含载脂蛋白B的脂蛋白对小鼠腹腔巨噬细胞内脂质的影响

将小鼠腹腔巨噬细胞和含载脂蛋白Ｂ的两种脂蛋白、这两种脂蛋白与硫酸葡聚糖或相应抗体形成的复合物一起培养２４ｈ后，用薄层层析法测定巨噬细胞内胆固醇酯的含量。结果发现，天然脂蛋白（ａ）对巨噬细胞内胆固醇酯含量影响不大（增加８％，Ｐ〉０．０５）；天然低密度脂蛋白具有促进巨噬细胞内脂质蓄积作用（增加３５．４％，Ｐ〈０．０５）；上述两种脂蛋白与硫酸葡聚糖一起共同作用，促进巨噬细胞内胆醇酯蓄积作用明显加强（分别     

巨噬细胞集落刺激因子对小鼠腹腔巨噬细胞清道夫受体途径和细胞内胆固醇酯积聚的影响

为探索巨噬细胞集落刺激因子、清道夫受体、氧化型低密度脂蛋白与动脉粥样硬化的关系，观察了重组人巨噬细胞集落刺激因子对小鼠腹腔巨噬细胞清道夫受体途径的影响以及重组人巨噬细胞集落刺激因子对氧化型低密度脂蛋白所致细胞内胆固醇酯积聚的影响．结果发现重组人巨噬细胞集落刺激因子能增加培养的小鼠腹腔巨噬细胞表面的清道夫受体数目，使之对氧化型低密度脂蛋白的结合和降解呈现剂量和时间依赖性增加，并使细胞内胆固醇酯积聚增多．表明巨噬细胞集落刺激因子可通过增加清道夫受体数目使小鼠腹腔巨噬细胞对氧化型低密度脂蛋白的结合和降解增多，从而增加细胞内胆固醇酯含量，促进动脉壁内泡沫细胞形成．     

低密度脂蛋白免疫复合物对单核细胞源性巨噬细胞胆固醇代谢及低密度脂蛋白受体表达的影响

目的 研究动脉粥样硬化过程中低密度脂蛋白免疫复合物对单核细胞源性巨噬细胞胆固醇代谢和低密度脂蛋白受体表达的影响。方法 以佛波酯刺激分化的THP-1细胞作为单核细胞源性巨噬细胞模型。通过胆固醇酶联法测定细胞内胆圊醇含量，逆转录-聚合酶链反应检测细胞低密度脂蛋白受体mRNA的表达，Western blot检测细胞低密度脂蛋白受体蛋白的表达。结果 与低密度脂蛋白组、阴性组及IgG免疫复合物组比较，低密度脂蛋白免疫复合物组细胞内胆固醇水平明显增高，并且低密度脂蛋白受体mRNA及蛋白的表达增加。结论 低密度脂蛋白免疫复合物能显著增加巨噬细胞内胆固醇含量，亦能够促进低密度脂蛋白受体mRNA与蛋白的表达，提示低密度脂蛋白免疫复合物在动脉粥样硬化进展过程中起重要的作用。     

巨噬细胞集落刺激因子对小鼠腹腔巨噬细胞清道夫受体途径和细胞内胆固醇酯积聚的影响

为探索巨噬细胞集落刺激因子，清道夫受体、氧化型低密度脂蛋白与动脉粥样硬化的关系，观察了重组人巨噬细胞集落刺激因子对小鼠腹腔巨噬细胞清道夫受体途径的影响以及重组人巨细胞内胆固醇酯积聚的影响。结果发现重组人巨噬细胞集落刺激因子能增加培养小鼠的腹腔巨噬细胞表面的清道夫受体数目，使之对氧化型低密度脂蛋白的结合和降解呈现剂量和时间依赖性增加，并使细胞内胆固醇酯积聚增多。     

