肺炎衣原体通过上调清道夫受体A1诱导THP-1源性泡沫细胞形成

目的观察清道夫受体A1和CD36在肺炎衣原体诱导THP-1源性泡沫细胞形成中的作用。方法给予不同浓度的肺炎衣原体(1×105～1×106IFU)感染THP-1源性巨噬细胞0～72h。运用油红O染色观察细胞质内脂滴的变化,酶荧光学法检测细胞内胆固醇酯含量的变化。分别运用逆转录聚合酶链反应和Western-Blot检测清道夫受体A1和CD36的mRNA和蛋白表达。结果高浓度的肺炎衣原体(5×105和1×106IFU)感染负荷低密度脂蛋白的THP-1源性巨噬细胞48h后,细胞质内的脂滴明显增多,胆固醇酯占总胆固醇百分比明显增加(>50%)。在负荷低密度脂蛋白的THP-1源性巨噬细胞上,肺炎衣原体感染呈浓度和时间依赖性地上调道夫受体A1 mRNA和蛋白表达,但不影响CD36 mRNA和蛋白表达。结论清道夫受体A1表达上调是肺炎衣原体诱导THP-1源性泡沫细胞形成的机制之一,这可能为进一步阐明肺炎衣原体感染促进动脉粥样硬化发生发展提供一个新的理论依据。     

核因子κB通过Sortilin促进荷脂THP-1巨噬细胞泡沫化

目的探究核因子κB(NF-κB)是否通过分拣蛋白Sortilin影响荷脂THP-1巨噬细胞脂质代谢和泡沫化。方法 NF-κB激活剂PMA或其特异性抑制剂PDTC孵育荷脂THP-1巨噬细胞,qRT-PCR检测Sortilin mRNA水平,Western blot检测Sortilin蛋白表达;沉默荷脂THP-1巨噬细胞Sortilin同时加入PMA孵育,胞内胆固醇流出行液体闪烁计数器检测,胞内脂质含量行高效液相色谱法检测,胞内脂滴情况用油红O染色显示。结果 PMA孵育处理荷脂THP-1巨噬细胞后Sortilin mRNA水平及蛋白表达上调,PDTC孵育处理后Sortilin mRNA水平及蛋白表达下调;PMA孵育处理荷脂THP-1巨噬细胞内脂质流出减少,脂质蓄积程度增加,泡沫细胞形成加剧,而PMA与Sortilin shRNA共处理后,其胞内脂质流出增多,胞内脂质蓄积程度减轻,泡沫细胞形成受抑。结论 NF-κB通过促进Sortilin表达,导致巨噬细胞内脂质流出受抑,胞内脂质蓄积程度加重,从而加剧泡沫细胞形成。     

小檗碱调节肝X受体α去乙酰化促进THP-1

目的 观察小檗碱对THP-1巨噬细胞源性泡沫细胞ATP结合盒转运体A1(ABCA1)表达及其细胞内胆固醇流出的影响,并探讨肝x受体α(LXR-α)去乙酰化在此过程中的作用.方法 用不同浓度的小檗碱(0、5、10、20 μmol/L)处理THP-1巨噬细胞源性泡沫细胞24 h,或以20 μmol/L小檗碱处理THP-1巨噬细胞源性泡沫细胞不同的时间(0、6、12、24 h).采用Western Blot检测ABCA1、LXR-α和乙酰化LXR-α的蛋白表达,液体闪烁计数法观察细胞内胆固醇的流出,高效液相色谱法测定细胞内胆固醇浓度.结果 与对照组比较,小檗碱呈浓度(0～20 μmol/L)和时间依赖性(0～24 h)上调巨噬细胞源性泡沫细胞ABCA1的表达和下调乙酰化LXR-α的表达,增加THP-1巨噬细胞源性泡沫细胞内胆固醇的流出,减少细胞内胆固醇的含量.上述效应在20 μmol/L小檗碱处理THP-1巨噬细胞24 h后达到最大值.结论 小檗碱能上调THP-1巨噬细胞源性泡沫细胞ABCA1的表达,并促进细胞内胆固醇流出,这种效应与调节LXR-α乙酰化有关.     

