BN1028_Heat_treatment_defects_2002-09
白藜芦醇与人血清白蛋白相互作用的光谱研究_吴秋华
第29卷,第1期 光谱学与光谱分析Vol 129,No 11,pp226-2302009年1月 Spectro sco py and Spectr al AnalysisJanuary ,2009白藜芦醇与人血清白蛋白相互作用的光谱研究吴秋华,周 欣,臧晓欢,王 春,张志恒,王 志*河北农业大学理学院,河北省生物无机化学重点实验室,河北保定 071001摘 要 在不同温度下,用荧光猝灭光谱、同步荧光光谱和紫外-可见吸收光谱,研究了白藜芦醇与人血清白蛋白(H SA)相互作用的光谱学行为。
根据不同温度下白藜芦醇对H SA 的荧光猝灭作用,利用Stern -Vo lmer 方程处理实验数据,结果表明白藜芦醇与H SA 的结合常数K A 为2139@105(25e ),1125@105(35e )和1110@105(45e )。
根据F Êrster 非辐射能量转移理论,求出了白藜芦醇与HSA 之间的结合距离为3102nm(25e ),3146nm(35e )和3179nm(45e )。
实验表明静态猝灭和非辐射能量转移是导致白藜芦醇对HSA 荧光猝灭的两大原因,通过计算热力学参数,可知该药物与人血清白蛋白的相互作用是一个吉布斯自由能降低的自发过程,且二者之间的主要作用力类型为疏水作用力。
并采用同步荧光光谱探讨了白藜芦醇对H SA 构象的影响。
关键词 白藜芦醇;H SA ;相互作用;荧光光谱中图分类号:O 43311 文献标识码:A DOI :1013964/j 1issn 11000-0593(2009)01-0226-05收稿日期:2007-10-06,修订日期:2008-01-08基金项目:人事部留学人员科技择优项目,河北省自然科学基金项目(B2006000413)和河北农业大学科研发展基金项目资助 作者简介:吴秋华,1969年生,河北农业大学理学院讲师 *通讯联系人 e -mail:wangzh i@heb 引 言白藜芦醇(r esv eratr ol,简称R es),化学名称为3,4,5-三羟基二苯乙烯,是含有芪类结构的酚化合物,主要存在于葡萄、虎杖、花生、桑椹等植物中,尤其在种皮中含量较高。
间苯二酚杯[4]芳烃为涂层的压电石英传感器测定液相多巴胺
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2002年第60卷第6期,1084～1087化学学报ACT A CHIMICA SINICAV ol.60,2002N o.6,1084～1087间苯二酚杯[4]芳烃为涂层的压电石英传感器测定液相多巴胺陈朗星 徐 华 何锡文Ξ①(南开大学化学系 天津300071)摘要 研究了以间苯二酚杯[4]芳烃为敏感涂层的石英压电晶体(PQC )传感器,在中性磷酸盐缓冲液体系中对神经传递质多巴胺和抗坏血酸的响应,发现间苯二酚杯[4]芳烃对多巴胺有很好的响应选择性,这归因于间苯二酚杯[4]芳烃与多巴胺分子结构相匹配,形成主客体超分子体系.以间苯二酚杯[4]芳烃为涂层的PQC 传感器对液相中多巴胺具有响应快、重现性好、灵敏度高的特点,线性响应范围为3.5～500μg Πg.关键词 间苯二酚杯[4]芳烃,石英压电晶体,传感器,多巴胺,主客体作用A Piezoelectric Q uartz Crystal Coated with R esorcincalix[4]arene for the Determination of Dopamine in LiquidCHE N ,Lang 2X ing X U ,Hua HE ,X i 2WenΞ(Department o f Chemistry ,Nankai Univer sity ,Tianjin 300071)Abstract The res orcincalix[4]arene derivative has been synthesized and studied as a sensitive material for thedetermination of dopamine in liquid through a piezoelectric quartz crystal (PQC )sens or.The PQC 2coated res orcincalix[4]arene is highly selective for dopamine in com paris on with ascorbic acid.The new selectivity dimension is attributed to host 2guest interactions between res orcincalix [4]arene and dopamine.The PQC is used for m onitoring of dopamine in liquid at phosphate bu ffer (pH =7.40)with fast response time ,g ood sensitivity and reproducibility.It exhibits a linear response in the range 3.5～500μg Πg of dopamine.K eyw ords res orcincalix[4]arene ,piezoelectric quartz crystal ,sens or ,dopamine ,host 2guest interaction 多巴胺(dopamine ,又名32羟酪胺或儿茶酚乙胺)是医学上研究较多的一种主要的神经传递质,在健康和疾病中起着重要角色,体内多巴胺含量多少与心脏病、血压、甲状腺荷尔蒙含量、神经肌肉失调和各种精神疾病有关[1].临床上多巴胺受体激动药可兴奋心脏,增加肾血流量.近年来,对中枢神经系统细胞内液体中多巴胺的高灵敏度选择性测定已引起广泛关注,测定方法主要有分光光度法、荧光法、放射性酶法、HP LC 法、电化学方法[1,2],测试的主要难点源于几种共存物质的干扰,如抗坏血酸(维生素C )和3,42二羟基苯乙酸[3,4]、肾上腺素、邻苯二酚、尿酸、乙酰氨基苯、过氧化氢等[5].用压电石英晶体传感器(piezoelectric quartz crystal sens or ,PQCS )测定多巴胺未见报道.压电石英晶体传感器因其装置可制成微型化、自动化、实时在线检测,灵敏度高,现已逐步运用于医学检测、环境监测和工业品质控制.挑选具有选择性高、稳定性好并对分析物敏感的涂层,能提高PQCS 的灵敏度.许多大环超分子化合物如冠醚、环糊精、穴配体、杯芳烃作为敏感材料涂层能增强响应信号强度,只有少量文献报道了化学涂层的PQCS 响应识别液相中有机分子[6～10].杯芳烃作为第三代①E 2mail :xiwenhe @Received August 18,2001;revised January 8,2002;accepted M arch 17,2002.国家自然科学基金资助项目(N o.29975014,20175009).主体超分子,金属离子和有机分子均可作为其客体[11],杯芳烃分子为敏感涂层的PQC 传感器已开始用于液相中有机分子的测定[9,10].考虑到多巴胺的分子结构,我们从合成的数种杯芳烃衍生物中选择了一种间苯二酚杯[4]芳烃为涂层,其环外含8个羟基,并具有碗状构型,希望其能与含氧基团的极性客体分子形成主客体化合物.本实验证实了采用间苯二酚杯[4]芳烃作压电石英晶片的涂层,能有效识别多巴胺与抗坏血酸,对液相中多巴胺具有响应快、重现性好、灵敏度高、线性响应范围广的特点.可用于实际装置中,定量检测溶液中多巴胺的浓度.1 实验部分1.1 试剂Na 2HPO 4,KH 2PO 4(A.R.,天津市化学试剂二厂);无水甲醇(A.R.,天津市天泰精细化学品有限公司),抗坏血酸(A.R.,北京芳草医药化工研制公司),盐酸多巴胺(化学对照品,中国药品生物制品检定所),盐酸多巴胺与抗坏血酸均用二次蒸馏水配制成浓度为0.1m ol ·dm -3的溶液,放于容量管中.盐酸多巴胺溶液在每次使用完后均立即充入氮气加以保护,并将管口密封,管外套上照相馆用的遮光纸,避免盐酸多巴胺见光或遇空气被氧化.抗坏血酸溶液每天配制(防止其氧化).间苯二酚杯[4]芳烃、盐酸多巴胺与抗坏血酸的结构见图式1.图式1 敏感涂层和分析物的结构式Scheme 1 The structures of sensitive material and analyte间苯二酚杯[4]芳烃的合成[12]:将27.52g(0125m ol )间苯二酚溶于125m L 95%乙醇,加入到500m L 三口瓶中,再加入125m L 水和62.5m L 浓HCl ,通氩气保护,开始搅拌,并将反应温度降低到15℃,逐滴加入11.01g (0.25m ol )乙醛,30min 内加完,在50℃反应搅拌1h 后,将温度降低到25℃,一直通氩气,搅拌反应4d ,将反应得到的沉淀物过滤,用水研磨固体产物后,过滤,用乙醇-水重结晶,得到产物20.5g (产率为60%),m.p.>360℃.结构式经IR 和NMR 光谱证实.1.2 仪器装置实验用压电晶体振荡器为JM5型AT 切双面被金晶振(北京707厂),石英电极的基频为9MH z ,直径为6mm ,晶体直径为12.0mm ,频率以N3165型频率计(台湾Sam po 公司)测定.频率测定用的振荡电路和数据采集系统由本实验室自行设计[13].图1 间苯二酚杯[4]芳烃为涂层的压电晶体传感器实验装置1—压电晶体传感器;2—微量注射器;3—恒温水浴;4—振荡电路;5—频率计;6—计算机;7—电磁搅拌器Figure 1 Experimental set 2up for the res orcincalix [4]arene 2coated PQC sens or1—piez oelectric crystal sens or ;2—microsyringe ;3—therm ostat ;4—oscillator ;5—frequency counter ;6—com puter ;7—electromagnetic stirrer1.3 敏感涂层的制备间苯二酚杯[4]芳烃不溶于氯仿,用无水甲醇配制溶液.涂膜时,先准确称取10mg 间苯二酚杯[4]芳烃溶于2m L 无水甲醇,间苯二酚杯[4]芳烃浓度为5mg ΠL.将石英晶片金电极平放于台上,用微量注射器分别将3.0μL 间苯二酚杯[4]芳烃的甲醇溶液滴于金电极的两面,待溶剂挥发后,用电吹风热风吹约30s ,得到很薄的间苯二酚杯[4]芳烃涂层.涂覆上间苯二酚杯[4]芳烃后,金电极在空气中的频率下降约8000～10000H z.1.4 测量方法实验测量装置如图1所示.图中描述了电脑控制的PQC 传感器测定液相中多巴胺的实验装置.将连接到振荡电路中的PQC 传感器,双面浸入到用二5801N o.6陈朗星等:间苯二酚杯[4]芳烃为涂层的压电石英传感器测定液相多巴胺 次蒸馏水配制的pH 值为7.40的0.0010m ol ·dm-3磷酸盐缓冲溶液中.测量池的温度用恒温水浴控制在(25±0.5)℃.压电晶体的振荡频率用自制的数据采集系统贮存在386微机中,整个测量池的体积约为16m L ,将0.2～50.0μL 浓度为0.10m ol ·dm -3的分析物用微量注射器注入到16m L pH 值为7.40的0.0010m ol ·dm -3磷酸盐缓冲溶液中,搅拌速度固定在一个位置上.每5s 记录一次频率变化数据,裸露的压电石英金电极和涂覆有间苯二酚杯[4]芳烃涂层的压电石英金电极在空气中的频率变化为±2H z ,可连续工作24h 以上.实验证明,加入分析物的体积为50μL 以下时,系统不会因样品体积的增加而导致超出实验误差以外的误差(±5H z ).当间苯二酚杯[4]芳烃为涂层的PQC 传感器双面放入液相中时,没有加入分析物时,频率变化的波动为±10H z.间苯二酚杯[4]芳烃涂层可连续使用1个月以上,不会从压电晶片上脱落下来.为了保证测定的重现性,实验系统尤其是检测池在测定过程中保持位置不变,每次测定时移取一定量的待测物加入检测池中.读取加入样品前后的稳定频率差值.然后用注射器抽出瓶中溶液,换入二次蒸馏水洗涤,重新注入pH 值为7.40的0.0010m ol ·dm -3磷酸盐缓冲溶液测下一个样品.每个样品平行测定3次以上,以保证准确性.2 结果与讨论2.1 间苯二酚杯[4]芳烃涂层的选择性图2为在pH =7.40的0.0010m ol ·dm -3磷酸盐缓冲液中,间苯二酚型杯[4]芳烃为涂层的PQC 传感器对多巴胺和抗坏血酸的吸附响应曲线.从图2可看出,PQC 传感器对多巴胺和抗坏血酸有很快的响应,加入分析物后,频率变化很快,5s 后响应值>90%,响应值在10s 达到稳定.我们对没有间苯二酚型杯[4]芳烃作涂层的空白石英晶片对多巴胺和抗坏血酸的响应进行了测定,发现空白石英晶片对多巴胺和抗坏血酸的响应频率变化在仪器噪声范围内(±10H z ),可以说明没有吸附.而涂有间苯二酚杯[4]芳烃的晶片对多巴胺的响应值Δf DA ≈2580H z ,对抗坏血酸的响应值Δf AA ≈280H z ,二者差别较大,选择性系数K =Δf DA ΠΔf AA ≈9.2,说明间苯二酚型杯[4]芳烃为涂层的PQC 传感器对多巴胺有较好的选择性,能有效识别多巴胺和抗坏血酸.间苯二酚型杯[4]芳烃对多巴胺的高选择性归因于主客体的分子识别,即间苯二酚型杯[4]芳烃的空穴结构与被测物多巴胺分子结构相匹配,多巴胺的中性苯环部分能进入到间苯二酚型杯[4]芳烃的空穴中,另外间苯二酚型杯[4]芳烃分子的8个羟基与多巴胺的胺基有氢键作用.而在pH 值7.4的缓冲溶液中,抗坏血酸的五元环带负电荷使其不能进入到间苯二酚型杯[4]芳烃的空穴中,但能和间苯二酚型杯[4]芳烃的羟基有氢键作用,因此间苯二酚型杯[4]芳烃的PQCS 对抗坏血酸有一定的响应.图2 PQC 传感器对盐酸多巴胺和抗坏血酸(均为312.5μg Πg )的吸附响应曲线Figure 2 The response of PQC sens or to 312.5μg Πg of dopamine and ascorbic acid respectively●—dopamine ;○—ascorbic acid2.2 吸附的重现性图3中列出了在pH =7.40的磷酸盐缓冲体系中,315μg Πg 多巴胺在涂有间苯二酚型杯[4]芳烃的晶片上的吸附重现性曲线,我们进行了9次重复性实验,测定结果的相对误差<5.0%.重现性能满足定量实验的要求.2.3 多巴胺的定量测定我们定义本测量体系的检测限为2倍噪声所对应的多巴胺的浓度.检测系统的噪声为±10H z ,2倍噪声40H z 所对应的多巴胺浓度为3.5μg Πg ,因此PQC 对多巴胺的检测限为3.5μg Πg.在pH =7.40的0.0010m ol ·dm -3磷酸盐缓冲体系中,我们测定间苯二酚型杯[4]芳烃涂层的PQC 对不同浓度的多巴胺的响应,由图4可知,当多巴胺的浓度在3.5～500μg Πg 范围内,随着多巴胺浓度的增加,其吸附引起的频率变化值成比例增加,对多巴胺的响应值进行了线性回归,在3.5～500μg Πg 范围内有非常好的线性关系,R =0.9998.6801 化学学报V ol.60,2002图3 涂有间苯二酚型杯[4]芳烃的晶片对多巴胺的吸附重现性曲线Figure 3 The reproducible response of PQC 2coated with res orcincalix[4]arene todopamine图4 多巴胺在间苯二酚型杯[4]芳烃为涂层的PQC 上的定量响应范围Figure 4 The response range of dopamine on PQC 2coated with res orcincalix[4]arene我们取10μL 0.10m ol ·dm -3多巴胺溶液和25μL 0.10m ol ·dm -3抗坏血酸溶液一起加入到体积为16m L ,pH =7.40的0.0010m ol ·dm -3磷酸盐缓冲液测量池中,对应的多巴胺和抗坏血酸的含量分别11.9和27.5μg Πg.用PQC 传感器测定了在1倍抗坏血酸存在下,多巴胺的含量,并与分子印迹聚合物敏感薄膜的循环伏安法[14]进行了对比实验.本方法测定多巴胺的含量为12.5μg Πg (n =5),回收率为106%,而分子印迹聚合物敏感薄膜的循环伏安法为11.4μg Πg (n =5),回收率为96%,两者测定结果基本一致.3 结论通过以间苯二酚杯[4]芳烃涂层的晶片和空白晶片,对多巴胺和抗坏血酸的吸附实验,表明由于间苯二酚杯[4]芳烃能与多巴胺形成主客体化合物,能较好识别多巴胺和抗坏血酸,本实验首次用压电传感器测定液相中的多巴胺,体系具有响应快、重现性好、灵敏度高、线性响应范围广的特点.杯芳烃的独特分子识别性能,使其在压电传感器中得到进一步应用.R eferences1Whitley ,R.J.;Meikle ,A.W.;Watts ,N. 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C.;K nobler ,C.B.;Cram ,D.J.J .Org .Chem .1989,54,1305.13Pan ,W.;Liu ,H.2Q.;X ing ,W.2L.;He ,X.2W.Chin .J .Anal .Chem .1997,25,1104(in Chinese ).(潘卫,刘鸿铨,邢婉丽,何锡文,分析化学,1997,25,1104.)14G uo ,H.2S.Ph .D .Dissertation ,T ianjin ,2001(inChinese ).(郭洪声,博士论文,南开大学,天津,2001.)(A0108183 SHE N ,H.;DONG,H.Z.)7801N o.6陈朗星等:间苯二酚杯[4]芳烃为涂层的压电石英传感器测定液相多巴胺 。
