─株特异抗人血浆Lp（a）单克隆抗体（A5g3）的制备及特征分析

用序列超速离心和柱层析分离纯化的人血浆脂蛋白（ａ）（Ｌｐ（ａ））免疫Ｂａｌｂ／ｃ小鼠，得到具有对Ｌｐ（ａ）高抗体活性的小鼠脾细胞，与小鼠ＮＳ１骨髓瘤细胞经聚乙二醇（ＰＥＧ）杂文融合，建立分泌特异抗人血浆Ｌｐ（ａ）的单克隆杂交瘤细胞系，对其中的─株单克隆细胞株──Ａ５ｇ３进行了初步分析。Ａ５ｇ３分泌的单克隆抗体类型为ＩｇＧ（２ａ），经间接ＥＬＩＳＡ法分析与Ｌｐ（ａ）有高度反应性，与低密度脂蛋白（ＬＤＬ）及血浆纤溶酶原（Ｐｇ）无反应，免疫印迹结果也表明，Ａ５ｇ３可与纯化的或血浆中的Ｌｐ（ａ）结合，与血浆中的其它成分和纯化的ＬＤＬ及Ｐｇ均不结合，因此Ａ５ｇ３是特异抗人血浆Ｌｐ（ａ）的单克隆抗体。Ａ５ｇ３与还原后的Ｌｐ（ａ）也不结合，说明其与Ｌｐ（ａ）的结合依赖于抗原的完整结构，具有此种特性的Ｌｐ（ａ）特异抗体国内少见报道。     

人主动脉粥样硬化病变中修饰脂蛋白的研究

目的研究人粥样硬化病变主动脉中丙二醛（ＭＤＡ）和４羟基壬烯醛（ＨＮＥ）修饰载脂蛋白Ｂ（ａｐｏＢ）的分布特点、含量和理化特性,并与脂蛋白（ａ）［Ｌｐ（ａ）］和ａｐｏＢ的分布进行比较。方法应用免疫组织化学、电镜、免疫电镜以及生物化学等方法进行定性或定量分析。结果ＭＤＡ和ＨＮＥ－ａｐｏＢ在细胞外基质中的分布与Ｌｐ（ａ）和ａｐｏＢ一致,但在泡沫细胞内则有不同。ＭＤＡ－ａｐｏＢ和ＨＮＥ－ａｐｏＢ在泡沫细胞内呈环状、孔状分布,与蜡样色素相似。病变动脉内膜低密度脂蛋白的超微结构、化学成分及电泳行为均与体外修饰脂蛋白相似,其醛类修饰ａｐｏＢ含量明显高于无病变者。结论脂蛋白的氧化性修饰是动脉粥样硬化发生的必要条件。     

脂蛋白（a）的研究近况—分子生物学与疾病的相关性

脂蛋白（ａｑ）「Ｌｐ（ａ）」是一种与低密度脂蛋白类似的脂蛋白，由ＬＤＬ成分和载脂蛋白（ａ）「Ａｐｏ（ａ）」组成。ＬＰ（ａ）的血浆浓度与Ａｐｏ（ａ）分子量大小成反比，主要由Ａｐｏ（ａ）基因的多态性决定，有个体差异和种族差异。     

氧化修饰的低密度脂蛋白和极低密度脂蛋白对单核细胞MCP—1…

为了研究氧化修饰的低密度脂蛋白（ＯＸ－ＬＤＬ）和氧化修饰的极低密度脂蛋白（ＯＸ－ＶＬＤＬ）对单核细胞的单核细胞趋化蛋白１（ＭＣＰ－１）ｍＲＮＡ和蛋白表达的影响，在单核细胞的培养基中分别加入２５μｇ／ｍｌ的ＬＤＬ，ＯＸ－ＬＤＬ，ＶＬＤＬ和ＯＸ－ＶＬＤＬ，培养２４小时后分别收集单核细胞及其条件培养基，对照异硫氰酸胍法提取单核细胞总ＲＮＡ，并用γ＾３２Ｐ末端标记ＭＣＰ－１寡核苷酸探针，进行ｓｌｏｔｂｌｏ     

