血管内皮细胞中三磷酸腺苷-结合盒转运子A1对炎性细胞因子表达的影响

目的研究在公认的ATP-结合盒转运子A1(ABCA1)诱导因素8-溴-环磷酸腺苷(8-Br-cAMP)刺激下,人血管内皮细胞ECV304中ABCA1基因对炎性细胞因子细胞间黏附分子-1(ICAM-1)、单核细胞化学趋向蛋白-1(MCP-1)及白介素-1β(IL-1β)的调节作用。以阐明ABCA1基因在动脉粥样硬化(AS)发生中的作用机制。方法培养人血管内皮细胞株ECV304,加入8-Br-cAMP(0.5 mmol/L)刺激3、6、12及24 h,以荧光定量逆转录聚合酶链式反应检测ABCA1、ICAM-1及MCP-1 mRNA表达量,Western blot蛋白印迹法和酶联免疫吸附测定法检测ABCA1I、CAM-1、MCP-1及IL-1β蛋白质表达量;ABCA1的反义寡核苷酸(100 nmol/L)转染人血管内皮细胞,给予上述的8-Br-cAMP,同样的方法测定上述指标的改变。结果人血管内皮细胞ECV304在给予8-Br-cAMP刺激6、12 h后,ABCA1I、CAM-1、MCP-1的mR-NA和蛋白质水平及IL-1β蛋白质水平均增高;给予反义寡核苷酸转染后,8-Br-cAMP刺激后36、h ABCA1I、CAM-1、MCP-1mRNA的表达降低,122、4 h ABCA1I、CAM-1、MCP-1及IL-1β蛋白质的表达水平降低。结论在8-Br-cAMP作用下,血管内皮细胞中ABCA1可增加炎性因子的表达,从而在AS早期的发生中发挥作用。     

Ox-LDL对血管内皮细胞中ABCA1及单核细胞趋化因子的影响

目的研究在致动脉粥样硬化（AS）因子氧化型低密度脂蛋白（Ox-LDL）刺激下,人血管内皮细胞ECV304中腺苷三磷酸结合盒转运体A1（ABCA1）基因对炎性因子单核细胞趋化蛋白1（MCP-1）的调节作用,从而阐明ABCA1基因在AS发生中的作用机制。方法培养人血管内皮细胞ECV304,加入Ox-LDL（30 ng/ml）刺激3、6、12、24 h,以荧光定量RT-PCR检测ABCA1、MCP-1 mRNA表达量,Western蛋白印迹法和ELISA法检测ABCA1、MCP-1及IL-1β蛋白表达量;ABCA1的反义寡核苷酸（100 nmol/L）转染人血管内皮细胞,给予上述的Ox-LDL,同样方法测定上述指标的改变。结果人血管内皮细胞ECV304在给予Ox-LDL刺激后,ABCA1、MCP-1的mRNA和蛋白质水平及IL-1β蛋白质水平均增高;给予反义寡核苷酸转染3、6 h后,ABCA1、MCP-1 mRNA的表达降低,12、24h后ABCA1、MCP-1及IL-1β蛋白质的表达水平降低。结论在Ox-LDL作用下,血管内皮细胞中的ABCA1可能通过增加IL-1β的表达,促进MCP-1释放,从而在AS的早期发生中发挥作用。     

阿托伐他汀调节巨噬细胞载脂蛋白A1天然反义转录表达的研究

目的:探讨阿托伐他汀调节人急性单核细胞白血病细胞1(THP1)诱导的巨噬细胞载脂蛋白A1(apoA1)天然反义转录(apoA1-NAT)表达的研究。方法:检索基因序列,设计引物,用不同浓度0、1、10、100 nmol/L阿托伐他汀干预THP1诱导的巨噬细胞,提取不同浓度和时间点(6、12、24、48 h)的RNA,检测apoA1-NAT、apoA1信使核糖核酸(mRNA)表达。同时检测与胆固醇代谢相关的三磷酸腺苷绑定的盒式转运体A1(ABCA1)、清道夫受体B1 (SRB1)及低密度脂蛋白受体(LDLR)mRNA的表达。结果:THP1诱导的巨噬细胞在10 nmol/L阿托伐他汀干预24 h,apoA1-NAT表达明显降低,同时apoA1 mRNA表达量显著升高(P0.01),ABCA1、SRB1及LDLR mRNA表达量也出现不同程度的升高(P均0.05)。结论:阿托伐他汀抑制巨噬细胞apoA1-NAT表达的同时,可升高apoA1、ABCA1、SRB1及LDLR mRNA表达,进而能促进巨噬细胞胆固醇的外流。     