动物食品中硫酸沙丁胺醇残留物的TLC-SERRS分析
动物食品中硫酸沙丁胺醇残留物的TLC-SERRS分析作者：曹萌许凤张宏莲梁鑫徐涛李骞来源：《食品安全导刊》2023年第11期摘要：目的：建立薄层色谱与表面增强共振拉曼光谱联用技术检测苯乙胺类拟肾上腺素硫酸沙丁胺醇药物残留的方法。
方法：使用微波加热法用硝酸银和柠檬酸钠制备表面增强剂银溶胶；对所制备的银溶胶进行参数测定，选择表面增强效果较好的银溶胶；应用表面增强共振拉曼光谱法检测硫酸沙丁胺醇时，在TLC板分离出的目标斑点上滴加芳香族重氮盐和表面增强剂银溶胶后再检测其拉曼光谱，并对市售猪肝进行样品检测。
结果：该方法可显著增强拉曼光谱的检测信号，增强因子可达到1.65×107。
结论：本方法专属性强、耐用性好，为苯乙胺类拟肾上腺素药物残留物的鉴别技术提供了一种快速、高效的新方法。
关键词：薄层色谱；表面增强共振拉曼光谱法；兽药残留Analysis of Residues of Salbutamol Sulfate in Animal Food Based on TLC-SERRSCAO Meng， XU Feng*， ZHANG Honglian， LIANG Xin， XU Tao， LI Qian （School of Pharmacy， Qiqihar Medical University， Qiqihar 161006， China）Abstract： Objective： To establish a method for the detection of phenethylamine-like adrenaline-like salbutamol sulfate residues by TLC and surface enhanced resonance Raman spectroscopy. Method： The surface strengthening agent silver sol was prepared from silver nitrate and sodium citrate by microwave heating method. The parameters of the prepared silver sol were determined， and the silver sol with better surface enhancement effect was selected. When salbutamol sulfate was detected by surface-to-enhanced resonance Raman spectroscopy， aromatic diazo salt and surface enhancer silver sol were added to the target spots isolated from TLC plate， and then the Raman spectra were detected. Result： This method can significantly enhance the detection signal of Raman spectrum， and the enhancement factor can reach 1.65×107. Conclusion： This method has strong specificity and good durability， and provides a rapid and efficient new method for the identification of phenethylamines and adrenergic drug residues.Keywords： thin layer chromatography; surface enhanced resonance Raman spectroscopy; veterinary drug residue硫酸沙丁胺醇，又称硫酸舒瑞灵，主要用于呼吸道疾病的治疗[1]。
使用β-环糊精交联聚合物作为吸附剂从水溶液中去除对羟基苯甲酸酯外文译文
.使用β-环糊精交联聚合物作为吸附剂从水溶液中去除对羟基苯甲酸酯摘要通过使用β-环糊精（β-CD）聚合物从水溶液中去除四种尼泊金酯，即对羟基苯甲酸甲酯，对羟基苯甲酸乙酯，对羟基苯甲酸丙酯，和对羟基苯甲酸苄酯，已经进行了研究。
不同的β-环糊精聚合物可通过使用不同摩尔比的两种交联剂制备，例如，六亚甲基二异氰酸酯（HMDI）和甲苯-2,6 -二异氰酸酯TDI）。
摩尔比为1:7的β-CD-HMID聚合物和摩尔比为1:4的β-CD-TDI是β-CD-HMID和β-CD-TDI系列聚合物中吸附尼泊金酯能力最强的聚合物，并随后在深入研究中被使用。
β-CD-HMDI对对羟基苯甲酸甲酯，对羟基苯甲酸乙酯，对羟基苯甲酸丙酯，和对羟基苯甲酸苄酯的吸附能力分别是0.0305，0.0376，0.1854和0.3026mmol/g。
与β-CD-HMDI相比，β-CD-TDI具有更高的吸附能力，对对羟基苯甲酸甲酯，对羟基苯甲酸乙酯，对羟基苯甲酸丙酯，和对羟基苯甲酸苄酯的吸附能力分别是0.1019，0.1286，0.2551和0.3699mmol/g。
研究的参数分别为吸附容量，保水性和可重用性。
两种交联剂无论是在聚合物的吸附能力和疏水性方面，还是在对不同尼泊金酯的吸附能力方面都进行了比较和讨论。
所有实验都是在同一种吸附技术下进行。
这些聚合物被应用于实际样品并呈阳性反应。
关键词：β- 环糊精聚合物，对羟基苯甲酸酯类，吸附1.1 介绍自1920年代，对羟基苯甲酸酯已在个人护理产品中使用发展，由于它们毒性低，成本低，抗菌活性广谱，在食品，医药和化妆品等产品中是最受欢迎和使用最广泛的防腐剂。
然而，有一小部分人口对对羟基苯甲酸酯过敏。
最近的研究还表明，对羟基苯甲酸酯具有雌激素激动剂活性，并已被发现在人类的乳腺肿瘤组织中有20纳克/克组织的平均浓度。
对羟基苯甲酸酯也可能会导致男性不育，因为它们可能会导致睾丸线粒体功能障碍。
由于其对人体健康的潜在影响，许多国家纷纷出台控制使用对羟基苯甲酸酯的立法。
在线固相萃取_高效液相色谱法测定水体中的多环芳烃
表 1 用于在线固相萃取和分离的梯度 Table 1 Gradient programs for online SPE and separation
在线固相萃取 Online SPE
时间 Time
（ min）
绞股蓝提取物对糖尿病肾病大鼠血液流变学和炎性因子影响的实验研究
绞股蓝提取物对糖尿病肾病大鼠血液流变学和炎性因子影响的实验研究目的初步探讨绞股蓝提取物对糖尿病肾病大鼠血液流变学和炎性因子的影响。
方法将40只Wistar大鼠随机分为空白对照组、模型组、绞股蓝大剂量组（5 mg/kg）和小剂量组（2.5 mg/kg）；模型组、大剂量组和小剂量组大鼠使用腹腔注射链脲佐菌素方法制作糖尿病肾病模型；绞股蓝大小剂量组按照上述剂量给予绞股蓝提取物灌胃，1次/d，持续10周，空白对照组和模型组则给予同剂量蒸馏水灌胃；实验结束时腹主动脉取血检测血液黏度、总胆固醇、三酰甘油及炎症因子。
结果绞股蓝提取物可以有效降低糖尿病肾病大鼠血液黏度、血浆黏度及总胆固醇和三酰甘油水平，与模型组比较差异具有统计学意义（P 0.05）。
见表1。
2.2 炎症因子给药10周后，绞股蓝大剂量组与小剂量组TNF-α、IL-6和CRP水平，均低于模型对照组，差异具有统计学意义（P 0.05）。
见表2。
3 讨论糖尿病肾病是糖尿病最常见、最严重的微血管并发症之一，高血糖、脂代谢紊乱、血流动力学及血流变性的异常等是糖尿病肾病发病的原因，同样也是导致糖尿病肾病进展的原因[6]。
血液流变学的异常是糖尿病微循环障碍的主要原因之一，微血管并发症是DN发生发展过程中的重要不利因素，血液流变学的异常往往在微血管并发症出现之前就已存，并且不同程度地促进了并发症的发生，糖尿病患者血液流变学异常时的主要表现为血液呈高凝状态、血液流速减慢和微血栓形成[7-8]。
本组研究显示，DN造模成功的大鼠同正常大鼠比较，均不同程度存在血液流变学的异常，主要是全血黏度（高切及低切）、血浆黏度、总胆固醇和三酰甘油的异常升高。
同时，近年来众多研究结果显示体内微炎症状态与DN 的发病密切相关，巨噬细胞、T细胞等免疫细胞在多种黏附因子、趋化因子和细胞因子的作用下在肾脏募集，引发局部炎症反应，加重血管损伤和基质纤维化[9]，上述免疫细胞通过分泌产生此类炎症因子来调节免疫应答作用，并最终参与了炎症反应。
A GENE-EXPRESSION SIGNATURE AS A PREDICTOR OF SURVIVAL IN BREAST CANCER
The New EnglandJournal of MedicineCopyr ight ©2002 by the Massachusetts Medical Societ yA GENE-EXPRESSION SIGNATURE AS A PREDICTOR OF SURVIVALIN BREAST CANCERM ARC J. VAN DE V IJVER, M.D., P H.D., Y UDONG D. H E, P H.D., L AURA J. VAN ’T V EER, P H.D., H ONGYUE D AI, P H.D.,A UGUSTINUS A.M. H ART, M.S C., D ORIEN W. V OSKUIL, P H.D., G EORGE J. S CHREIBER, M.S C., J OHANNES L. P ETERSE, M.D.,C HRIS R OBERTS, P H.D., M ATTHEW J. M ARTON, P H.D., M ARK P ARRISH,D OUWE A TSMA, A NKE W ITTEVEEN,A NNUSKA G LAS, P H.D., L EONIE D ELAHAYE, T ONY VAN DER V ELDE, H ARRYB ARTELINK, M.D., P H.D.,S JOERD R ODENHUIS, M.D., P H.D., E MIEL T. R UTGERS, M.D., P H.D., S TEPHEN H. F RIEND, M.D., P H.D.,AND R ENÉ B ERNARDS, P H.D.A BSTRACTBackground A more accurate means of prognos-tication in breast cancer will improve the selection ofpatients for adjuvant systemic therapy.Methods Using microarray analysis to evaluate ourpreviously established 70-gene prognosis profile, weclassified a series of 295 consecutive patients with pri-mary breast carcinomas as having a gene-expressionsignature associated with either a poor prognosis ora good prognosis. All patients had stage I or II breastcancer and were younger than 53 years old; 151 hadlymph-node–negative disease, and 144 had lymph-node–positive disease. We evaluated the predictivepower of the prognosis profile using univariable andmultivariable statistical analyses.Results Among the 295 patients, 180 had a poor-prognosis signature and 115 had a good-prognosis sig-nature, and the mean (±SE) overall 10-year survivalrates were 54.6±4.4 percent and 94.5±2.6 percent, re-spectively. At 10 years, the probability of remainingfree of distant metastases was 50.6±4.5 percent in thegroup with a poor-prognosis signature and 85.2±4.3percent in the group with a good-prognosis signature.The estimated hazard ratio for distant metastases inthe group with a poor-prognosis signature, as com-pared with the group with the good-prognosis signa-ture, was 5.1 (95 percent confidence interval, 2.9 to9.0; P<0.001). This ratio remained significant when thegroups were analyzed according to lymph-node sta-tus. Multivariable Cox regression analysis showed