母牛分支杆菌制剂对小鼠腹腔巨噬细胞产生一氧化氮水平的影响

一氧化氮（ＮＯ）在活化巨噬细胞抗某些肿瘤细胞和微生物上起重要作用。用不同剂量的母牛分支杆菌制剂免疫ＢＡＬＢ／ｃ小鼠，检测小鼠腹腔巨噬细胞产生一氧化氮的水平，结果显示受免疫小鼠腹腔巨噬细胞产生一氧化氮水平明显高于未免疫小鼠（Ｐ＜０．０１－０．００１）。不同剂量母牛分支杆菌制剂免疫的ＢＡＬＢ／ｃ小鼠，腹腔巨噬细胞产生的一氧化氮水平差别不显著（Ｐ＞０．０５）。因此认为，母牛分支杆菌制剂作为结核病免疫治疗剂其作用之一是活化巨噬细胞产生一氧化氮，从而达到杀菌的目的。     

蛋白激酶C抑制剂对小鼠腹腔巨噬细胞清道夫受体功能的影响

目的 观察细胞内蛋白磷酸化水平对清道夫受体功能的影响。方法 用蛋白激酶C抑制荆星形孢菌素处理小鼠腹腔巨噬细胞，利用蛋白质印迹试验和放射自显影方法观察药物对细胞表面受体表达的影响，并分别测定对照组和处理组细胞对碘标记的氧化型低密度脂蛋白的结合、降解以及细胞内脂质蓄积的程度。结果 0．4μmol／L蛋白激酶C抑制剂星形孢菌素可以促进细胞结合碘标记的氧化型低密度脂蛋白，增加细胞表面受体的表达，但抑制细胞降解碘标记的氧化型低密度脂蛋白，同时抑制细胞内胆固醇酯的蓄积。结论 以上表明清道夫受体功能与细胞内蛋白质磷酸化水平密切相关。     

低密度脂蛋白受体基因敲除鼠极低密度脂蛋白和中间密度脂蛋白组分诱导J774巨噬细胞胆固醇酯蓄积

为探讨低密度脂蛋白受体基因敲除鼠血浆极低密度脂蛋白和中间密度脂蛋白组分致动脉粥样硬化的作用,采用密度梯度序列超速离心法从低密度脂蛋白受体基因敲除鼠血浆分离极低密度脂蛋白和中间密度脂蛋白组分,用日立７４５０ 自动分析仪测定其脂含量,琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳测定其电泳迁移率和载脂蛋白组成,并与鼠腹腔巨噬细胞共同孵育,观察它与巨噬细胞的相互作用。结果发现, 低密度脂蛋白受体基因敲除鼠血浆极低密度脂蛋白和中间密度脂蛋白组分与Ｊ７７４ 巨噬细胞孵育后,细胞胆固醇酯水平［（７３±０ ．５） μｍｏｌ（ｇ．ｃｅｌｌｐｒｏｔｅｉｎ）］ 非常显著地大于空白对照组（ Ｐ＜ ０．００５） , 是天然低密度脂蛋白诱导细胞胆固醇酯［（８±７） μｍｏｌ（ｇ．ｃｅｌｌｐｒｏｔｅｉｎ）］蓄积的９ 倍。结果提示,低密度脂蛋白受体缺失鼠血浆非修饰极低密度脂蛋白和中间密度脂蛋白组分可被Ｊ７７４ 巨噬细胞摄取,转巨噬细胞为泡沫细胞。     

一氧化氮在低密度脂蛋白氧化中的双向作用

为观察一氧化氮与低密度脂蛋白氧化的关系,揭示一氧化氮在动脉粥样硬化发病和防治中的作用,给小鼠腹腔注射云芝多糖,再用干扰素γ、脂多糖和Ｌ－精氨酸刺激小鼠腹腔巨噬细胞,然后检测一氧化氮释放量与脂氢过氧化物浓度。结果发现,一氧化氮与低密度脂蛋白氧化间有一个量的相关关系（ｒ＝０．７５３,Ｐ〈０．０５）,一定范围内（≤５０μｍｏｌ／Ｌ）的一氧化氮释放量与脂氢过氧化的量呈负相关,即抑制细胞对低密度脂蛋白的氧化;当一氧化氮产生过多时（≥９０μｍｏｌ／Ｌ）,脂氢过氧化物的量随之增加,则促进低密度脂蛋白的氧化。结果提示,一氧化氮在低密度脂蛋白氧化中发挥双重作用,它对低密度脂蛋白的氧化起抑制或促进作用取决于它的释放量。     

