泳方法从部分纯化的辣根组织中区分出 2种不同的 HRP,之后此酶…     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

前β_1-HDL与细胞HDL受体结合活性的研究

研究发现肝细胞、动脉平滑肌细胞、巨噬细胞、成纤维细胞、内皮细胞以及卵巢组织等的细胞膜上均存在可特异性结合高密度脂蛋白 (high densitylipoprotein,HDL)的受体[1～ 3] .肝细胞 HDL受体介导胆固醇由 HDL 流入细胞转化清除 ,肝外细胞HDL受体则介导细胞内过量胆固醇流出 [4 ,5] .前 β1-HDL是外周细胞流出胆固醇的最初接受体 [6] ,而 α-HDL主要作为细胞流出胆固醇酯化及转运至肝脏清除的载体[4 ] .胆固醇逆向转运 (reverse cholesteroltransport,RCT)的过程实质上就是 HDL由前β1-HDL到 α- HDL递变成熟的代谢过程 [4 ] ,因…     

NB901的细胞毒作用及对拓扑异构酶Ⅱ活性的影响

DNA拓扑异构酶是抗肿瘤药物的一个重要靶点,近年来已发现许多不同类型的化合物对该靶酶有作用[1]．NB901是一种新合成的单核着类似物[2],具有较强的细胞毒作用[3]．本实验采用中性红（NR）染色法观察了NB901体外细胞毒作用和DNA拓扑异构酶（topoisomerase）Ⅱ活性的影响．1实验材料NB901为北京医科大学药学院合成,分子量280,淡黄色粉末,易溶于水．中性红（C15H17N4Cl）,北京化工厂生产,AR级．蛋白酶K,牛血清白蛋白（BSA）及腺苷三磷酸（ATP）均购自Sigma公司．细胞系：人肝癌细胞BEL7402及人巨细胞肺癌细胞PGT由北京…     

应用异型双功能交联剂制备酶-抗体结合物

酶标免疫测定法(ELISA)中最关键的化合物是酶-抗体结合物,将酶和抗体交联起来需用交联剂。本文作者使用了N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯(简称SPDP)将辣根过氧化物酶(HRP)和兔抗小鼠IgG(兔IgG)交联起来。我们试验了SPDP/HRP,SPDP/IgG和HRP/IgG的不同比例,以期获得活性高的酶-抗体结合物。此外还研究了从结合物中去除自由HRP和自由IgG的方法。用SDS-PAGE及硝酸纤维膜电泳转移法证明本法制备的结合物不含HRP及IgG的自身聚合物。用ELISA法鉴定结合物制品时,一般稀释度可达到1:10,000以上,有的可达到1:20,000(当结合物浓度A_(280nm)=1.0,底物显色A_(492nm)=1.0时)。     

HRP技术对鲫鱼尾部神经分泌系统的研究

本实验采用辣根过氧化物酶（HRP）逆行追踪技术对鲫鱼尾部神经分泌系统进行了研究,结果表明,尾部神经分泌细胞可分为大（60－70μm）、中（40－50μm）、小（20－30μm）三种类型,在尾部脊髓的背侧面和腹侧面都有分布,HRP的引入采用Griffin（1979）提出的缓释胶法,结果证明这种方法在鱼类神经解剖的束路示踪方而是可行的。     

兔抗麻雀IgY酶标抗体的制备

目的制备辣根过氧化物酶（HRP）标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。     

玉米精细胞及体细胞原生质体表膜蛋白的比较

以低渗冲击法（改良两步法）及Ｐｅｒｃｏｌｌ密度梯度离心,成功分离纯化生活玉米（Ｚｅａｍ ａｙｓ）精细胞;以混合酶解法制备玉米叶原生质体和愈伤组织原生质体;以ＮＨＳ－生物素标记完整精细胞及原生质体表膜蛋白,进行ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ,并以辣根过氧化物酶标亲和素检测被标记的表膜蛋白。结果表明,在精细胞中标记蛋白有４ 种,分子量分别为４８、５９、６７、７９ ｋＤ;叶片原生质体中有５ 种,分子量分别为５４、５８、６６、７１、７８ ｋＤ;愈伤组织原生质体中仅有２ 种,分子量６７ 和８０ ｋＤ。其中４８ ｋＤ蛋白为精细胞所特有,５４ ｋＤ 和７１ ｋＤ蛋白为叶片细胞所特有     