thatthe prognosis profile was a strong independent factor in predicting disease outcome.Conclusions The gene-expression profile we stud-ied is a more powerful predictor of the outcome of dis-ease in young patients with breast cancer than stand-ard systems based on clinical and histologic criteria. (N Engl J Med 2002;347:1999-2009.)Copyright © 2002 Massachusetts Medical Society.From the Divisions of Diagnostic Oncology (M.J.V., L.J.V., D.W.V., J.L.P., D.A., A.W., A.G., L.D.), Radiotherapy (A.A.M.H., H.B.), Medical Oncology (S.R.), Biometrics (T.V.), Surgical Oncology (E.T.R.), and Molecular Car-cinogenesis (R.B.), Netherlands Cancer Institute, Amsterdam; the Center for Biomedical Genetics, Amsterdam (R.B.); and Rosetta Inpharmatics, Kirk-land, Wash. (Y.D.H., H.D., G.J.S., C.R., M.J.M., M.P., S.H.F.). Address re-print requests to Dr. Bernards at the Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands, or at r.bernards@nki.nl.Drs. van de Vijver, He, and van ’t Veer contributed equally to this article.DJUVANT systemic therapy substantiallyimproves disease-free and overall survival inboth premenopausal and postmenopausalwomen up to the age of 70 years with lymph-node–negative or lymph-node–positive breast cancer.1,2 It is generally agreed that patients with poor prognostic features benefit the most from adjuvant therapy.3,4 The main prognostic factors in breast can-cer are age, tumor size, status of axillary lymph nodes, histologic type of the tumor, pathological grade, and hormone-receptor status. A large number of other factors have been investigated for their potential to pre-dict the outcome of disease, but in general, they have only limited predictive power.5Using complementary DNA (cDNA) microarrays to analyze breast-cancer tissue, Perou et al. identified tumors with distinct patterns of gene expression that they termed “basal type” and “luminal type.”6 These subgroups differ with respect to the outcome of dis-ease in patients with locally advanced breast cancer.7 In addition, microarray analysis has been used to dis-tinguish cancers associated with BRCA1 or BRCA2 mutations8,9 and to determine estrogen-receptor sta-tus6,9,10 and lymph-node status.11,12Using inkjet-synthesized oligonucleotide microar-rays, we recently identified a gene-expression profile Athat is associated with prognosis in patients with breast cancer.9 We analyzed only tumors that were less than 5 cm in diameter from lymph-node–negative patients who were younger than 55 years of age. We found that a classification system based on 70 genes outperformed all clinical variables in predicting the likelihood of dis-tant metastases within five years. We estimated that the odds ratio for metastases among tumors with a gene signature associated with a poor prognosis, as com-pared with those having a signature associated with a good prognosis, was approximately 15 using a cross-validation procedure. Even though these results were encouraging, a limitation of the study was that the re-sults were derived from and evaluated in two groups of patients selected on the basis of outcome: distant metastases had developed in one group within five years, and the other group remained disease-free for at least five years. Therefore, to provide a more accurate estimate of the risks of metastases associated with the two gene-expression signatures and to substantiate that the gene-expression profile of breast cancer is a clin-ically meaningful tool, we studied a cohort of 295 young patients with breast cancer, some of whom were lymph-node–negative and some of whom were lymph-node–positive.METHODSSelection of PatientsT umors from a series of 295 consecutive women with breast can-cer were selected from the fresh-frozen–tissue bank of the Nether-lands Cancer Institute according to the following criteria: the tumor was primary invasive breast carcinoma that was less than 5 cm in di-ameter at pathological examination (pT1 or pT2); the apical axillary lymph nodes were tumor-negative, as determined by a biopsy of the infraclavicular lymph nodes; the age at diagnosis was 52 years or younger; the calendar year of diagnosis was between 1984 and 1995; and there was no previous history of cancer, except nonmelanoma skin cancer. All patients had been treated by modified radical mas-tectomy or breast-conserving surgery, including dissection of the axillary lymph nodes, followed by radiotherapy if indicated. Among the 295 patients, 151 had lymph-node–negative disease (results on pathological examination, pN0) and 144 had lymph-node–positive disease (pN+). T en of the 151 patients who had lymph-node–neg-ative disease and 120 of the 144 who had lymph-node–positive dis-ease had received adjuvant systemic therapy consisting of chemo-therapy (90 patients), hormonal therapy (20), or both (20). Sixty-one of the patients with lymph-node–negative disease were also part of the previous study used to establish the prognosis profile.9 All patients were assessed at least annually for a period of at least five years. Follow-up information was extracted from the medical registry of the Netherlands Cancer Institute. The median duration of follow-up was 7.8 years (range, 0.05 to 18.3) for the 207 patients without metastasis as the first event and 2.7 years (range, 0.3 to 14.0) for the 88 patients with metastasis as the first event. The me-dian follow-up among all 295 patients was 6.7 years (range, 0.05 to 18.3). There were no missing data. The study was approved by the medical-ethics committee of the Netherlands Cancer Institute. Clinicopathological variables were determined as described pre-viously.9 The level of expression of estrogen receptors was estimated on the basis of the hybridization results on the microarray experi-ments, which is a reliable assay for estrogen-receptor status.9 On the basis of this assay, there were 69 estrogen-receptor–negative tumors (defined by an intensity ratio of less than ¡0.65 U on a logarithmic scale, corresponding to staining of less than 10 percent of nuclei on immunohistochemical analysis) and 226 estrogen-receptor–positive tumors in the cohort. The histologic grade was assessed according to the method described by Elston and Ellis13; vascular invasion was assessed as absent, minor (one to three vessels), or major (more than three vessels).Isolation of RNA and Microarray Expression ProfilingThe isolation of RNA, labeling of complementary RNA (cRNA), hybridization of labeled cRNA to 25,000-gene arrays, and assess-ment of expression ratios were all performed as previously de-scribed.9,14 In brief, tumor material was snap-frozen in liquid nitro-gen within one hour after surgery. Frozen sections were stained with hematoxylin and eosin; only samples that had more than 50 percent tumor cells were selected. Thirty 30-µm