NF-κB"诱骗"寡核苷酸对结肠癌SW480细胞IL-8表达的影响

目的 探讨核转录因子κB (NF-κB)"诱骗(decoy)"寡核苷酸(ODNs)对结肠癌SW480细胞IL-8表达的影响.方法 体外培养SW480细胞,LPS(10 μg/L )刺激3 h后,用脂质体lipofectin2000介导NF-κB decoy ODNs转染 6 h,ELISA法检测上清液中IL-8,RT-PCR检测细胞IL-8 mRNA,并设对照组、LPS组、SODN组、lipofectin2000组.结果 转染NF-κB decoy ODNs细胞的 IL-8 mRNA和IL-8的表达高于对照组(P<0.01),但明显低于其他组(P<0.01). 结论 NF-κB decoy ODNs明显抑制SW480细胞IL-8的表达.     

白细胞介素-1β诱导关节软骨细胞发生过早衰老的机制

目的 研究软骨降解因子白细胞介素(IL)-1β是否诱导关节软骨细胞发生过早衰老,以及caveolin-1是否介导IL-1β诱导的细胞衰老.方法 培养的关节软骨细胞在IL-1β(10 ng/ml)刺激后,流式细胞术分析细胞周期,染色法测定与衰老相关的β-半乳糖苷酶(SA-β-Gal)活性,测定累计的细胞群倍增次数反映细胞增殖寿命,Southem印迹测定平均端粒长度,细胞转染caveolin-1的反义寡核苷酸和外源性caveolin-1基因改变细胞内caveolin-1的表达水平,caveolin-1蛋白表达量采用Westem印迹分析.结果 IL-1p可诱导关节软骨细胞停滞在G0/G1期的比例明显升高,而处于S期的细胞比例减少.IL-1β单次刺激可使软骨细胞变得大而平、细胞内SA-β-Gal活性增高,IL-1β反复刺激可使细胞内平均端粒长度缩短加速、增殖寿命缩短.反义寡核苷酸抑制caveolin-1表达,可以阻抑IL-1β诱导的软骨细胞衰老;外源性基因增高细胞内caveolin-1表达,可诱导关节软骨细胞发生过早衰老.结论 IL-1β可诱导关节软骨细胞发生应激诱导的过早衰老和端粒依赖的复制性衰老,该作用是由caveolin-1介导的.提示IL-1β可能通过增高关节软骨细胞内caveolin-1表达,诱导细胞发生过早衰老,从而促进骨关节炎进展.     