[AAGG]异源四倍体新棉种的核型及同功酶分析

本文对山西农业大学棉花育种组创育的[AAGG]异源四倍体新棉种及其二倍体亲本亚洲棉(G．arboreum)、比克氏棉(G．bickii)进行了核型分析,并对[AG]异源二倍体和[AAGG]异源四倍体的过氧化物同功酶进行了分析。研究结果：异源四倍体[AAGG]的体细胞染色体数目为2n=4x=52,表现为双亲染色体数目之和,其核型公式为2n=4x=52=50m(6SAT) 2sm,按stebbins的核型分析原则,异源四倍体[AAGG]属1A型。过氧化物酶同功酶分析表明：[AG]棉种酶谱表现为双亲不完全互补型。并具有新式酶带。因此过氧化物酶同功酶可以作为鉴别远缘杂种的生物技术之一。     

ToRCH系列酶标诊断试剂的研制

报道了用辣根过氧化物酶标记的抗人ＩｇＧ和抗人ＩｇＭ（ｕ链）单克隆抗体作第二抗体,用自己培养、纯化的弓形体（Ｔｏ）、风疹病毒（ＲｕＶ）、巨细胞病毒（ＣＭＶ）和单纯疱疹病毒（ＨＳＶ１２）的虫体和病毒抗原包被酶标板,研制出检测ＴｏＲＣＨ系列的特异性ＩｇＧ和ＩｇＭ的间接ＥＬＩＳＡ试剂。质量检定结果表明,该试剂特异性强、本底低,能有效消除ＲＦ因子等干扰因素的影响;灵敏度达１∶１６０～６４０;精密性好,变异系数（Ｃ．Ｖ）在１．４％～９．０％;试剂稳定,３７℃存放４ｄ,各项指标的变化率不超过１５％。     

Apolipoprotein AI efficiently binds to and mediates cholesterol and phospholipid efflux from human but not rat aortic smooth muscle cells

Aortic smooth muscle cells (SMC) from several animal species have been reported to resist depletion of cellular cholesterol by the major apolipoprotein of HDL, apoAI. Resistance of SMC to this protective action of apoAI, if present in humans, could contribute to the overaccumulation of arterial wall cholesterol seen in atherosclerosis. We investigated the ability of human aortic medial SMC to bind and be depleted of cholesterol and phospholipids by apoAI. In contrast to rat aortic SMC, but similar to human fibroblasts, human SMC were readily depleted of cholesterol by apoAI, measured by a marked depletion of intracellular cholesterol available for esterification, and an increase in cholesterol efflux to the medium. Human SMC were also actively depleted of the phospholipids phosphatidylcholine and sphingomyelin by apoAI. In contrast, rat SMC released only a small fraction of these cellular phospholipids to apoAI-containing medium. (125)I-labeled apoAI bound with high affinity and specificity to human SMC, but failed to bind to rat SMC. Similar levels of expression of class B, type I scavenger receptor (SR-BI) and caveolin in human and rat SMC suggested these proteins do not account for the differences in apoAI binding or lipid efflux seen in these cells. An enhancer of apolipoprotein-mediated cholesterol efflux, tyrosyl radical-oxidized HDL, markedly amplified the depletion of cholesterol available for esterification in human SMC compared to HDL, but had no enhanced effect in rat SMC. These results show that human SMC bind and are readily depleted of cellular lipids by apoAI, and suggest that apoAI-mediated cholesterol efflux from arterial SMC may contribute significantly to the circulating pool of HDL cholesterol in vivo. The marked difference in apoAI binding to human and rat arterial SMC provides an excellent model to study the nature of the apoAI-cell binding interaction.     

氧化修饰HDL对培养人主动脉平滑肌细胞胆固醇流出的影响

大量研究显示,高密度脂蛋白(highdensitylipoprotein,HDL)具有抗动脉粥样硬化(atherosclerosis,AS)作用.这是由于HDL能够促进外周组织如血管壁内皮细胞、平滑肌细胞(smoothmusclecell,SMC)及巨噬细胞储集的胆固醇流出,并将其转移到肝脏通过胆汁分泌而排出体外[1],这一过程...     