sections were used for the isolation of RNA. T otal RNA was isolated with RNAzolB and dis-solved in RNase-free water. Then 25 µg of total RNA was treated with DNase with use of the Qiagen RNase-free DNase kit and RNeasy spin columns, the RNA was then dissolved in RNase-free water to a final concentration of 0.2 µg per microliter, and cRNA was generated by in vitro transcription with the use of T7 RNA polymerase and 5 µg of total RNA and labeled with Cy3 or Cy5 (Cy Dye, Amersham Pharmacia Biotech). Five micrograms of Cy-labeled cRNA from one breast-cancer tumor was mixed with the same amount of reverse-color Cy-labeled product from a pool that consisted of an equal amount of cRNA from each patient. Labeled cRNAs were fragmented to an average size of approxi-mately 50 to 100 nucleotides by heating the samples to 60°C in the presence of 10 mM zinc chloride and adding a hybridization buffer containing 1 M sodium chloride, 0.5 percent sodium sarcosine, 50 mM morpholino-ethane sulfonic acid (pH 6.5), and formamideFigure 1 (facing page). Pattern of Expression of Genes Used to Determine the Prognosis and Clinical Characteristics of 295 Patients with Breast Cancer.Panel A shows the pattern of expression of the 70 marker genes (also referred to as prognosis-classifier genes9) in a series of 295 consecutive patients with breast carcinomas. Each row represents the prognostic profile of the 70 marker genes for one tumor, and each column represents the relative level of expression of one gene. The tumors are numbered from 1 to 295 on the y axis, and the genes are numbered from 1 to 70 on the x axis. The genes in the horizontal direction are arrayed in the same order as in our previous study.9 Red indicates a high level of expression of messenger RNA (mRNA) in the tumor, as compared with the reference level of mRNA, and green indicates a low level of expression. The dotted line is the previously determined threshold between a good-prognosis signature and a poor-prognosis signature. Tumors are rank-ordered according to their correlation with the previously determined average profile in tumors from patients with a good prognosis. Panel B shows the time in years to distant metastases as a first event for those in whom this occurred, and the total duration of follow-up for all other patients. Panel C shows the lymph-node status (blue marks indicate lymph-node–positive disease, and white lymph-node–negative disease), the number of patients with distant metasta-ses as a first event (blue marks), and the number of patients who died (blue marks).GENE-EXPRESSION SIGNATURE AS A PREDICTOR OF SURVIVAL IN BREAST CANCER150200250100500¡0.500.5201030405060700510Reporter Genes ABCRatio (log scale)L y m p h -N o d e S t a t u sMetastasisTotal follow-upM e t a s t a s i sD e a t hYearsXxxxxxxxT u m o r s w i t h P o o r -P r o g n o s i s S i g n a t u r e T u m o r s w i t h G o o d -P r o g n o s i s S i g n a t u r e(final concentration, 30 percent at 40°C); the final volume was 3 ml. The microarrays included the 24,479 biologic oligonucleotides as well as 1281 control probes. After hybridization, the slides were washed and scanned with a confocal laser scanner (Agilent T echnol-ogies). Fluorescence intensities on scanned images were quantified, and the values were corrected for the background level and nor-malized.Validation StrategyWe wished to investigate the prognostic value of the gene-expres-sion profile in a consecutive series of patients with breast cancer. We included 61 of the 78 patients with lymph-node–negative disease who were involved in the previous study that determined the 70-gene prognosis profile.9 Leaving them out would have resulted in selection bias, since the previous study included a disproportionate-ly large number of patients in whom distant metastases developed within five years. We included these 61 patients in the study, but we used the “leave-one-out” cross-validated classification established in our previous study to predict the outcomes among these patients. In this approach, the classification of the left-out sample was based on its correlation with the mean levels of expression of the remain-ing samples from the patients with a good-prognosis signature, with the sample in question excluded from the gene-selection process.9 This approach minimizes to some extent the possibility of overesti-mating the value of the prognosis profile while it keeps the con-secutive series complete. We also provide validation results taking only the new samples into account.Correlation of the Microarray Data with the Prognosis ProfileFor each of the 234 tumors from patients who were not included in the previous study, we calculated the correlation coefficient of the level of expression of the 70 genes with the previously determined average profile of these genes in tumors from patients with a good prognosis (C1).9 A patient with a correlation coefficient of more than 0.4 (the threshold in the previous study of 78 tumors that re-sulted in a 10 percent rate of false negative results) was then assigned to the group with a good-prognosis signature, and all other patients were assigned to the group with a poor-prognosis signature. For the 61 patients with lymph-node–negative disease who were included in the previous study, we used a cutoff value of 0.55 (corresponding to the threshold that resulted in a 10 percent rate of false negative results in the cross-validated classification in our previous study).9 Study DesignStudy design, patient selection, RNA isolation from tumor ma-terial, histopathological analyses, clinical annotation, and clinical in-terpretation were carried out at the Netherlands Cancer Institute. RNA amplification and microarray hybridization were carried out at Rosetta Inpharmatics. Bioinformatic and statistical analyses were performed jointly by authors at both locations. All raw data were available to all the investigators.Statistical AnalysisIn the analysis of the probability that patients would remain free of distant metastases, we defined distant metastases as a first event to be a treatment failure; data on all other patients were censored on the date of the last follow-up visit, death from causes other than breast cancer, the recurrence of local or regional disease, or the development of a second primary cancer, including contralateral breast cancer. Data on patients were analyzed from the date of sur-gery to the time of the first event or the date on which data were censored, according to the method of Kaplan and Meier, and the curves were compared with use of the log-rank test. Values are expressed as means ±SE, calculated according to the method of Tsiatis.15We used proportional-hazards regression analysis16 to adjust the association between the correlation coefficient (C1) and metastases for other variables. All SEs were calculated with use of the sandwich estimator.17 The histologic grade, extent of vascular invasion, and number of axillary-lymph-node metastases (0 vs. 1 to 3 or 0 vs. »4) were used as variables. The linearity of the relation between the relative hazard ratio and the diameter of the tumor, age, and level of expression of estrogen receptors was tested with use of the Wald test for nonlinear components of restricted cubic splines.18 No evi-dence of nonlinearity was found (P=0.83 for age, P=0.75 for tumor diameter, P=0.65 for the number of positive nodes, and P=0.27 for the level of expression of estrogen receptors). We eval-uated whether the hazard ratio was proportional using the method of Grambsch and Therneau.19 In addition, we determined the dif-ference between the relative hazard ratio before and after five years of follow-up with respect to the prognosis signature using the Wald test. All calculations were performed with the S Plus 2000 or S Plus 6 statistical package.RESULTSCategorization of Gene-Expression SignaturesT otal RNA from each tumor was isolated and used to generate cRNA, which was labeled and hybridized to microarrays containing approximately 25,000 hu-T ABLE 1. A SSOCIATION BETWEEN C LINICAL C HARACTERISTICSAND THE P ROGNOSIS S IGNATURE.C HARACTERISTICP OOR-P ROGNOSISS IGNATURE(N=180)G OOD-P ROGNOSISS IGNATURE(N=115)PV ALUEno. of patients (%)Age<0.001 <40 yr52 (29)11 (10)40–44 yr41 (23)44 (38)45–49 yr55 (31)43 (37)»50 yr32 (18)17 (15)No. of positive nodes0.60 091 (51)60 (52)1–363 (35)43 (37)»426 (14)12 (10)Tumor diameter0.012«20 mm84 (47)71 (62)>20 mm96 (53)44 (38)Histologic grade<0.001 I (good)19 (11)56 (49)II (intermediate)56 (31)45 (39)III (poor)105 (58)14 (12)Vascular invasion0.38 Absent108 (60)77 (67)1–3 Vessels18 (10)12 (10)>3 Vessels54 (30)26 (23)Estrogen-receptor status<0.001 Negative66 (37) 3 (3)Positive114 (63)112 (97)Surgery0.63 Breast-conserving therapy97 (54)64 (56) Mastectomy83 (46)51 (44) Chemotherapy0.79 No114 (63)71 (62)Yes66 (37)44 (38)Hormonal therapy0.63 No157 (87)98 (85)Yes23 (13)17 (15)GENE-EXPRESSION SIGNATURE AS A PREDICTOR OF SURVIVAL IN BREAST CANCERman genes.9 Fluorescence intensities of scanned images were quantified and normalized. We calculated the ra-tio of these values to the intensity of a reference pool made up of equal amounts of cRNA from all tumors.The gene-expression ratios of the previously deter-mined 70 marker genes for all 295 tumors in this study are shown in Figure 1A. The 115 tumors with values above the previously determined threshold 9 were as-signed to the good-prognosis category, and the 180below the threshold were assigned to the poor-prog-nosis category. Figure 1B shows the time to distant metastases as a first event as well as the total duration of follow-up for all patients who did not have distant metastases as a first event. Figure 1C shows lymph-*The patients selected either had had distant metastases as a first event within five years or had remained free of disease for at least five years.†Odds ratios were calculated with use of a two-by-two contingency table. CI denotes confidence interval.‡P values were calculated with use of Fisher’s exact test.§In this analysis, patients who were part of the previous study of gene-expression profiling were excluded from the series of consecutive patients.T ABLE 2. O DDS R ATIO FOR D ISTANT M ETASTASES WITHIN F IVE Y EARS AS A F IRST E VENT ,A CCORDING TO THE P ROGNOSIS S IGNATURE .G ROUP *N O . OF P ATIENTS D ISTANT M ETASTASES WITHIN 5 Y RD ISEASE -FREE>5 Y R O DDS R ATIO (95% CI)†P V ALUE ‡no. of patientsPatients with lymph-node–negative disease <0.001Patients in previous study 7815.0 (3.3–56)Poor-prognosis signature 3118Good-prognosis signature326Consecutive series (new patients only)§6715.3 (1.8–127)0.003Poor-prognosis signature 1123Good-prognosis signature132Patients with lymph-node–positive disease Consecutive series11313.7 (3.1–61)<0.001Poor-prognosis signature 2842Good-prognosis signature241All new patients in the consecutive series 18014.6 (4.3–50)<0.001Poor-prognosis signature 3965Good-prognosis signature373*Distant metastasis was a first event. Plus–minus values are means ±SE.T ABLE 3. R ATE OF O VERALL S URVIVAL AND THE P ROBABILITY T HAT P ATIENTSW OULD R EMAIN F REE OF D ISTANT M ETASTASES AT 5 AND 10 Y EARS ,A CCORDING TO THE P ROGNOSIS S IGNATURE .*G ROUP N O . OF P ATIENTSF REE OF D ISTANT M ETASTASES O VERALL S URVIVAL 5 YR10 YR5 YR10 YRpercentAll patientsGood-prognosis signature 11594.7±2.185.2±4.397.4±1.594.5±2.6Poor-prognosis signature18060.5±3.850.6±4.574.1±3.354.6±4.4Patients with lymph-node–negativediseaseGood-prognosis signature 6093.4±3.286.8±4.896.7±2.396.7±2.3Poor-prognosis signature9156.2±5.544.1±6.371.5±4.849.6±6.1Patients with lymph-node–positivediseaseGood-prognosis signature 5595.2±2.682.7±7.898.2±1.892.0±4.8Poor-prognosis signature8966.3±5.256.7±6.476.5±4.659.5±6.3Figure 2.0.00.20.40.60.81.0120246810YearsAll PatientsP r o b a b i l i t y o f R e m a i n i n g M e t a s t a s i s -f r e eGood signaturePoor signatureP<0.001Good signaturePoor signatureP<0.0010.00.20.40.60.81.0120246810YearsAll PatientsO v e r a l l S u r v i v a lP<0.001120.00.20.40.60.81.0120246810YearsO v e r a l l S u r v i v a lGood signaturePoor signatureP<0.0010.00.20.40.60.81.00246810YearsGood signaturePoor signatureM e t a s t a s i s -f r e eGENE-EXPRESSION SIGNATURE AS A PREDICTOR OF SURVIVAL IN BREAST CANCERP<0.0010.00.20.40.60.81.0120246810YearsLymph-Node–Negative PatientsM e t a s t a s i s -f r e eGood signaturePoor signatureP<0.001Lymph-Node–Negative Patients0.00.20.40.60.81.0120246810YearsO v e r a l l S u r v i v a lGood signaturePoor signature0.00.20.40.60.81.0120246810YearsGene-Expression ProfilingGood signaturePoor signatureP<0.001P=0.050.00.20.40.60.81.0120246810YearsSt. Gallen CriteriaLow riskHigh risk0.00.20.40.60.81.0120246810YearsNIH, High RiskM e t a s t a s i s -f r e eGood signaturePoor signatureP<0.001P<0.001M e t a s t a s i s -f r e eM e t a s t a s i s -f r e eM e t a s t a s i s -f r e e0.00.20.40.60.81.0120246810YearsSt. Gallen, High RiskGood signaturePoor signatureGENE-EXPRESSION SIGNATURE AS A PREDICTOR OF SURVIVAL IN BREAST CANCERP=0.230.00.20.40.60.81.0120246810YearsNIH Consensus CriteriaM e t a s t a s i s -f r e eLow riskHigh riskM e t a s t a s i s -f r e eP=0.050.00.20.40.60.81.0120246810YearsNIH, Low RiskGood signaturePoor signatureM e t a s t a s i s -f r e eGood signaturePoor signature P=0.110.00.20.40.60.81.0120246810YearsSt. Gallen, Low RiskThe prognosis profile was also strongly associated with the outcome in the group of 144 patients with lymph-node–positive disease (Table 3 and Fig. 2E and 2F). In this group, the hazard ratio for distant metastases was 