干扰素-γ对单核-巨噬细胞源性泡沫细胞ACAT-1表达的影响

目的观察干扰素-γ对THP-1、THP-1源性巨噬细胞泡沫细胞酰基辅酶A胆固醇酰基转移酶-1(ACAT-1)表达的影响.方法将THP-1细胞与PMA孵育48小时使之分化为巨噬细胞,继以100mg/ml Ox-LDL处理24小时使之形成泡沫细胞.将THP-1细胞、巨噬细胞、泡沫细胞分别用300U/ml IFN-γ干预24小时.RT-PCR检测ACAT-1mRNA水平,Western Blot检测其蛋白表达.结果 THP-1细胞分化成巨噬细胞以及形成泡沫细胞时ACAT-1mRNA及蛋白含量增加(P＜0.05),而后两种细胞之间无显著性差异.与各自的对照组相比,经IFN-γ作用后三种细胞ACAT-1mRNA及蛋白表达均增强(P＜0.05).结论 IFN-γ诱导单核、巨噬细胞、泡沫细胞ACAT-1的mRNA及蛋白表达,这可能是其促进动脉粥样硬化的机制之一.     

载脂蛋白A1对胆固醇逆转运及三磷酸腺苷结合盒转运体A1表达的影响

目的 观察载脂蛋白A1(apoAl)对人急性单核细胞白血病细胞株(THP-1)源性泡沫细胞内胆固醇、胆固醇酯含量和三磷酸腺苷结合盒转运蛋白A1(ABCAl)表达的影响和机制.方法 以50 ng/ml的佛波酯诱导人THP-1,使其分化为巨噬细胞,再经50μg/ml氧化型低密度脂蛋白充分诱导使其转变为负脂的泡沫细胞;然后用不同浓度apoAl(5μg/ml、10μg/ml、15μg/ml、20 μg/ml)孵育泡沫细胞24 h,以apoAl(10μg/ml)孵育泡沫细胞6 h、12 h、24 h.采用油红O染色观察细胞内脂滴的变化,用氧化酶法检测细胞内总胆固醇、游离胆固醇和胆固醇酯的含量;用反转录聚合酶链反应检测不同浓度apoAl干预泡沫细胞后ABCA1 mRNA的表达;用免疫荧光法检测经不同浓度和不同时间apoAl和ABCAl多克隆抗体干预泡沫细胞后ABCA1蛋白的表达.结果 (1)THP-1经佛波酯(50 ng/ml)和氧化型低密度脂蛋白(50μg/ml)分别干预48 h后可成功诱导为富含脂质的泡沫细胞;(2)apoA1可促进THP-1巨噬细胞源性泡沫细胞内胆固醇逆向转运,减少细胞内胆固醇含量,呈浓度依赖性和时间依赖性;(3)随着apoA1干预浓度增加,THP-1巨噬细胞源性泡沫细胞内ABCA1 mRNA的表达变化不显著,但蛋白表达增加,0 μg/ml与5 μg/ml、10μg/ml、15μg/ml、20μg/ml apoAl干预浓度下油红O染色阳性细胞率分别为(55±4)%与(43±9)%、(33±4)%、(28±1)%、(26±2)%,差异有统计学意义(均P<0.05);(4)ABCAl抗体干预可增加THP-1巨噬细胞源性泡沫细胞ABCA1蛋白的表达.结论 apoA1-ABCA1通路参与泡沫细胞胆固醇逆向转运,apoA1起关键作用,apoA1增加ABCA1蛋白的表达可能与降低ABCA1蛋白的降解有关.     

