MPTP诱导小鼠黑质区铁摄取和DMT1表达增加

铁在帕金森病(Parkinson‘s disease,PD)的发病机制中起着非常关键的作用,为了探讨PD中铁升高的机制,本实验观察了1-甲基4-苯基—1,2,3,6-四氢吡啶(MPTP)处理小鼠黑质(substantia nigra,SN)内铁摄取及新的铁转运蛋白二价金属离子转运蛋白1(DMT1)的表达变化。结果表明：(1)MPTP处理组小鼠SN内铁染色增高,注射MPTP7d组明显高于3d组。(2)MPTP处理组小鼠,酪氨酸羟化酶(TH)免疫阳性细胞数目显著减少。(3)MPTP处理组小鼠,“-IRE”型DMT1表达在各组中均增加,而“ IRE”型DMT1仅在MPTP处理后7d才出现变化。上述结果提示,这种新发现的哺乳动物跨膜铁转运蛋白表达增加可能是引起MPTP处理小鼠SN中铁升高的关键因素,铁的升高进一步导致DA神经元的死亡。     

肥胖对小鼠十二指肠 DMT1和 FPN1表达的影响

目的：观察肥胖对小鼠十二指肠二价金属离子转运体（divalent metal transporter 1,DMT1）mRNA、膜铁转运蛋白（ferroportin1,FPN1）mRNA及蛋白表达的变化,探讨肥胖影响铁吸收的机制。方法 C57BL／6J小鼠随机分为正常对照组和肥胖模型组,每组6只,通过喂养高脂饲料喂养建立肥胖模型,对照组采用普通饲料饲养,实验干预期14周。建模完成后,采用实时荧光定量PCR方法检测小鼠十二指肠DMT1、FPN1 mRNA 的表达,用Western blot检测小鼠十二指肠FPN1蛋白表达。结果与对照组小鼠相比,肥胖模型组小鼠十二指肠DMT1、FPN1 mRNA表达以及FPN1蛋白表达水平降低,差异具有统计学意义（ P ＜0.05）。结论肥胖会下调机体十二指肠DMT1、FPN1的表达,导致铁吸收不良,为进一步研究肥胖引起铁缺乏机制提供理论和实验依据。     

氧化低密度脂蛋白对THP-1巨噬细胞吞噬和分泌功能的影响

目的:探讨氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)对巨噬细胞源性泡沫细胞吞噬功能和炎症相关因子分泌功能的影响。方法:利用佛波酯(phorbol ester,PMA)诱导THP-1细胞分化形成巨噬细胞,之后采用ox-LDL处理48小时后,诱导其形成泡沫细胞。利用中性红吞噬实验,分析泡沫细胞形成前后吞噬功能的变化;通过ELISA法,检测细胞培养上清中肿瘤坏死因子α(tumor necrosis factorα,TNF-α)含量,观察ox-LDL对THP-1巨噬细胞功能的影响。结果:细胞形态学结果表明,我们成功利用ox-LDL诱导THP-1巨噬细胞形成泡沫细胞;进一步发现ox-LDL诱导THP-1巨噬细胞表面的清道夫受体CD36表达升高,并促进细胞吞噬功能增加,进一步促进细胞内胆固醇含量显著升高(P0.05);同时,ox-LDL能够刺激巨噬细胞大量分泌TNF-α(P0.05)。结论:ox-LDL通过增强清道夫受体CD36表达,提高巨噬细胞的吞噬功能,引起大量胆固醇聚集,产生细胞毒性损伤,并促进TNF-α炎性因子的大量分泌。     

LDL、oxLDL对THP-1巨噬细胞PCSK9、LDLR表达的影响研究

为研究低密度脂蛋白(LDL)、氧化修饰的低密度脂蛋白(oxLDL)对THP-1源性巨噬细胞中PCSK9、 LDLR表达的影响及两者之间的关系.分别用0mg/L、10mg/L、20mg/L、30mg/L的LDL和0mg/L、10mg/L、20mg/L、30mg/L的oxLDL处理THP-1巨噬细胞,油红O染色检测细胞荷脂情况,免疫荧光检测THP-1巨噬细胞上PCSK9蛋白表达及分布情况,RT-PCR、 Western blot检测THP-1巨噬细胞上PCSK9、 LDLR mRNA、蛋白质的表达.结果发现,不同浓度LDL处理THP-1巨噬细胞后,随着LDL浓度的增大,细胞内脂滴数目略有增多.免疫荧光染色发现,PCSK9在THP-1巨噬细胞上的表达随LDL浓度的增加而增多,胞浆内定位于某一特定细胞器中;RT-PCR、 Western blot检测发现,LDL可以呈浓度依赖性下调THP-1巨噬细胞中LDLR的表达和上调PCSK9的表达.不同浓度oxLDL处理THP-1巨噬细胞后,随oxLDL浓度的增大,脂滴颗粒明显增加;oxLDL处理对THP-1巨噬细胞上PCSK9、 LDLR mRNA、蛋白质的表达影响均不明显.研究结果表明:THP-1巨噬细胞上,同时有PCSK9和LDLR的表达,且PCSK9定位于胞浆中某一特定细胞器;oxLDL对THP-1巨噬细胞LDLR和PCSK9表达没有影响;LDL能够降低THP-1巨噬细胞表面LDLR的表达,同时上调PCSK9表达,初步说明在THP-1巨噬细胞中,两者有一定的相关性.     