血浆氧化型低密度脂蛋白水平与低密度脂蛋白循环免疫复合物的相关性

为探讨血浆氧化型低密度脂蛋白水平与低密度脂蛋白循环免疫复合物间关系。分别建立了以抗人IgG为包被抗体、酶标抗载脂蛋白B为检测抗体的低密度脂蛋白循环免疫复合物酶联免疫吸附试验;以抗氧化型低密度脂蛋白为包被抗体、酶标抗载脂蛋白B为检测抗体的氧化型低密度脂蛋白酶联免疫吸附试验。同时测定60例冠心病患者及50例对照人群低密度脂蛋白循环免疫复合物、氧化型低密度脂蛋白水平并进行相关性分析。结果发现,冠心病患者甘油三酯、脂蛋白(a)和载脂蛋白B水平均显著升高;高密度脂蛋白胆固醇和载脂蛋白AI水平降低;且低密度脂蛋白循环免疫复合物(2.74±0.73比1.38±0.78,p<0.001)、氧化型低密度脂蛋白水平(595.5±194.8比440.3±175.0μg/L,p<0.001)均显著升高。低密度脂蛋白循环免疫复合物水平分别同血浆总胆固醇、甘油三酯、低密度脂蛋白胆固醇、脂蛋白(a)及载脂蛋白B正相关;同载脂蛋白A_Ⅰ负相关。氧化型低密度脂蛋白水平亦分别同血浆总胆固醇、低密度脂蛋白胆固醇、脂蛋白(a)及载脂蛋白B正相关。氧化型低密度脂蛋白水平与低密度脂蛋白循环免疫复合物亦呈正相关(r=0.313,p<0.005)。结果提示,氧化型低密度脂蛋白及循环免疫复合物升高是动脉粥样硬化发生的危险因素。     

肉苁蓉总苷对D-半乳糖所致衰老模型小鼠免疫功能的影响

目的　研究肉苁蓉总苷对 D-半乳糖致衰老小鼠抗衰老作用的免疫学机制。方法　通过 3H- Td R掺入法测定淋巴细胞转化能力、中性红试验测定小鼠腹腔巨噬细胞吞噬功能、免疫荧光法进行淋巴细胞亚群的检测、放免法测定外周血 IL- 2含量、微量 NAG酶释放法测定 NK细胞活性。结果　 D-半乳糖致衰小鼠免疫功能明显下降 ,表现为淋巴细胞转化能力、外周血 IL - 2含量、CD4 T细胞和 CD8 T细胞含量、腹腔巨噬细胞吞噬功能和 NK细胞活性均明显降低 (P<0 .0 5) ;肉苁蓉总苷能提高模型小鼠淋巴细胞转化能力、外周血 IL - 2含量、腹腔巨噬细胞吞噬功能、NK细胞活性、CD4 T和 CD8 T细胞含量 (P<0 .0 5)。结论　肉苁蓉总苷明显增强 D-半乳糖致衰老小鼠的免疫机能 ,使其恢复或接近了青年小鼠状态 ,具有延缓衰老的作用     

Effects of noninsulin-dependent diabetes mellitus on the uptake of very low density lipoproteins by thioglycolate-elicited mouse peritoneal macrophages