白细胞介素37对THP-1源巨噬细胞泡沫化的影响

目的:研究白细胞介素(白介素)-37对人THP-1源巨噬细胞泡沫化的影响及机制。方法:体外培养THP-1,用佛波酯(PMA)刺激24h使其分化为巨噬细胞后,进行以下处理:①用IL-37和(或)ox-LDL刺激THP-1源巨噬细胞24h;②用不同浓度的IL-37和ox-LDL同时刺激24h;③用ox-LDL刺激THP-1源巨噬细胞24h,再加IL-37干预24h,收集细胞。采用油红染色观察各组泡沫细胞所占细胞总数的比例,TC试剂盒测定细胞内TC的含量,利用RT-PCR、Western blot实验方法从mRNA水平和蛋白水平测定泡沫细胞清道夫受体CD36和SRA和ABCA-1的表达。结果:与对照组比较,IL-37能够明显减少泡沫细胞的形成,降低泡沫细胞TC的聚集[(303.10±8.90)ng/mg∶(150.40±7.36)ng/mg,P0.01],显著抑制巨噬细胞清道夫受体CD36和SRA的表达,并增强ABCA-1的表达,且作用效果与溶度呈正相关。对ox-LDL诱导的泡沫细胞,给予IL-37后仍能够显著上调ABCA1的表达,显著下调SRA、CD36的表达。结论:白细胞介素-37可抑制THP-1源巨噬细胞的泡沫化,其作用机制可能是通过IL-37抑制清道夫受体CD36和SRA、促进ABCA-1的表达实现的。     

去甲肾上腺素对THP-1源性巨噬细胞SR-A1和转化生长因子-β1表达的影响

目的:探讨去甲肾上腺素对人THP-1源性巨噬细胞SR-A1和转化生长因子(TGF)-β1表达的影响.方法:不同浓度的去甲肾上腺素(10 pmol/L～10 μmol/L)作用THP-1源性巨噬细胞24 h,运用逆转录多聚酶链反应检测SR-A1和TGF-β1 mRNA的表达,Western-Blot检测SR-A1蛋白的表达,酶联免疫吸附试验检测TGF-β1蛋白的表达.结果:1 μmol/L及10 μmol/L的去甲肾上腺素作用THP-1源性巨噬细胞后,SR-A1 mRNA和蛋白表达上调,100 nmol/L 、1 μmol/L及10 μmol/L的去甲肾上腺素引起巨噬细胞中 TGF-β1 mRNA和蛋白水平均下降(均P＜0.05).结论:应激浓度的去甲肾上腺素能增加THP-1源性巨噬细胞SR-A1的表达,降低TGF-β1的表达.     

内脂素对人单核细胞株源性巨噬细胞ATP结合盒转运蛋白A1的调控

目的:观察过氧化物酶体增殖物激活受体γ(PPARγ)信号转导通路在内脂素调控人单核细胞株(THP-1)源性巨噬细胞ATP结合盒转运蛋白A1(ABCA1)表达中的作用,探讨内脂素诱导泡沫细胞形成的机制和途径。方法:THP-1单核细胞诱导分化为巨噬细胞,随机分组,给予不同浓度和不同作用时间的内脂素进行干预,分别运用油红O染色观察细胞浆脂滴变化,反转录聚合酶链反应(RT-PCR)法和蛋白免疫印迹(Westernblot)法检测各组细胞PPARγ及ABCA1mRNA和蛋白表达,酶荧光学法检测细胞内TC和游离胆固醇(FC)含量,TC与FC之差为胆固醇酯(CE)含量。结果:与对照组比较,内脂素干预组细胞浆脂滴明显增多,细胞内FC和CE含量增加(P<0.05),PPARγ及ABCA1mRNA和蛋白表达降低(P<0.05);相关分析显示,内脂素呈浓度和时间依赖性下调PPARγ及ABCA1mRNA和蛋白的表达。结论:内脂素可能通过PPARγ信号转导通路下调ABCA1表达,减少细胞内FC流出,使细胞内CE合成增加,从而诱导泡沫细胞形成。这可能为内脂素致动脉粥样硬化发病机制的研究提供了一个新的理论依据。     

黄杆菌裂解产物对巨噬细胞源性泡沫细胞形成的作用及机制研究

目的:探讨黄杆菌裂解产物对巨噬细胞源性泡沫细胞形成的作用及可能的机制。方法:将黄杆菌裂解产物与THP-1细胞进行共培养,观察其对THP-1巨噬细胞及泡沫细胞形成的影响,并检测CD14、TLR4、LDLr三个受体mRNA的表达水平。结果:黄杆菌裂解产物促进THP-1单核细胞向巨噬细胞分化,并促进巨噬细胞源性泡沫细胞的形成,上调CD14、TLR4、LDLr三个受体mRNA的表达水平。结论:黄杆菌裂解产物促进单核巨噬细胞源性泡沫细胞的形成,这可能与其上调LDLr表达有关。     