氧化修饰脂蛋白刺激人动脉平滑肌细胞DNA合成

动脉平滑肌细胞(SMC)是动脉粥样硬化(As)斑块中的主要细胞, 它的增殖在As的形成过程中极为重要. 在建立人主动脉SMC体外培养法的基础上, 通过 ³H-TdR掺入实验观察了人低密度脂蛋白(LDL)、极低密度脂蛋白(VLDL)及高密度脂蛋白(HDL)和相应的氧化修饰型脂蛋白对培养人SMC DMA合成的影响.结果发现,HDL对 ³H-TdR掺入SMC DNA无影响(P>0.05); LDL和VLDL ³H-TdR掺入量明显增加(P<0.05);OX-LDL, OX-VLDL及OX-HDL均使 ³H-TdR掺入DNA显著增加(P<0.01).结果表明,LDL和OX-LDL, OX-VLDL及OX-HDL均能刺激SMC DNA合成,促进SMC增殖.     

氧化修饰高密度脂蛋白对培养人动脉平滑肌细胞细胞周期蛋白D_1基因表达的影响

动脉平滑肌细胞（ｓｍ ｏｏｔｈ ｍ ｕｓｃｌｅ ｃｅｌｌ,ＳＭＣ）是动脉粥样硬化（ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ,ＡＳ）斑块中的主要细胞,它的增殖在ＡＳ形成过程中极其重要．利用体外培养的人主动脉ＳＭＣ,观察了天然高密度脂蛋白（ｎａｔｉｖｅ ｈｉｇｈ ｄｅｎｓｉｔｙ ｌｉｐｏｐｒｏｔｅｉｎ,Ｎ－ＨＤＬ）及氧化修饰ＨＤＬ（ｏｘｉｄｉｚｅｄ ＨＤＬ,ＯＸ－ＨＤＬ）对培养人主动脉ＳＭＣ ｃｙｃｌｉｎ Ｄ１（细胞周期蛋白Ｄ１）基因转录表达的影响．结果表明：（１）Ｎ－ＨＤＬ对ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达无影响（Ｐ＞ ０．０５）;（２）ＯＸ－ＨＤＬ使ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达显著增强（Ｐ＜０．０１）,其表达量随时间（２、１２、２４ ｈ）延长而增加．上述结果表明,ＯＸ－ＨＤＬ的致ＡＳ作用可能与其刺激ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达增加有关．     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

天然及氧化修饰脂蛋白对人动脉平滑肌细胞原癌基因表达的影响

动脉平滑肌细胞（ＳＭＣ）的增殖在动脉粥样硬化（ＡＳ）的形成过程中极其重要。我们在建立人主动脉ＳＭＣ体外培养方法的基础上,观察了ＬＤＬ,ＶＬＤＬ及ＨＤＬ和相应的氧化修饰型脂蛋白对培养人ＳＭＣｓｉｓ,ｊｕｎ,Ｈ－ｒａｓ原癌基因及Ｒｂ抗癌基因转录表达的影响。结果表明：（１）ＨＤＬ对ＳＭＣｓｉｓ,ｊｕｎ,ｒａｓ基因表达无影响;（２）ＬＤＬ和ＶＬＤＬ有使这些基因表达增加的趋势;（３）ｏｘ－ＬＤＬ,ｏｘ－ＶＬＤＬ和ｏｘ－ＨＤＬ具有使ＳＭＣｓｉｓ,ｊｕｎ,和ｒａｓ基因表达显著增强的作用（Ｐ＜０．０１）,且其作用较相应的天然脂蛋白大（Ｐ＜０．０１）;（４）天然和氧化修饰型脂蛋白对Ｒｂ基因表达均无影响。据上述结果推测：ＬＤＬ,ＶＬＤＬ,ｏｘ－ＬＤＬ,ｏｘ－ＶＬＤＬ和ｏｘ－ＨＤＬ的致ＡＳ作用可能与刺激ＳＭＣｓｉｓ,ｊｕｎ和ｒａｓ原癌基因表达增加有关。     

Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture.

Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18-24 h incubation in the presence of LDL at 100 microgram LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 microgram LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 microgram LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 microgram/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.     