The lipid-laden foam cells from atherosclerotic lesions appear to be derived from macrophages which have accumulated lipids from plasma lipoproteins. When examined in vitro, thioglycolate-elicited mouse peritoneal macrophages do not accumulate lipids when exposed to normal low density lipoproteins (LDL), but take up very low density lipoproteins (VLDL) or chemically modified LDL with resultant lipid accumulation. Patients with noninsulin-dependent diabetes mellitus (NIDDM) have an increased incidence of atherosclerosis, due in part to disturbances in lipoprotein metabolism. We investigated the possibility that VLDL isolated from patients with NIDDM are taken up by mouse peritoneal macrophages more avidly than normal. Two groups of patients with NIDDM were studied; one group was normotriglyceridemic and the other group was hypertriglyceridemic. The VLDL from both normotriglyceridemic and hypertriglyceridemic patients were enriched in cholesterol and triglyceride compared to that from normal subjects. Thioglycolate-elicited mouse peritoneal macrophages bound and degraded greater amounts of VLDL isolated from patients with NIDDM (both normo- and hypertriglyceridemic) than of VLDL from normal subjects. While normal VLDL caused a marked increase in cellular triglyceride and cholesteryl ester contents in macrophages, VLDL isolated from patients with NIDDM resulted in an even greater cellular accumulation of lipids. These results suggest that the VLDL of patients with NIDDM have alterations in their composition and metabolic behavior. The increased uptake of VLDL by macrophages may contribute to the enhanced atherosclerosis present in NIDDM.     

Effect of plasma lipoproteins on cholesterol accumulation in macrophages: comparison of lipoproteins from normal and homozygous familial hypercholesterolemic subjects

Total cholesterol (TC) content of mouse peritoneal macrophages (MPM) increased when incubated with increasing concentrations of normal low density (N-LDL) or very low density (N-VLDL) lipoprotein. Incubation with increasing concentrations of normal high density lipoprotein (N-HDL) caused a decrement in cellular mass of TC in MPM. Incubation of MPM with serum from normal subjects as well as from subjects with homozygous familial hypercholesterolemia (HFH) resulted in a 25% increment in cellular mass of TC, due to an increment in both free cholesterol (FC) and cholesteryl ester (CE) fractions. Accumulation of TC in MPM, due mainly to elevation of CE, was observed when the macrophages were incubated in the presence of LDL or VLDL derived from either group of subjects. N-LDL caused a higher increment in cellular CE compared to HFH-LDL. However, the presence of HFH-VLDL in the medium caused elevation in the cellular TC and CE content to a higher level than did N-VLDL. The presence of N-HDL as well as of HFH-HDL in the medium resulted in a similar decrement in the cholesterol content of MPM. The decrement was expressed in both FC and CE fractions. The present study shows different abilities of normal and HFH plasma lipoproteins to cause cholesterol accumulation in MPM.     

Large variations in human foam cell formation in individuals: a fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids

The transformation of monocyte-derived macrophages into lipid-laden foam cells constitutes a characteristic and crucial event in the development of the earliest atherosclerotic lesions. We investigated whether the propensity to form foam cells varies among individuals. We developed a fully autologous foam cell assay based on a recently developed novel culture technique for human monocyte-derived macrophages (Wintergerst ES, Jelk J, Asmis, R. Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity, Histochem. Cell Biol. 1998;110:231-241). Thin layer chromatography and laser densitometry were used to determine cholesterol, triglyceride and cholesteryl ester levels in human macrophages. Aggregated LDL obtained by vortexing was found to be a reproducible stimulus of foam cell formation in human macrophages. In our hands, Cu(2+)-oxidized LDL also induced cholesteryl ester accumulation, but only when vortexed. We found that foam cell formation in an individual varied by less than 25% over a 10-month period. In contrast, we observed a sevenfold difference in foam cell formation among eight male volunteers. The transfer of foam cells into culture medium with freshly thawed autologous serum resulted in a 75% regression within 1 week, independent of the amount of cellular cholesteryl esters accumulated. Foam cell formation correlated neither to serum nor to cellular cholesterol and triglyceride levels. The propensity to form foam cells could therefore represent a novel indicator of individual risk of atherogenesis.     

Unmodified low density lipoprotein causes cholesteryl ester accumulation in J774 macrophages.