氧化修饰的低密度脂蛋白和极低密度脂蛋白对单核细胞MCP-1表达的影响

为了研究氧化修饰的低密度脂蛋白（ＯＸ┐ＬＤＬ）和氧化修饰的极低密度脂蛋白（ＯＸ┐ＶＬＤＬ）对单核细胞的单核细胞趋化蛋白１（ＭＣＰ┐１）ｍＲＮＡ和蛋白表达的影响。在单核细胞的培养基中分别加入２５μｇ／ｍｌ的ＬＤＬ、ＯＸ┐ＬＤＬ、ＶＬＤＬ和ＯＸ┐ＶＬＤＬ，培养２４小时后分别收集单核细胞及其条件培养基。参照异硫氰酸胍法提取单核细胞总ＲＮＡ，并用γ３２Ｐ末端标记ＭＣＰ┐１寡核苷酸探针，进行ｓｌｏｔｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析。同时用夹心ＥＬＩＳＡ检测其条件培养基中ＭＣＰ┐１蛋白含量。结果显示单核细胞能表达ＭＣＰ┐１，ＯＸ┐ＬＤＬ和ＯＸ┐ＶＬＤＬ能明显增强单核细胞表达ＭＣＰ┐１。而ＬＤＬ和ＶＬＤＬ的作用却很轻微。说明单核细胞能通过表达ＭＣＰ┐１，从而进一步招引其它的单核细胞迁入内皮下间隙，而ＯＸ┐ＬＤＬ和ＯＸ┐ＶＬＤＬ能加强这种作用。     

载脂蛋白（a）基因结构与功能研究

脂蛋白（ａ）［Ｌｐ（ａ）］是一种低密度脂蛋白（ＬＤＬ）样的脂蛋白。ＬＤＬ样脂蛋白是致动脉粥样硬化的主要危险因素。ＬＤＬ样脂蛋白与Ｌｐ（ａ）不同之处就在于前者比后者多个载脂蛋白（ａ）［ａｐｏ（ａ）］。ａｐｏ（ａ）基因结构与血纤维蛋白溶酶原同源。ａｐｏ（ａ）与血纤维蛋白溶酶原活性竞争可以说明一些与Ｌｐ（ａ）有关的病理生理学问题。现已识别６个高度相关的基因，至少在重叠的基因组克隆附近发现４个相关基因。目     

抗apoAl、B和Lp（α）混合单抗与单一单抗包被的比较

抗ａｐｏＡｌ、Ｂ和Ｌｐ（α）混合单抗与单一单抗包被的比较赵满仓，刘菊林，李健斋，蒋雷（空军石家庄医院检验科，石家庄０５００８１）（卫生部北京老年医学研究所，北京１００７３０）：ａＰｏＡ１、Ｂ、ＬＰ（ａ）分别为高密度脂蛋白（ＨＤＬ）、低密度脂蛋白（ＬＤ...     

粘附分子对CD3介导的胸腺细胞[Ca^2＋]i应答的影响

ＬＦＡ－１和ＩＣＡＭ－１广泛表达于各胸腺细胞亚群，但ＩＣＡＭ－１在ＰＮＡ￣＋细胞的表达下调。本文报道：用抗ＬＦＡ－１／ＩＣＡＭ－１和抗ＣＤ３单抗，分析了粘附分子ＬＦＡ－１／ＩＣＡＭ－１对抗ＣＤ３诱导的胸腺细胞［Ｃａ￣（２＋）］ｉ应答的影响。结果显示，可溶性抗ＬＦＡ－１／ＩＣＡＭ－１可抑制ＣｏｎＡ刺激的胸腺细胞增殖，且以抗ＬＦＡ－１抗体的作用更为显著，在ＣｏｎＡ或抗ＣＤ３诱导的胸腺细胞［Ｃａ￣（２＋）］ｉ应答中，抗ＬＦＡ－１单抗可明显抑制［Ｃａ￣（２＋）ｉ升高。但如果用二抗交联ＣＤ３和ＬＦＡ－１，胸腺细胞［Ｃａ￣（２＋）ｉ则显著高于单独交联ＣＤ３时的水平（Ｐ＜０．０１），而ＣＤ３与ＩＣＡＭ－ｌ交联却无此效应，此外，仅交联ＬＦＡ－１或ＩＣＡＭ－１也无诱导［Ｃａ￣（２＋）］ｉ应答的作用。提示在ＬＦＡ－ｌ与ＩＣＡＭ－１介导的胸腺细胞与胸腺基质细胞相互作用中，ＬＦＡ－１可为ＴＣＲ／ＣＤ３途径介导的胸腺细胞活化提供复合刺激信号。     