High-density-lipoprotein-induced cholesterol efflux from arterial smooth muscle cell derived foam cells: functional relationship of the cholesteryl ester cycle and eicosanoid biosynthesis

Eicosanoids have been implicated in the regulation of arterial smooth muscle cell (SMC) cholesteryl ester (CE) metabolism. These eicosanoids, which include prostacyclin (PGI2), stimulate CE hydrolytic activities. High-density lipoproteins (HDL), which promote cholesterol efflux, also stimulate PGI2 production, suggesting that HDL-induced cholesterol efflux is modulated by eicosanoid biosynthesis. To ascertain the role of endogenously synthesized eicosanoids produced by arterial smooth muscle cells in the regulation of CE metabolism, we examined the effects of cyclooxygenase inhibition on CE hydrolytic enzyme activities, cholesterol efflux, and cholesterol content in normal SMC and SMC-derived foam cells following exposure to HDL and another cholesterol acceptor protein, serum albumin. Alterations of these activities were correlated with cholesterol efflux in response to HDL or bovine serum albumin (BSA) in the presence or absence of aspirin. HDL stimulated PGI2 synthesis and CE hydrolases in a dose-dependent manner. Eicosanoid dependency was established by demonstrating that HDL-induced acid cholesteryl ester hydrolase (ACEH) activity was blocked by aspirin. CE enrichment essentially abrogated HDL-induced PGI2 production in cells which also exhibited decreased lysosomal and cytoplasmic CE hydrolase activities. In CE-enriched cells whose cytoplasmic CE pool was metabolically labeled with [3H]oleate or cLDL containing [3H]cholesteryl linoleate, aspirin did not alter HDL- or BSA-induced net CE hydrolysis or efflux, respectively. Finally, aspirin treatment did not alter the mass of either free or esterified cholesterol content of untreated or CE-enriched SMC following exposure to acceptor proteins. These data demonstrated that CE enrichment significantly reduced HDL-induced activation of CE hydrolytic activity via inhibition of endogenous PGI2 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

巨噬细胞、内皮细胞对高密度脂蛋白的氧化修饰

为了探讨高密度脂蛋白 (HDL)在体内发生氧化修饰的部位及机制 ,分别观察了培养人动脉平滑肌细胞 (SMC)、动脉内皮细胞 (EC)及巨噬细胞 (MΦ)与HDL共同温育过程中 ,HDL的琼脂糖电泳相对迁移率 (REM)、硫代巴比妥酸反应物质 (TBARS)以及溶血卵磷脂 卵磷脂 (LPC PC)值等氧化指标的变化 .结果发现 ,HDL与 3种细胞温育 12h时 ,HDL几乎不发生氧化修饰 ,而HDL与MΦ和EC温育 2 4h后 ,其REM、TBARS、LPC PC值均显著上升 (p <0 0 1) ;而HDL与SMC温育后 ,其REM、TBARS值无显著改变 ,LPC PC值增加 (p <0 0 1) .结果提示 ,活体内HDL可能主要在动脉壁的内皮细胞及巨噬细胞的作用下发生氧化修饰     

天然和氧化修饰型LDL、VLDL及HDL对培养人动脉平滑肌细胞原癌基因fos及myc表达的影响

动脉平滑肌细胞（ＳＭＣ）是动脉粥样硬化（ＡＳ）斑块中的主要细胞,它的增殖在ＡＳ形成过程中极其重要。脂蛋白和氧化修饰型脂蛋白对ＳＭＣ增殖的影响以及ＳＭＣ增殖与原癌基因异常表达的关系是当前ＡＳ发病机制研究的热点之一。我们在建立人主动脉ＳＭＣ体外培养方法的基础上,观察了ＬＤＬ,ＶＬＤＬ及ＨＤＬ和相应的氧化修饰型脂蛋白对培养人ＳＭＣｆｏｓ,ｍｙｃ,ｅｒｂ－Ｂ原癌基因转录表达的影响。结果表明：①ＨＤＬ对ＳＭＣｆｏｓ,ｍｙｃ基因表达无影响;②ＬＤＬ和ＶＬＤＬ有使这些基因表达增加的趋势,但与对照比较差异不显著（Ｐ＞０．０５）;③ＯＸ－ＶＬＤＬ,ＯＸ－ＶＬＤＬ和ＯＸ－ＨＤＬ有使ＳＭＣｆｏｓ,ｍｙｃ基因表达显著增强的作用（Ｐ＜０．０１）,且其作用较相应的天然脂蛋白大（Ｐ＜０．０１）．上述结果说明：ＬＤＬ,ＶＬＤＬ,ＯＸ－ＬＤＬ,ＯＸ－ＶＬＤＬ和０Ｘ－ＨＤＬ的致ＡＳ作用可能与刺激ＳＭＣｆｏｓ和ｍｙｃ癌基因表达增加有关。