Cholesteryl ester (CE)-loaded macrophages (foam cells) are a prominent feature of atherosclerotic plaques. Previous studies have shown that human monocytes or resident mouse peritoneal macrophages accumulate CE in response to low density lipoprotein (LDL) only when the LDL has been appropriately chemically modified. By contrast, we report here that J774 macrophages accumulate large amounts of CE when incubated with unmodified LDL. The CE is stored in oil red O-positive droplets, which have the typical appearance of foam cell inclusions by electron microscopy. The fatty acid moieties of the cellular CE are enriched in oleate unlike those of LDL-CE, which are enriched in linoleate, indicating that the LDL-CE undergoes hydrolysis and reesterification by acyl CoA:cholesterol acyltransferase. Studies with 125I-labeled LDL at both 4 degrees C and 37 degrees C indicate that the LDL is internalized by a specific receptor that has several characteristics in common with the apolipoprotein B/E (apo B/E) receptor. However, in comparison with fibroblasts, the LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity in J774 cells are relatively resistant to down-regulation by LDL or 25-hydroxycholesterol, leading to receptor-mediated CE storage. In addition, J774 cells appear to accumulate CE from LDL internalized by nonspecific means. Thus, macrophage-like cells can accumulate CE in response to unmodified LDL by both nonspecific and receptor-mediated processes.     

Cytotoxic cholesterol is generated by the hydrolysis of cytoplasmic cholesteryl ester and transported to the plasma membrane.

The present study examines the fate and effects of free cholesterol (FC) generated by the hydrolysis of cytoplasmic cholesteryl esters (CE) in model macrophage foam cells. J774 or elicited mouse peritoneal macrophages (MPM) were enriched with CE by incubating with acetylated low density lipoprotein (acLDL) and FC/phospholipid dispersions, thus creating model foam cells. Treatment of the foam cells with the acyl coenzyme-A:cholesterol acyltransferase (ACAT) inhibitor, CP-113,818, in the absence of any extracellular cholesterol acceptors, resulted in cellular toxicity. This was accompanied by an increase in the amount of FC available for oxidation by an exogenous cholesterol oxidase. Furthermore, cellular toxicity was proportional to the size of the oxidase susceptible pool of FC over time. Morphological analysis and in situ DNA fragmentation assay demonstrated the occurrence of apoptosis in the ACAT inhibited cells. Co-treatment with the hydrophobic amine U18666A, an intracellular cholesterol transport inhibitor, led to a dose dependent reduction in cytotoxicity and apoptosis, and blocked the movement of FC into the oxidase susceptible pool. In addition, treating model foam cells with CP-113,818 plus chloroquine, a compound that inhibits the function of acidic vesicles, also diminished cellular toxicity. Staining with the cholesterol binding dye filipin revealed that the macrophages treated with CP-113,818 contained a cholesterol oxidase accessible pool of FC in the plasma membrane. These results suggest that FC generated by the hydrolysis of cytoplasmic CE is transported through acidic vesicles to the plasma membrane, and accumulation of FC in this pool triggers cell death by necrosis and apoptosis.     

Inhibition of mast cell-dependent conversion of cultured macrophages into foam cells with antiallergic drugs

Degranulation of isolated, rat peritoneal mast cells in the presence of low density lipoprotein (LDL) induces cholesteryl ester accumulation in cocultured macrophages with ensuing foam cell formation. This event occurs when the macrophages phagocytose LDL particles that have been bound to the heparin proteoglycans of exocytosed granules. In an attempt to inhibit such foam cell formation pharmacologically, rat peritoneal mast cells that had been passively sensitized with anti-ovalbumin-IgE were treated with 2 mast cell-stabilizing antianaphylactic drugs, MY-1250 or disodium cromoglycate (DSCG). Both drugs were found to inhibit antigen (ovalbumin)-triggered release of histamine from the mast cells, revealing mast cell stabilization. In cocultures of rat peritoneal macrophages and passively sensitized mast cells, addition of MY-1250 before addition of the antigen resulted in parallel reductions in histamine release from mast cells, uptake of [(14)C]sucrose-LDL, and accumulation of LDL-derived cholesteryl esters in the cocultured macrophages. Similarly, when passively sensitized mast cells were stimulated with antigen in the presence of DSCG and the preconditioned media containing all substances released from the drug-treated mast cells were collected and added to macrophages cultured in LDL-containing medium, uptake and esterification of LDL cholesterol by the macrophages were inhibited. The inhibitory effects of both drugs were mast cell-specific because neither drug inhibited the ability of macrophages to take up and esterify LDL cholesterol. Analysis of heparin proteoglycan contents of the incubation media revealed that both drugs had inhibited mast cells from expelling their granule remnants. Thus, both MY-1250 and DSCG prevent mast cells from releasing the heparin proteoglycan-containing vehicles that bind LDL and carry it into macrophages. This study suggests that antiallergic pharmacological agents could be used in animal models to prevent mast cell-dependent formation of foam cells in vivo.     