LDL受体对清道夫受体活性的影响

应用经ＰＭＡ诱导衍生的ＴＨＰ－１巨噬细胞为模型,以单克隆抗体Ｃ７Ｂ封闭ｏｘＬＤＬ上的ＬＤＬ受体结合位点,结果发现,正常细胞在摄取ｏｘＬＤＬ时ＬＤＬ受体与清道夫受体起协同作用;但Ｃ７Ｂ作用于蓄积了脂质的ＴＨＰ－１巨噬细胞时,对细胞脂质蓄积程度无明显影响,清道夫受体活性不但不降低反而有所升高,说明由于脂质蓄积ＬＤＬ受体的作用减弱。     

人血浆HDL对动脉粥样硬化家兔肝细胞膜LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化(As)模型家兔通过静脉注射人血浆HDL制剂,观察HDL对As家兔肝细胞膜LDL受体活性的影响.结果发现,摄取高胆固醇饲料的As家兔,其肝细胞膜LDL受体K_d值虽无明显变化但B_max值显著减小（P＜0.01,与正常对照组比较);注射HDL制剂后,As家兔肝细胞膜LDL受体K_d值仍无明显改变,但B_max值却显著回升（P＜0.01,与高脂组比较).表明人血浆HDL具有增加As家兔肝细胞膜LDL受体活性的作用.     

人血浆HDL对动脉粥样硬化家兔肝细胞膜 LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化（Ａｓ） 模型家兔通过静脉注射人血浆ＨＤＬ 制剂, 观察ＨＤＬ 对Ａｓ家兔肝细胞膜ＬＤＬ受体活性的影响． 结果发现, 摄取高胆固醇饲料的Ａｓ 家兔, 其肝细胞膜ＬＤＬ 受体 Ｋｄ 值虽无明显变化但Ｂｍａｘ 值显著减小（ Ｐ＜ ０－０１ , 与正常对照组比较） ; 注射ＨＤＬ 制剂后, Ａｓ 家兔肝细胞膜ＬＤＬ受体Ｋｄ 值仍无明显改变, 但Ｂｍａｘ 值却显著回升（ Ｐ＜ ０－０１ , 与高脂组比较） ． 表明人血浆ＨＤＬ 具有增加Ａｓ 家兔肝细胞膜ＬＤＬ 受体活性的作用．     

氧化低密度脂蛋白抗体对ApoE-/-小鼠动脉粥样硬化形成的影响

目的探讨在氧化低密度脂蛋白抗体免疫ApoE基因敲除(ApoE-/-)小鼠体内ACAT1的变化及其与动脉粥样硬化形成的关系。方法采用油红染色、免疫组化染色(包括染CD68,a-SMA及Masson)方法确定各组小鼠斑块大小及各组分的含量,Western blot及Real-Time PCR检测ACAT1蛋白及mRNA在小鼠肝脏的表达。结果抗体免疫组斑块面积明显小于对照组(P0.05),ACAT1水平明显低于对照组(P0.05)。结论氧化低密度脂蛋白抗体可能通过抑制ACAT1的表达来减少动脉粥样斑块的形成,可为临床提供新的治疗途径和思路。     

ApoAⅠ促进THP-1源性泡沫细胞ApoE分泌的研究

目的通过建立动脉粥样硬化损伤部位细胞模型,观察ApoAI对THP—l源性泡沫细胞ApoE分泌的影响,探讨ApoAI与ApoE之间是否存在相互关系以及这种关系对动脉粥样硬化的影响。方法采用聚乙二醇-6000（PEG-6000）沉淀法制备人血浆HDL,再利用凝胶过滤柱层析法分离ApoA I;佛波酯（PMA）诱导THP-1单核细胞分化为巨噬细胞,氧化型低密度脂蛋白（oxidized low density lipoprotein,OXLDL）使分化后THP-1细胞荷脂,建立动脉粥样硬化细胞模型;ELISA检测不同浓度ApoAI（0,5,10,15,25ug／m1）及不同孵育时间（0．3,6,12,24h）,THP—1源性泡沫细胞ApoE的分泌;RT—PCR检测细胞ApoEmRNA的表达情况。结果ApoAI增加THP-1源性泡沫细胞ApoE的分泌量;且ApoE蛋白质的分泌随着ApoAI孵育时间增加而增加,在24hApoE的分泌量达最大;在剂量试验中,ApoE的分泌量随着ApoAI剂量的增加有增加趋势,ApoAI浓度为25μmol／L时,ApoE分泌量最大;而却oE基因的表达不受ApoAI的影响。结论-定浓度的ApoAI促进THP-1源性泡沫细胞ApoE的分泌,且具有时间及剂量依赖性。而在基因水平上,ApoAI对ApoEmRNA表达没有影响。     