脂多糖对大鼠肺泡巨噬细胞Toll样受体4和髓样分化蛋白-2基因表达及炎性因子分泌的影响

目的 探讨脂多糖刺激大鼠肺泡巨噬细胞株NR8383细胞Toll样受体4(TLR4)、髓样分化蛋白-2(MD-2)mRNA表达和炎性因子分泌的影响,以及MD-2小干扰RNA(MD-2 siRNA)对脂多糖刺激下NR8383细胞炎性因子分泌的作用.方法 体外培养NR8383细胞,以不同浓度脂多糖(0.01～10 mg/L)刺激2 h,以1 mg/L脂多糖刺激2～24 h.运用脂质体Lipofectamine 2000将MD-2siRNA转染至细胞.半定量RT-PCR方法检测细胞TLR4和MD-2 mRNA的表达,酶联免疫吸附法检测细胞培养上清中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6和IL-1β的含量.统计学处理采用单因素方差分析、独立样本t检验和Pearson相关分析.结果 对照组NR8383细胞TLR4和MD-2mRNA的相对表达量分别为0.52±0.05和0.44±0.09,0.01 mg/L浓度脂多糖刺激前后表达量无明显改变,0.1 mg/L浓度时表达增加,随脂多糖浓度的升高表达量进一步增加,10 mg/L刺激后相对表达量分别为0.72±0.06和0.65±0.10(F=17.26、6.04,P<0.01);TNF-αIL-6和IL-1β含量具有类似改变趋势,对照组分别为(25.8±3.4)ng/L、(62.4±4.7)ng/L和(31.6±1.7)ng/L;在10 mg/L脂多糖刺激下分别增加至(58.9±5.3)ng/L、(96.5±3.9)ng/L和(55.4±5.4)ng/L(F=29.55、54.47、31.45,P<0.01).在1 mr/L脂多糖刺激下,TLR4和MD-2 mRNA的表达量在2 h后明显增加,6 h达高峰,8 h开始回落,至24 h时仍高于基础值(F=5.28、4.11,P<0.01);TNF-α、IL-6和IL-1β含量具有类似变化(F=10.64、11.23、17.58,P<0.01),其中TNF-α和IL-1β含量在6 h达高峰,持续至8 h,IL-6含量在8 h达高峰,持续至12 h.TLR4和MD-2 mRNA表达呈正相关(r=0.513,P<0.01).MD-2 siRNA对NR8383细胞MD-2基因的干扰效率为67%,在脂多糖刺激下干扰组细胞培养上清液中TNF-α、IL-1β和IL-6水平未见明显升高.结论 高浓度脂多糖可较长时间上调大鼠肺泡巨噬细胞株NR8383细胞TLR4和MD-2基因的表达,并促进TNF-α、IL-6和IL-1β的分泌.MD-2 siRNA可抑制脂多糖刺激下NR8383细胞分泌TNF-α、IL-1 β和IL-6.     

转化生长因子β1反义寡核苷酸抑制单侧肾切除糖尿病大鼠系膜增生

目的 探讨转化生长因子（TGF）β1反义寡核苷酸对单侧肾切除糖尿病大鼠系膜增生的作用。方法 采用人工合成TGF－β1反义、正义及错配的3组寡核苷酸经脂质体包裹柏转染单侧肾切除糖尿病大鼠肾小球系膜细胞（MC）。用细胞计数检测转染后MC的生长抑制率，用RT－PCR和ELISA方法检测TGF－β1 mRNA和蛋白的表达水平，并检测IV型胶原mRNA表达水平的变化。结果 TGF－β1反义寡核苷酸可抑制MC的存活，TGF－β1 mRNA及蛋白的表达水平下降，IV型胶原mRNA的水平也下降。结论 TGF－β1反义寡核苷酸不仅抑制TGF－β1 mRNA及蛋白的表达，而且还可抑制细胞外基质IV型胶原mRNA的表达，抑制MC的肥大，从而抑制单侧肾切除糖尿病大鼠系膜的增生。     

芍药苷对脂多糖诱导的THP-1细胞炎症因子分泌和ABCA1表达的影响

目的观察芍药苷(PF)对脂多糖(LPS)诱导的THP-1细胞炎症因子和三磷酸腺苷结合盒转运体A1(ABCA1)表达的影响。方法用含PF(10-8、10-7、10-6、10-5、10-4mol/L)培养基预处理细胞0.5 h,再用含LPS(1mg/L)培养基共同培养细胞24 h。ELISA检测细胞培养液上清中白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、白细胞介素8(IL-8)和肿瘤坏死因子α(TNF-α)水平,Western blot检测细胞中ABCA1蛋白的表达。结果与对照组比较,LPS组细胞上清液中IL-1β、IL-6、IL-8和TNF-α的水平显著性升高(P0.05),ABCA1蛋白表达显著性下调(P0.05)。与LPS组比较,LPS+PF(10~(-6)、10~(-5)和10~(-4)mol/L)组上清液中IL-1β、IL-6、IL-8和TNF-α的水平显著性降低(均P0.05),ABCA1蛋白表达显著性上调(P0.05),呈现浓度依赖性。结论芍药苷抑制LPS诱导的THP-1细胞炎症因子分泌和ABCA1表达下调。     