4.5 (95 percent confidence interval, 2.0 to 10.2; P<0.001).Multivariable AnalysisTable 4 shows the results of the multivariable analy-sis of the risk of distant metastases as the first event. The only independent predictive factors were a poor-prognosis signature, a larger diameter of the tumor, and the nonuse of adjuvant chemotherapy. During the period in which these patients were treated, most pre-menopausal patients with lymph-node–positive dis-ease received adjuvant chemotherapy, whereas the ma-jority of patients with lymph-node–negative disease did not receive adjuvant treatment. Patients who re-ceived adjuvant chemotherapy in this series had a high-er likelihood of remaining free of distant metastases (hazard ratio for distant metastases, 0.37; 95 percent confidence interval, 0.20 to 0.66; P<0.001). The poor-prognosis signature was by far the strongest pre-dictor of the likelihood of distant metastases, with an overall hazard ratio of 4.6 (95 percent confidence in-terval, 2.3 to 9.2; P<0.001).DISCUSSIONWe previously identified a gene-expression profile of 70 genes that is associated with the risk of early dis-tant metastases in young patients with lymph-node–negative breast cancer.9 In the present study we tested this profile in a series of 295 consecutive patients who were treated at the hospital of the Netherlands Cancer Institute. The profile performed best as a predictor of the appearance of distant metastases during the first five years after treatment. This finding is not unexpect-ed, since the tumors on which the profile was based had all metastasized within five years. The prognosis profile is also a strong predictor of the development of distant metastases in patients with lymph-node–pos-itive disease. This finding is important, since the pres-ence of lymph-node metastases is by itself a strong predictor of poor survival. Since most patients with lymph-node–positive breast cancer in our study re-ceived adjuvant chemotherapy or hormonal therapy (120 of 144 patients), we could not evaluate the prog-nostic value of the profile in patients with untreated lymph-node–positive disease. There is, however, no indication of an effect of adjuvant chemotherapy on the prognostic value of the profile (data not shown). Figure 3 shows the Kaplan–Meier estimates of the probability that patients would remain free of distant metastases among the 151 patients with lymph-node–negative cancer, according to whether the patients were classified with the use of gene-expression pro-filing (Fig. 3A), the St. Gallen criteria3 (Fig. 3B), or the National Institutes of Health (NIH) consensus criteria4 (Fig. 3C). The St. Gallen and NIH criteria classify patients as at low risk or high risk on the basis of various histologic and clinical characteristics. This comparison shows that the prognosis profile assigned many more patients with lymph-node–negative dis-ease to the low-risk (good-prognosis signature) group than did the traditional methods (40 percent, as com-pared with 15 percent according to the St. Gallen cri-teria and 7 percent according to the NIH criteria). Moreover, low-risk patients identified by gene-expres-sion profiling had a higher likelihood of metastasis-free survival than those classified according to the St. Gallen or NIH criteria, and high-risk patients iden-tified by gene-expression profiling tended to have a higher rate of distant metastases than did the high-risk patients identified by the St. Gallen or NIH criteria. This result indicates that both sets of the currently used criteria misclassify a clinically significant number of patients. Indeed, the high-risk group defined ac-cording to the NIH criteria included many patients who had a good-prognosis signature and a good out-come (Fig. 3E). Conversely, the low-risk group iden-tified by the NIH criteria included patients with a poor-prognosis signature and poor outcome (Fig. 3G). Similar subgroups were identified within the high-risk and low-risk groups identified according to the St. Gallen criteria (Fig. 3D and 3F, respectively). Since both the St. Gallen and the NIH subgroups contain misclassified patients (who can be better identified through the prognosis signature), these patients would be either overtreated or undertreated in current clin-ical practice.Our data indicate that the ability to metastasize to distant sites is an early and inherent genetic property of breast cancer. Our findings argue against the widely accepted idea that metastatic potential is acquired rel-atively late during multistep tumorigenesis.20 If the metastatic ability of breast cancer is determined early in tumorigenesis, early prognostic testing could be un-dertaken, an approach that would clearly be beneficial. On the other hand, an early onset of metastatic capa-bility theoretically limits the benefit of early detection and treatment. Furthermore, our findings suggest that the molecular mechanism leading to hematogenous (distant) metastases is distinct from the mechanism of lymphogenic (regional) spread of tumor cells. Our conclusion that the prognosis profile is independent of lymphogenic metastases is based on its strong pre-dictive power with respect to hematogenous metasta-ses, regardless of the presence or absence of lymph-node involvement.Our data indicate that classification of patients into high-risk and low-risk subgroups on the basis of the prognosis profile may be a useful means of guiding。
NUVAN PROSTRIP产品说明书
INTENDED FOR SALE TO AND USE BY PEST MANAGEMENT PROFESSIONALSONLYHow it works: The NUVAN PROSTRIP utilizes controlled release technology to slowly diffuse a deep penetrating vapor in enclosed spaces for up to 4 months. The clean, odorless vapor is evenly distributed throughout the enclosed treatment area, killing visible and hidden insects on contact and preventing new insect infestations (while you’re away).ACTIVE INGREDIENT:Dichlorvos (2,2-dichlorovinyl dimethyl phosphate)...............................................18.6%INERT INGREDIENTS:.........................................................................................81.4%Total 100.0%This product contains the toxic inert ingredient Bis (2-ethylhexyl) adipate (DEHA) at 19.8%.KEEP OUT OF REACH OF CHILDRENWARNINGSee Back Panel for Additional PrecautionsEPA Reg. No. 5481-553 EPA Est. No. ______________Net Weight: __________4100 E. Washington Blvd.Los Angeles, CA 90023 U.S.A.1-888-462-6822Hazards to Humans and Domestic AnimalsWARNING: May be fatal if swallowed. Do not get in mouth. Harmful if inhaled. Causes moderate eye irritation. Avoid breathing vapors. Avoid contact with eyes or clothing. Wear chemical resistant gloves. After prolonged storage, a small amount of liquid may form on strip. Do not get liquid in eyes. Wash thoroughly with soap and water after handling and before eating, drinking, chewing gum, using tobacco, or using the toilet. Remove and wash contaminated clothing before reuse. Not to be taken internally by humans or animals.FIRST AIDIF SWALLOWED ∙Call a poison control center or doctor immediately for treatment advice.