白介素-1对单核细胞向泡沫细胞分化过程中ACAT-1蛋白表达和活性的影响

目的探讨白介素-1（IL-1）对单核细胞向泡沫细胞分化过程中脂酰辅酶A-胆固醇酰基转移酶-1（ACAT-1）蛋白表达和活性的影响。方法200nmol／L佛波酯（PMA）诱导THP-1单核细胞48h使其转化为巨噬细胞,流式细胞术检测CDl4的表达;巨噬细胞与80mg／L氧化型低密度脂蛋白（Ox-LDL）共孵育24h,使之向泡沫细胞分化,油红O染色观察细胞质内脂质沉积;Westernblot检测各组细胞（单核细胞组、巨噬细胞组、泡沫细胞组、泡沫细胞＋IL-1组、泡沫细胞＋IL-1／Anti-ILl组）ACAT-l的蛋白表达,液相闪烁计数法检测ACAT-1的酶活性。结果单核细胞株THP-1与200nM的PMA共孵育48h后,分化为巨噬细胞,CDld阳性表达率为85．7％;巨噬细胞与Ox-LDL共孵育24h后,油红O染色胞浆内可见大量吞噬的脂质小滴。与单核细胞组相比,巨噬细胞组、泡沫细胞组和泡沫细胞＋IL-1组ACAT-1蛋白表达上调,活性升高（P〈0．05）,泡沫细胞＋IL-1／Anti-ILl组蛋白表达上调及活性升高不明显（P〉0．05）。结论IL-1对单核细胞向泡沫细胞分化过程中ACAT-1蛋白表达及酶活性有上调作用,IL-1单克隆抗体可以拮抗这种效应。     

1-Naphthyl isocyanate and 1-naphthylamine as metabolites of 1-naphthylisothiocyanate

Abstract: The importance of the bioactivation of 1-naphthylisothiocyanate was studied. Forty minutes after 1-naphthylisothiocyanate administration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1-naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1-naphthyl isocyanate, 1-naphthylamine and the parent compound, 1-naphthylisothiocyanate. When rats were given cycloheximide, which attenuates 1-naphthylisothiocyanate toxicity, 30 min before 1-naphthylisothiocyanate (300 mg/kg), 1-naphthyl isocyanate concentration was significantly lower than in rats receiving only 1-naphthylisothiocyanate. The appearance of 1-naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepatotoxicity, enhanced 1-naphthyl isocyanate and 1-naphthylamine formation. It is suggested that 1-naphthyl isocyanate, 1-naphthylamine and the highly reactive sulfur released from 1-naphthylisothiocyanate might be involved in the hepatotoxic effect of 1-naphthylisothiocyanate.     

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1)

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.     

Inhibition of procarcinogen-bioactivating human CYP1A1, CYP1A2 and CYP1B1 enzymes by melatonin

Abstract:  Administration of melatonin to rodents decreases the incidence of tumorigenesis initiated by benzo[ a ]pyrene or 7,12-dimethylbenz[ a ]anthracene, which requires bioactivation by cytochrome P450 enzymes, such as CYP1A1, CYP1A2 and CYP1B1, to produce carcinogenic metabolites. The present study tested the hypothesis that melatonin is a modulator of human CYP1 catalytic activity and gene expression. As a comparison, we also investigated the effect of melatonin on the catalytic activity of CYP2A6, which is also a procarcinogen-bioactivating enzyme. Melatonin (3–300 μ m ) decreased 7-ethoxyresorufin O -dealkylation catalyzed by human hepatic microsomes and recombinant CYP1A1, CYP1A2 and CYP1B1, whereas it did not affect coumarin 7-hydroxylation catalyzed by hepatic microsomes or recombinant CYP2A6. Melatonin inhibited CYP1 enzymes by mixed inhibition, with apparent K _i values (mean ± S.E.M.) of 59 ± 1 (CYP1A1), 12 ± 1 (CYP1A2), 14 ± 2 (CYP1B1) and 46 ± 8 μ m (hepatic microsomes). Additional experiments indicated that melatonin decreased benzo[ a ]pyrene hydroxylation catalyzed by hepatic microsomes and CYP1A2 but not by CYP1A1 or CYP1B1. Treatment of MCF-10A human mammary epithelial cells with melatonin (up to 300 μ m ) did not affect basal or benzo[ a ]pyrene-inducible CYP1A1 or CYP1B1 gene expression. Consistent with this finding, melatonin did not influence reporter activity in aryl hydrocarbon receptor-dependent pGudluc6.1-transfected MCF-10A cells treated with or without benzo[ a ]pyrene, as assessed in an in vitro cell-based luciferase reporter gene assay. Overall, melatonin is an in vitro inhibitor of human CYP1 catalytic activity, and it may be useful to develop potent analogues of melatonin as potential cancer chemopreventive agents that block CYP1-mediated chemical carcinogenesis.     