氧化修饰极低密度脂蛋白对单核和巨噬细胞趋化蛋白1mRNA表…

近年来的研究发现，单核细胞趋化蛋白１（ＭＣＰ－１）是一种极为强烈的单核细胞趋向因子。本研究着重观察了极低密度脂蛋白（ＶＬＤＬ）和氧化修饰的极低密度脂蛋白（ＯＸ－ＶＬＤＬ）对单核细胞和巨噬ＭＣＰ－１ｍＲＮＡ表达的影响。在单核核和巨噬细胞的培养基中分别中入２５μｇ／ｍｌ的ＶＬＤＬ或ＯＸ－ＶＬＤＬ，培养２４ｈ提取单核细胞和巨噬细胞总ＲＮＡ用ＭＣＰ－１的寡核苷酸探针，进行Ｎｏｒｔｈｅｍｂｌｏｔ分析。结果显     

离体培养动脉内皮细胞结合,内移和降解脂蛋白（a）作用的研究

本文研究离体培养动脉内皮细胞通过ＬＤＬ受体结合，内移和降解脂蛋白（ａ）［Ｌｉｐｏｐｒｏ－ｔｅｉｎ（ａ），Ｌｐ（ａ）］的作用，实验取第３－４代离体培养的动脉内皮细胞分为两组，对照组培养液中不加碘标的脂蛋白（ａ）［＾１２５Ｉ－Ｌｐ（ａ）］，实验组培养液中加＾１２５Ｉ－Ｌｐ（ａ）最终浓度分别为０．２５、０．５、１、１０和５０μｇ／ｍｌ。用γ计数器测定内皮细胞对Ｌｐ（ａ）的结合、内移和降解值，并测定内皮细     

Galectin-1, an endogenous lectin produced by arterial cells, binds lipoprotein(a) [Lp(a)] in situ: relevance to atherogenesis

Lipoprotein(a) [Lp(a)], a modified LDL molecule, is implicated in atherogenesis. Mechanisms of the accumulation of [Lp(a)] in atherosclerotic vessels is lacking in literature. We sought to investigate the complementarities of the carbohydrate structures on Lp(a) and LDL with galectin-1(a carbohydrate binding protein) and whether endogenous galectin-1 binds Lp(a) in situ. We investigated T-antigen structures on Lp(a) and LDL by enzyme-linked lectin assay using T-antigen specific lectins, galectin-1 and jacalin. Both jacalin and galectin-1 bound strongly to Lp(a) and to a much lesser extent, to LDL. Galectin-1 recognition of the lipoproteins was abolished when the O-linked sugars were selectively removed. Localization of endogenous galectin-1 within histological sections of human internal mammary artery and in vitro binding of Lp(a) to the tissues was analyzed by immunohistochemical staining. The Lp(a)-binding pattern was found to overlap with the localization of galectin-1. The poor Lp(a)-binding on inhibiting tissue galectin-1 with lactose, suggested the binding of Lp(a) to galectin-1. This may be suggestive of a mechanism by which Lp(a) accumulates within arterial walls in atherogenesis.     

阿魏酸钠拮抗氧化型脂蛋白(a)对人动脉平滑肌细胞的作用

目的:研究脂蛋白(a)氧化前后致人动脉平滑肌细胞(SMC)增殖及细胞内游离钙浓度([Ca²⁺]_i)的变化,观察阿魏酸钠(SF)对其的影响。方法:Lp(a)经体外Cu²⁺氧化法氧化,硫代巴比妥酸(TBARS)比色法检测氧化程度,培养的人动脉SMC中分别加入不同浓度SF,作用12h后再与天然和氧化型Lp(a)共同孵育,以MTT比色法、流式细胞仪检测细胞增殖状况,采用荧光探针Fura-2/AM检测细胞[Ca²⁺]_i。结果:氧化型Lp(a)促人动脉SMC增殖的同时亦明显增加了[Ca²⁺]_i水平,作用较天然Lp(a)明显,SF(40,80mg/L)可显著抑制氧化型Lp(a)所致的细胞增殖和[Ca²⁺]_i增加,并呈剂量效应关系,而对天然Lp(a)所致的细胞增殖和[Ca²⁺]_i增加无明显影响。结论:氧化型Lp(a)通过升高[Ca²⁺]_i而显著促动脉SMC增殖可能是其致动脉粥样硬化的机制之一,SF拮抗这种作用可能与其抗氧化能力有关。     

Evidence of dependence of lipoprotein(a) on triglyceride and high-density lipoprotein metabolism