Impaired mobilisation of cholesterol from stored cholesteryl esters in human (THP-1) macrophages

The formation of macrophage-derived foam cells is central to the development of fatty streaks within the arterial wall, and to the progression of atherosclerosis. The unregulated deposition of cholesteryl esters, as lipid droplets within the cytoplasm of these cells, is responsible for the formation of foam cells; this process is thought to be regulated by the balance between cholesterol esterification, by acyl CoA:cholesterol acyltransferase (ACAT), and hydrolysis, by neutral cholesteryl ester hydrolase (nCEH). This study examines the importance of the balance between these two enzymes in determining the efflux of cholesterol from human (THP-1) macrophages. The presence of modified lipoprotein, or of 25-hydroxycholesterol, markedly increased cholesterol esterification in these cells and these effects were potently inhibited by the presence of the ACAT inhibitor, 447C88. In the absence of HDL, an acceptor particle, there was little or no hydrolysis of the cholesteryl ester pool and no efflux of cholesterol to the extracellular milieu; addition of HDL led to a partial (36%) reduction in cholesteryl esters, an effect which was not enhanced by the inhibition of ACAT. This suggested that the stored cholesteryl esters in human (THP-1) macrophages, unlike those in mouse peritoneal macrophages, were relatively resistant to removal by efflux to HDL. Efflux of newly synthesised free cholesterol from these macrophages was increased by HDL in a saturable manner, suggesting that the lack of reduction of stored cholesteryl esters was due to impaired mobilisation of cholesteryl esters to free cholesterol via nCEH. Indeed, nCEH activity in these macrophages was much lower than in mouse peritoneal macrophages, and appeared to be down-regulated in the presence of 25-hydroxycholesterol or modified lipoproteins; this loss of nCEH activity was prevented by the ACAT inhibitor 447C88. The efflux of stored cholesteryl esters from THP-1 macrophages therefore appears to be limited by the activity of nCEH.     

槟榔碱对泡沫细胞胆固醇流出和ABCA1表达的影响

目的 探讨槟榔碱对氧化型低密度脂蛋白(ox-LDL)诱导的鼠源性巨噬细胞性泡沫细胞形成的影响及可能机制.方法 采用体外培养的鼠源性巨噬细胞株作为研究对象,加入ox-LDL (50 mg/L)诱导泡沫细胞,同时加入不同浓度的槟榔碱处理,油红0染色进行形态学观察,高效液相色谱法测定细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)的水平,3H标记的胆固醇测定胆固醇流出率.采用实时定量PCR和蛋白印迹法分别检测细胞中三磷酸腺苷结合盒转运体A1( ABCA1)的表达.结果 ox-LDL处理48 h后,与正常巨噬细胞相比,油红O染色阳性细胞显著增加,细胞体积明显增大,形态多呈圆形或不规则形;槟榔碱(10-6、10-5和10-4mol/L)组油红O染色阳性细胞数比泡沫细胞模型组显著减少,细胞体积明显缩小.与泡沫细胞模型组相比,槟榔碱(10-5和10-4mol/L)显著性降低细胞内TC、FC、CE水平及CE/TC,显著性增加细胞胆固醇流出率和ABCA1的表达.结论 槟榔碱抑制ox-LDL诱导的鼠源性巨噬细胞性泡沫细胞形成,上调泡沫细胞中ABCA1的表达.