心肌营养素-1对血管紧张素Ⅱ诱导心肌细胞肥大的影响

目的:研究心肌营养素-1(CT-1)对血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大的影响。方法:利用AngⅡ刺激心肌细胞,导致细胞肥大,应用CT-1反义脱氧寡核苷酸进行干预。检测心肌细胞大小及3H-亮氨酸掺入率的变化;以逆转录聚合酶链反应(RT-PCR)检测CT-1 mRNA及β-肌球蛋白重链(β-MHC)mRNA表达,免疫组织化学法检测心肌细胞CT-1蛋白的表达。结果:经AngⅡ刺激后,在相差显微镜下可见心肌细胞面积变大,3H-亮氨酸掺入率、CT-1蛋白的表达增高;CT-1和β-MHC mRNA水平的表达增加。利用Fugene6可将CT-1反义脱氧寡核苷酸转染入心肌细胞。CT-1反义脱氧寡核苷酸组心肌细胞面积较肥大组明显减小[(81.257±3.995)mm2∶(127.214±5.693)mm2],3H-亮氨酸掺入量[(1653.33±17.91)cpm∶(1971.50±42.16)cpm]及CT-1蛋白[(3.16±0.17)%∶(3.51±0.29)%]明显减少;CT-1[(0.2137±0.0227)∶(0.4023±0.0160)]和β-MHC mRNA[(0.5032±0.0261)∶(0.773 4±0.0486)]表达亦显著减少(P0.05~0.01)。各指标Fugene 6组、错义组与肥大组无明显差异。结论:AngⅡ致心肌细胞肥大过程中伴有CT-1表达增加,应用CT-1反义脱氧寡核苷酸后可使之下降,说明CT-1参与了心肌细胞的肥大机制。     

强直性脊柱炎CD4+T细胞白细胞介素-17核孤儿受体的表达及意义

目的 探讨强直性脊柱炎(AS)患者外周血CD4+T细胞白细胞介素(IL)-17蛋白水平和IL-17、核孤儿受体(RORγt)mRNA水平及意义.方法 取AS患者和健康人外周血单个核细胞(PBMCs),免疫磁珠分选CD4+T细胞,加佛波脂(PMA)腐子霉素(Ion)作用,经固定/透膜处理进行细胞内染色,流式细胞术(FCM)检测CD4+T细胞IL-17蛋白水平;用或不用非特异性刺激剂[抗CD3单抗(A-CD3)、抗CD28单抗(A-CD28)]刺激CIM+T细胞12 h和24 h后,应用实时荧光定量反转录聚合酶链反应(RT-PCR)检测IL-17、RORγt mRNA水平.组间比较采用方差分析,相关性分析采用Pearson相关分析.结果 CD4+T细胞IL-17蛋白水平AS活动组(1.26±0.44)较稳定组(0.56±0.21)和对照组(0.45±0.13)显著增高(P<0.01),同时IL-17、RORγt mRNA水平活动组较稳定组和对照组显著增高(P<0.01). 用A-CD3/A-CD28刺激后,AS患者CD4+T细胞IL-17、RORγtmRNA水平显著高于未刺激组(P<0.05),12 h刺激组又高于24 h刺激组(P<0.01).结论 AS患者外周血CD4+T细胞IL-17、RORγt表达水平显著增高,IL-17、RORγt表达水平可能与AS病情活动相关.     