∙Have person sip a glass of water if able to swallow.∙Do not induce vomiting unless told to by a poison control center or doctor. ∙Do not give anything by mouth to an unconscious person.IF INHALED ∙Move person to fresh air.∙If person is not breathing, call 911 or ambulance, then give artificial respiration, preferably mouth-to-mouth, if possible.∙Call a poison control center or doctor for further treatment advice.IF IN EYES ∙Hold eye open and rinse slowly and gently with water for 15-20 minutes.∙Remove contact lenses, if present, after the first 5 minutes, then continue rinsing eye. ∙Call a poison control center or doctor for treatment advice.IF ON SKIN OR CLOTHING ∙Take off contaminated clothing.∙Rinse skin immediately with plenty of water for 15-20 minutes. ∙Call a poison control center or doctor for treatment advice.NOTE TO PHYSICIANProbable mucosal damage may contraindicate the use of gastric lavage. POISONING SYMPTOMS: This product is a cholinesterase inhibitor. Symptoms include weakness, headache, tightness in chest, blurred vision, non-reactive pin point pupils, salivation, sweating, nausea, vomiting, diarrhea, and abdominal cramps. TREATMENT: Atropine is the specific therapeutic antagonist of choice against parasympathetic nervous stimulation. If there are signs of parasympathetic stimulation, atropine sulfate should be injected at 10 minute intervals in doses of 1 to 2 milligrams until complete atropinization has occurred. Morphine is contraindicated. 2-PAM is also antidotal and may be administered in conjunction.Have the product container or label with you when calling a poison control center or doctor, or going for treatment.FOR THE FOLLOWING EMERGENCIES, PHONE 24 HOURS A DAY:For Medical Emergencies phone:...........................................................................................1-888-681-4261 For Transportation Emergencies, including spill, leak or fire, phone: CHEMTREC®………..1-800-424-9300 For Product Use Information phone: AMVAC®……...……................................................…..1-888-462-6822 STOP! READ ENTIRE LABEL BEFORE USEIt is a violation of Federal law to use this product in a manner inconsistent with its labeling. Wearing gloves, remove the insecticide strip from sealed bag when ready to use and place strip in provided holder if treatment area is routinely accessed by people or pets. Do not allow children to handle new or used strips. Place (Hang) in desired location away from windows and out of reach of children and pets. (To apply strips to vertical surfaces, use the enclosed stickers). Drafts, weather and other conditions may affect the performance, but treatment usually lasts for four (4) months. One 16 g strip will treat 100 to 200 cu. ft.Record the date of installation and replace with a new, fresh, full-strength strip at the end of four (4) months or when effectiveness diminishes. Do not use in hospitals or clinic rooms, such as patient rooms, wards, nurseries, operating or emergency areas. Within homes, use only in closets, wardrobes and cupboards. Do not use in closets and wardrobes of rooms where infants, children and the sick or aged are or will be present for any extended period of confinement. Do not use in kitchens (except cupboards), restaurants or areas where food is prepared or served. Do not use in any area of the home where people will be present for extended periods of time. Do not use in the food/feed areas of food/feed processing or food/feed manufacturing or food/feed service establishments. Food/feed areas of these establishments where the product is not permitted for use are areas for receiving, storing, packing (canning, bottling, wrapping, boxing), preparing, edible waste storage and enclosed processing systems(mills, dairies, edible oils, syrups) of food. Serving areas where food is exposed and the facility is in operation are also considered food areas. Non-food/feed areas of these establishments where the product can be used are areas such as garbage rooms, lavatories, floor drains (to sewer), entries and vestibules, offices, locker rooms, machine rooms, boiler rooms, utility equipment, garages, mop closets, and product storage (after canning or bottling). This product controls flies, gnats, mosquitoes, moths, silverfish, cockroaches, spiders, beetles, earwigs and other pests as listed.Only available in the following sizes: 16 grams (0.56 oz.), 10.5 grams (0.37 oz.) and 5.25 grams (0.19 oz.) pest strip sizes.NUVAN PROSTRIP is intended for sale to and use by pest management professionals only. This product is NOT intended for sale to or use by homeowners. Place strips in areas including garages, sheds, attics, crawl spaces, pantries, cupboards, closets, museums, museum collections, animal buildings, barns, storage units, trash bins, grain bins, pest traps, utility boxes, vacation homes, cabins, out-houses, mobile homes, recreational vehicles, boats, farm houses, and ranch houses. Also use strips to control pests around non-perishable packaged, bagged or bulk raw and processed agricultural commodities (including corn, soybeans, grains, cocoa beans, and peanuts). When properly placed, strips provide control of pests for up to four months depending upon local conditions. Replace strip with a fresh strip as needed. See below for more detailed use instructions.Each 16 gram strip treats100 to 200 cubic feet of space. Always use the appropriate number of strips for the space intended to be treated. Place strips uniformly in the area to be treated to facilitate a uniform treatment of the space. Strip provides continuous control of pests in treated areas through controlled release technology.Within homes, use only in closets, wardrobes and cupboards. Also for use in storage units, garages, attics, crawl spaces, boathouses, museum collections, garbage cans, trash dumpsters, animal buildings, milk rooms, catch basins, bulk raw grain, storage bins, enclosed utility boxes, reptile houses and terrariums.HOUSEHOLD, INDUSTRIAL AND COMMERCIAL USESWithin Homes, Use Only in Closets, Wardrobes and CupboardsGarages, Storage Units, Attics, Crawl Spaces and BoathousesNUVAN PROSTRIP kills flies, mosquitoes, gnats and other insects as listed for up to four (4) months. Wearing gloves, remove the insecticide strip from sealed bag when ready to use and place strip in provided holder if treatment area is routinely accessed by people or pets. Hang or place, out of reach of children and pets, in an open space of an enclosed area, away from windows. Do