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．     

油酸对THP-1巨噬细胞源性泡沫细胞三磷酸腺苷结合盒转运体A1表达和胆固醇流出的影响

以THP 1巨噬细胞源性泡沫细胞为研究对象 ,观察油酸对THP 1巨噬细胞源性泡沫细胞胆固醇流出和三磷酸腺苷结合盒转运体A1(ABCA1)表达的影响 ,以探讨油酸对动脉粥样硬化发生发展的影响。用液体闪烁计数器检测细胞内胆固醇流出 ,高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量 ,运用逆转录多聚酶链反应和Western印迹分别检测ABCA1mRNA与ABCA1蛋白的表达 ,采用流式细胞术检测细胞平均ABCA1荧光强度。实验显示油酸引起THP 1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯呈时间依赖性增加 ,而ABCA1蛋白水平、细胞平均ABCA1荧光强度以及apoA I介导的胆固醇流出呈时间依赖性减少 ,细胞内胆固醇增多 ,但ABCA1mRNA没有明显变化。结果表明 ,油酸减少THP 1巨噬细胞源性泡沫细胞ABCA1蛋白水平 ,降低细胞内胆固醇流出 ,增加细胞内胆固醇聚积。     

Influence of age and sex on levels of anti-oxidized LDL antibodies and anti-LDL immune complexes in the general population

Most studies of antibodies to oxidized LDL have been undertaken in patients with different diseases and cardiovascular risk factors. However, very few studies have researched the distribution and determining factors of antibodies to oxidized LDL in the general population. A total of 1,354 persons (817 females and 537 males) aged 5-65 years were included in this study. They were selected randomly from the population census of Málaga, in southern Spain. The females had lower levels of total cholesterol and triglycerides and higher levels of HDL-cholesterol and a very significant increase (P < 0.0001) in levels of anti-oxidized LDL [low density lipoprotein modified by malondialdehyde (MDA-LDL)] antibodies but no difference in levels of immune complexes consisting of LDL and IgG antibodies (anti-LDL immune complex). Younger persons (16-35 years) had higher levels of anti-oxidized LDL (MDA-LDL) antibodies than persons older than 35 years (P = 0.05). Levels of immune complexes were significantly higher (P = 0.05) in persons aged 5-15 years than in persons older than 40 years. A very weak association was found between levels of anti-oxidized LDL (MDA-LDL) antibodies and anti-LDL immune complexes. The higher prevalence of anti-oxidized LDL (MDA-LDL) antibodies in females and young persons is in agreement with studies that found an inverse association between atherosclerosis and the level of these antibodies.     

Decreased DMT1 and increased ferroportin 1 expression is the mechanisms of reduced iron retention in macrophages by erythropoietin in rats

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague-Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then (55)Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT-PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo-treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo-treated rats.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols

Statins do not always decrease coronary heart disease mortality, which was speculated based on increased serum plant sterols observed during statin treatment. To evaluate plant sterol atherogenicity, we fed low density lipoprotein-receptor deficient (LDLr(+/-)) mice for 35 weeks with Western diets (control) alone or enriched with atorvastatin or atorvastatin plus plant sterols or stanols. Atorvastatin decreased serum cholesterol by 22% and lesion area by 57%. Adding plant sterols or stanols to atorvastatin decreased serum cholesterol by 39% and 41%. Cholesterol-standardized serum plant sterol concentrations increased by 4- to 11-fold during sterol plus atorvastatin treatment versus stanol plus atorvastatin treatment. However, lesion size decreased similarly in the sterol plus atorvastatin (-99% vs. control) and the stanol plus atorvastatin (-98%) groups, with comparable serum cholesterol levels, suggesting that increased plant sterol concentrations are not atherogenic. Our second study confirms this conclusion. Compared with lesions after a 33 week atherogenic period, lesion size further increased in controls (+97%) during 12 more weeks on the diet, whereas 12 weeks with the addition of plant sterols or stanols decreased lesion size (66% and 64%). These findings indicate that in LDLr(+/-) mice 1) increased cholesterol-standardized serum plant sterol concentrations are not atherogenic, 2) adding plant sterols/stanols to atorvastatin further inhibits lesion formation, and 3) plant sterols/stanols inhibit the progression or even induce the regression of existing lesions.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron''s damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.