Cytochrome 1A1 and 1B1 gene diversity in the Zanzibar islands

Amodiaquine (AQ) is a 4‐aminoquinoline widely used in the treatment of malaria as part of the artemisinin combination therapy (ACT). AQ is metabolised towards its main metabolite desethylamodiaquine mainly by cytochrome P450 2C8 (CYP2C8). CYP1A1 and CYP1B1 play a minor role in the metabolism but they seem to be significantly involved in the formation of the short‐lived quinine‐imine. To complete the genetic variation picture of the main genes involved in AQ metabolism in the Zanzibar population, previously characterised for CYP2C8, we analysed in this study CYP1A1 and CYP1B1 main genetic polymorphisms. The results obtained show a low frequency of the CYP1A1*2B/C allele (2.4%) and a high frequency of CYP1B1*6 (approximately 42%) followed by CYP1B1*2 (approximately 27%) in Zanzibar islands. Genotype data for CYP1A1 and CYP1B1 show a low incidence of fast metabolisers, revealing a relatively safe genetic background in Zanzibar’s population regarding the appearance of adverse effects.     

CYP1A1 , GSTM1 , GSTT1 and NQO1 polymorphisms and colorectal adenomas in Japanese men

AIM: To investigate the role of functional genetic poly-morphisms of metabolic enzymes of tobacco carcinogens in the development of colorectal adenomas. METHODS: The study subjects were 455 patients with colorectal adenomas and 1052 controls with no polyps who underwent total colonoscopy in a preretirement health examination at two Self Defense Forces hospitals. The genetic polymorphisms studied wereCYP1A1*2A (rs 4646903), CYP1A1*2C (rs 1048943), GSTM1 (null or non-null genotype), GSTT1 (null or non-null genotype) and NQO1 C609T (rs 1800566). Genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR method using genomic DNA extracted from the buffy coat. Cigarette smoking and other life-style factors were ascertained by a self-administered questionnaire. The associations of the polymorphisms with colorectal adenomas were examined by means of OR and 95%CI, which were derived from logistic regression analysis. Statistical adjustment was made for smoking, alcohol use, body mass index and other factors. The gene-gene interaction and effect modification of smoking were evaluated by the likelihood ratio test. RESULTS: None of the five polymorphisms showed a significant association with colorectal adenomas, nor was the combination of GSTM1 and GSTT1 . A borderline significant interaction was observed for the combination of CYP1A1*2C and NQO1 (P = 0.051). The OR associated with CYP1A1*2C was significantly lower than unity among individuals with the NQO1 609CC genotype. The adjusted OR for the combination of the CYP1A1*2C allele and NQO1 609CC genotype was 0.61 (95%CI: 0.42-0.91). Although the interaction was not statistically significant (P = 0.24), the OR for individuals carrying the CYP1A1*2C allele and GSTT1 null genotype decreased significantly compared with those who had neither CYP1A1*2C allele nor GSTT1 null genotype (adjusted OR: 0.69, 95%CI: 0.49-0.97). Smoking did not modify the associations of the individual polymorphisms with colorectal adenomas. There w     

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.     

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.     

Interleukin 1 (IL 1)

Interleukin 1 is an essential factor of macrophage dependent T cell activation and has a large quantity of other biological activities. This paper gives a review of present knowledge of Interleukin 1. In addition to biochemical properties, the IL 1 production and IL 1 activities, methods for determining of IL 1 and inhibitory factors of IL 1 induced T cell proliferation are described.     

Pandemic H1N1 influenza

The 2009 H1N1 influenza A virus that has targeted not only those with chronic medical illness, the very young and old, but also a large segment of the patient population that has previously been afforded relative protection - those who are young, generally healthy, and immune naive. The illness is mild in most, but results in hospitalization and severe ARDS in an important minority. Among those who become critically ill, 20-40% will die, predominantly of severe hypoxic respiratory failure. However, and potentially in part due to the young age of those affected, intensive care with aggressive oxygenation support will allow most people to recover. The volume of patients infected and with critical illness placed substantial strain on the capacity of the health care system and critical care most specifically. Despite this, the 2009 pandemic has engaged our specialty and highlighted its importance like no other. Thus far, the national and global critical care response has been brisk, collaborative and helpful - not only for this pandemic, but for subsequent challenges in years ahead.