Lipoprotein(a) [Lp(a)] is a complex lipoprotein consisting of a low-density lipoprotein (LDL)-like ApoB₁₀₀-containing core particle covalently bound to apo(a), a large functionally complex glycoprotein. The mechanisms of Lp(a) metabolism and its interactions with cell-surface lipoprotein receptors are incompletely understood. In this study, we investigated the relationship of Lp(a) to other lipoproteins at high and normal levels of serum triglycerides (TGs). We measured serum lipid and Lp(a) particle concentrations in 148 unselected primary- and secondary-prevention patients. Subjects with TG > 200 mg/dL were classified as having high TG in accordance with National Cholesterol Education Program Adult Treatment Panel III guidelines. Our analysis revealed mean TG levels of 100 and 270 mg/dL in the normal and high TG groups, respectively. Lp(a)-C, Lp(a)-P, and Lp(a) cholesterol content per particle [Lp(a)-C/Lp(a)-P] did not differ between groups. At normal TG levels, stepwise multiple linear regression revealed that Lp(a)-P correlated with Lp(a)-C (P < 10^?6), ApoAI (P = .0001), the high-density lipoprotein cholesterol subfraction ratio (HDL₂-C/HDL₃-C; P = .002), and dense very-low-density lipoprotein cholesterol (VLDL₃-C; P = .04), overall model R = 0.74. At high TG levels, Lp(a)-P very strongly correlated primarily with HDL₂-C/HDL₃-C and TG-related variables with minimal dependence on Lp(a)-C (P = .09), overall model R = 0.96. These findings provide evidence of shared metabolic mechanisms for Lp(a), HDL, TG, and very low-density lipoprotein at high serum TG. Future studies are needed to elucidate common mechanisms, enzymes, and receptors involved in Lp(a) and HDL/TG metabolism with a focus on how these mechanisms are modified in the setting of hypertriglyceridemia.     

Human PlasmaAnti-α-galactoside Antibody Forms Immune Complex with Autologous Lipoprotein(a)

Anti-α-galactoside antibody (anti-Gal) from human plasma that bound to α-galactoside-bearing guar galactomannan gel and was eluted with specific sugar (affinity-purified anti-Gal ; APAG) invariably contained apo(a) and apo B subunits in a proportion close to that in plasma lipoprotein(a) [ Lp(a)]. Since LDL does not contain apo(a), result suggested Lp(a) as a component of APAG. Lp(a) in APAG was complexed with anti-Gal since plate-coated anti-apo(a) captured Lp(a) along with the antibody. Association of Lp(a) with anti-Gal in APAG was considerably lower in presence of anti-Gal-specific sugar, suggesting that Lp(a) occupied the sugar-binding site of anti-Gal. Content of Lp(a)-bound anti-Gal in APAG, though a minor fraction of total antibody, increased steadily with total Lp(a) content of plasma. Further, Lp(a) released from immune complex-rich fraction of plasma by anti-Gal- specific sugar was proportional to total plasma Lp(a). Anti-Gal titre decreased with increasing Lp(a) concentration among 114 plasma samples. Results indicate the potential of anti-Gal molecules with its binding site partially occupied by Lp(a) molecule(s) to a) use the remaining binding site(s) to recognize other macromolecules or cells and b) transport Lp(a) across Fc receptor-bearing cells.     

Oxidized phospholipids and lipoprotein-associated phospholipase A2 as important determinants of Lp(a) functionality and pathophysiological role

Lipoprotein(a) [Lp(a)] is composed of a low density lipoprotein (LDL)-like particle to which apolipoprotein (a) [apo(a)] is linked by a single disulfide bridge. Lp(a) is considered a causal risk factor for ischemic cardiovascular disease (CVD) and calcific aortic valve stenosis (CAVS). The evidence for a causal role of Lp(a) in CVD and CAVS is based on data from large epidemiological databases, mendelian randomization studies, and genome-wide association studies. Despite the well-established role of Lp(a) as a causal risk factor for CVD and CAVS, the underlying mechanisms are not well understood. A key role in the Lp(a) functionality may be played by its oxidized phospholipids (OxPL) content. Importantly, most of circulating OxPL are associated with Lp(a); however, the underlying mechanisms leading to this preferential sequestration of OxPL on Lp(a) over the other lipoproteins, are mostly unknown. Several studies support the hypothesis that the risk of Lp(a) is primarily driven by its OxPL content. An important role in Lp(a) functionality may be played by the lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme that catalyzes the degradation of OxPL and is bound to plasma lipoproteins including Lp(a). The present review article discusses new data on the pathophysiological role of Lp(a) and particularly focuses on the functional role of OxPL and Lp-PLA2 associated with Lp(a)     

Lipoprotein(a) and inflammation- pathophysiological links and clinical implications for cardiovascular disease