甲型H1N1与季节性H1N1流感病毒血凝素基因比较分析

目的分析泰安市2008～2009年度季节性流感与2009年度甲型H1N1流感病原学检测结果 ,比较季节性H1N1与甲型H1N1血凝素基因变异情况。方法选择国家级流感监测哨点医院以及暴发疫情的疫点,采集流感样病例的鼻咽拭子标本,通过RealtimePCR进行病毒检测,用MDCK细胞进行病毒分离,通过RT-PCR扩增血凝素HA1片段的基因并测序,利用生物信息学进行序列分析。结果 2008～2009年共检测鼻咽拭子标本283份,分离出流感病毒33株,分离阳性率为11.67%,其中季节性H1N1亚型31株。2009年5月1日～12月31日,检测鼻咽拭子标本996份,流感核酸检测阳性417份,阳性率为41.86%,其中甲型H1N1337份,季节性H1N1亚型1份。6株季节性H1N1病毒均在多个氨基酸位点上发生变异,与疫苗株A/Brisbane/59/2007(H1N1)比较,有11个位点发生了突变,其中5个位点位于抗原决定簇上;测序成功的6株甲型H1N1病毒在多个氨基酸位点发生变异,与疫苗株A/California/07/2009(H1N1)比较,有6个位点发生突变,其中1个位点位于抗原决定簇的B区。结论 2008～2009年度季节性H1N1为优势株,甲流暴发后,甲型H1N1成为绝对优势毒株。季节性H1N1分离株有多处氨基酸替换,抗原决定簇B区变异频繁;甲型H1N1病毒分离株的基因有变异,但关键位点第222位仍为D(天冬氨酸),与疫苗株相比抗原决定簇的关键位点变化不大。     

Characterization of MTHFR,GSTM1, GSTT1, GSTP1, and CYP1A1 genotypes in childhood acute leukemia

The role of methylenetetrahydrofolate reductase (MTHFR C677T), glutathione S-transferases (GSTM1 and GSTT1 null, GSTP1 Ile105Val), and cytochromes p450 (CYP1A1*2A) genotypes in the etiology of childhood leukemia was simultaneously investigated. 144 Turkish children with acute lymphoblastic leukemia (ALL) and 33 with acute nonlymphoblastic leukemia (ANLL) were studied and compared with 185 healthy pediatric controls. The frequency of MTHFR genotype was insignificantly higher in ALL (7.7%) and ANLL (6.3%) than in controls (4.4%). Equal distribution of the GSTM1 null genotype was detected between ALL patients and controls (55%), while its incidence was slightly higher in ANLL patients (61.3%). Although GSTT1 null genotype was insignificantly lower in ALL patients (20.9%) than controls (22.7%), it was significantly underrepresented in ANLL patients (6.5%) (P = 0.05, OR 0.24, 95% CI 0.05-1.03). The homozygous frequency of GSTP1 genotype did not differ significantly between groups of ALL (3.7%), ANLL patients (9.1%) and controls (4.9%). Homozygous CYP1A1*2A genotype was underrepresented in ALL patients (1%) as compared to control (4.8%) but the differences did not reach to statistical significance (OR 0.21; 95% CI 0.03-1.72). Homozygosity for this genotype was not detected in ANLL patients. No particular association was noted between different combinations of combined genotypes and risk of development of childhood ALL and ANLL. These results suggested that there are no significant associations between the studied genotypes and the risk of developing either form of acute leukemia except GSTT1 null and homozygosity for CYP1A1 genotypes that may play protective roles in the development of ANLL in Turkish children.