The role of lipoprotein(a) (Lp[a]) as a significant and possibly causal cardiovascular disease (CVD) risk factor has been well established. Many studies, mostly experimental, have supported inflammation as a mediator of Lp(a)-induced increase in CVD risk. Lp(a), mainly through oxidized phospholipids bound to its apolipoprotein(a) part, leads to monocyte activation and endothelial dysfunction. The relationship between Lp(a) and inflammation is bidirectional as Lp(a) levels, besides being associated with inflammatory properties, are regulated by inflammatory stimuli or anti-inflammatory treatment. Reduction of Lp(a) concentration, especially by potent siRNA agents, contributes to partial reversion of the Lp(a) related inflammatory profile. This review aims to present the current pathophysiological and clinical evidence of the relationship between Lp(a) and inflammation.     

Quantitative analysis of apolipoproteins and lipoprotein particles in patients with head injuries

By head-injured patients, apo A-I and apo A-II concentrations were more decreased in HDL3 than in HDL2. Then, the plasmatic concentrations of the main lipoprotein particles present in HDL fraction were modified. For example, a significant decrease of Lp A-I: A-II particles was observed and this variation was similar to that of total apo A-I (r = 0.78). On the other hand, the concentration of Lp A-I particles was slightly modified, apo C-III concentration was markedly decreased whereas apo E concentration was significantly increased (p less than 0.05); in plasma samples obtained 10 days after a severe head injury, apo E reached three times the normal value.     

INDICATIONS OF AN AUTOIMMUNE COMPONENT IN LP(a) ASSOCIATED DISORDERS

The exceptionally strong independent association found between Lp(a) lipoprotein [Lp(a)] levels and atherosclerotic disorders indicate that Lp(a) is a factor of considerable importance in the pathogenesis of atherosclerosis. The association between Lp(a) and diabetes, rheumatoid arthritis and renal diseases suggest that Lp(a) may be involved in immunological mechanisms. Lp(a) has a great tendency to aggregate and bind to glucosaminoglycans, fibrin and fibronectin and is preferentially retained in the extracellular matrix during development of atherosclerosis and is in vitro phagocytosed by macrophages, probably as small aggregates. It was previously found that the Lp(a) level is significantly related to the HLA class II genotype in male patients with early coronary artery disease. In this paper additional results of interleukin determinations in relation to HLA type and Lp(a) levels are presented and discussed. It is suggested that an autoimmune process, perhaps triggered by a concomitant intracellular infection may occur, especially in patients with inherited high Lp(a) levels in combination with certain inherited HLA class II genotypes.     

Lipoprotein(a) particle concentration and lipoprotein(a) cholesterol assays yield discordant classification of patients into four physiologically discrete groups

There is little known about the relative predictive value of different lipoprotein(a) [Lp(a)] assays in clinical use, although each has been shown to predict similar incremental risk over conventional clinical and lipid risk factors. Thus, we examined the classification behavior of two commonly used Lp(a) assays and their associations with other lipid parameters. Serum lipid and Lp(a) concentrations were measured in 144 primary and secondary prevention patients. Lp(a) cholesterol [Lp(a)-C] was measured with the Vertical Auto Profile (upper limit of normal, 10 mg/dL). Lp(a) particle concentrations [Lp(a)-P] were measured with an isoform-independent molar assay (upper limit of normal, 70 nmol/L). The subjects were divided into the following four groups on the basis of their Lp(a)-C and Lp(a)-P levels: normal Lp(a)-P and Lp(a)-C; high Lp(a)-P and normal Lp(a)-C; normal Lp(a)-P and high Lp(a)-C; and high Lp(a)-P and Lp(a)-C. The proportion of subjects with values above the upper limit of normal was similar with both assays (P = .15). However, the Lp(a)-C and Lp(a)-P assays discordantly classified 23% of the study's subjects. In addition, the four Lp(a)-defined groups displayed differences in their relationships with other lipoproteins. The two groups with elevated Lp(a)-C showed significant associations with higher high-density lipoprotein cholesterol, apolipoprotein AI, and high-density lipoprotein cholesterol/apolipoprotein AI ratios. Triglycerides were also noted to be above normal in discordant and normal within concordant Lp(a) groups. Finally, the amount of cholesterol per Lp(a) particle [Lp(a)-C/Lp(a)-P] varied widely across the four groups. These findings suggest that the four Lp(a)-defined groups are physiologically discrete. Further investigation is warranted to assess which parameters among Lp(a)-P, Lp(a)-C, and Lp(a)-C/Lp(a)-P can be used to more accurately characterize Lp(a)-associated cardiovascular risk.