not allow children to handle new or used strips. One 16 gram strip will treat approximately 100 to 200 cubic feet. Replace with a new strip at the end of four (4) months or as needed.Garbage Cans, Trash Dumpsters and Trash ReceptaclesFor control of flying and crawling pests, place the appropriate number of strips within trash receptacles such as compactors, garbage chutes, garbage rooms, garbage bins and others. Strip provides control of pests within trash receptacles for up to four months.Animal BuildingsUse NUVAN PROSTRIP to control pests in animal buildings including stalls, barns, milk rooms, kennels, pens, poultry houses, manure storage and other such areas. Do not contaminate milk. Place strip in manure collection areas such as beneath egg layers and broilers but do not allow strip to be placed in areas directly occupied by poultry. Place the appropriate number of strips for the space to be treated. Strip will control pests for up to four months.Milk RoomsNUVAN PROSTRIP controls flies, mosquitoes and gnats in milk rooms and bulk storage rooms. DO NOT contaminate milk or milking equipment.Agricultural CommoditiesUse NUVAN PROSTRIP to control pests of non-perishable agricultural commodities, grains, bulk foodstuffs, packaged foods and other similar items. Do not contaminate foods. Place the appropriate number of strips for the headspace (volume) to be treated. Strip will control pests for up to four months.Catch BasinsUse NUVAN PROSTRIP to control mosquitoes and other pests in Catch Basins where water accumulates and mosquitoes may breed. Place the appropriate number of Strips for the space to be treated. Place strip by suspending about ten inches above the water surface to control mosquitoes. Strip may control mosquitoes for up to four months.Enclosed Utility Boxes and Utility ChasesUse NUVAN PROSTRIP to control ants (including fire ants), spiders, wasps, hornets and other pests within electric junction boxes, plumbing control boxes, pump houses and other such utility boxes as needed. Place the proper number of strips for the space to be treated. Strip will control pests for up to four months.Reptile Houses and Terrariums Occupied by People for Less than 4 Hours PerDayUse NUVAN PROSTRIP to control mites on reptiles and other pests in reptile enclosures, displays, reptile houses and terrariums. Place strip such that the reptile and desired animals within the enclosure or terrarium will not come in direct contact with the strip. Do not over treat. Strip is NOT intended for treatment of aviaries or birdcages. Place the appropriate number of strips in a suitable place to provide control. Strip must be removed after the 48 hour treatment interval.Museum Collections Occupied by People for Less than 4 Hours Per DayUse NUVAN PROSTRIP to protect museum collections and other similar materials from damaging pests when this is a concern. Place strip in close proximity or within collection boxes using care to avoid contact with collectible and valuable items.For Control of Bedbugs and Bedbug EggsNUVAN PROSTRIP may be used to control crawling bedbug nymphs and adults exposed to product vapors for 48 hours. In difficult to treat areas, a minimum treatment time of 72 hours will provide better results. Strip may be used to control bedbugs that have entered various items in an infested structure including, but not limited to: electronics, appliances, footwear, art work, collectibles, plush toys, clocks, radios, wall hangings, telephones, computers, printers, mattresses, box springs, books, lamps, furniture and other such items. Place infested items in a suitable plastic bag, poly sheeting that is completely sealed by adhesive tape on all sides, container or room that is closed to contain the strip treatment. Plastic bags or poly sheeting should be at least 2 mils thick. Any mattress or box springs to be treated must be fully enclosed with a single strip within a large plastic bag or within poly sheeting with all edges taped closed. Do not sleep or lay on mattress while in treatment bag. The closed volume for treatment should not exceed the volume to be treated for the size of the strip used. Take care to avoid direct contact of the strip with the surface of items being treated. Seal items in the containment for a minimum of 48 hours to kill bedbug nymphs and adults. To kill any bedbug eggs, if suspected to be present, seal items in the treated space for seven days. Seal plastic bags and poly sheeting with as much air space around the treated article as is practical as this will enhance the exposure to the product vapors. Proper seal can be attained by any appropriate manner such as the use of tape, twist ties or other means. Professionals should test for adequate seal by testing for the escape of air from the sealed bag. Identify sealed treatment by a label indicating a pesticide treatment is in process that should not be disturbed by unauthorized persons. The label should include the date, pest professional person or company responsible and contact telephone number. When treatment is completed, remove treated items from the treatment in a well ventilated area and air out for a period of not less than two hours. Discard plastic treatment bag or treatment poly sheeting and used pest strip in trash. Do not allow children to handle plastic treatment bags or treatment poly sheeting or new or used insecticide strips.How much space will one strip treat?Strip Size Treats Approx. Equivalent to Area Measuring16 grams 100 to 200 cu. ft.8' x 8' x 3’ = 192 cu. ft.Always use the appropriate size and number of strips for the space being treated. If more than one is required, distribute them within the space equally.HOUSEHOLD USESWithin Homes, Use Only in Closets, Wardrobes and CupboardsNUVAN PROSTRIP gives continuous protection against moths, silverfish, cockroaches and other insects as listed in closets, wardrobes and cupboards for up to four (4) months and leaves no smell (odor). Wearing gloves, remove the insecticide strip from sealed bag when ready to use and place strip in provided holder if treatment area is routinely accessed by people or pets. Hang or stand on wardrobe or closet rail, ensuring that strip does not contact clothes or fabrics and that air can circulate around the strip. Use one 16 g strip to treat 100 to 200 cu. ft. for up to four (4) months. If more than one is required, distribute them within the space equally. Keep the space closed. Record the date of installation so that the old strip may be replaced with a new, fresh, full-strength strip at the end of the four (4) month period or when effectiveness diminishes.Do not use more than the number of strips recommended for the space to be treated. Do not use in closets and wardrobes of rooms where infants, children and the sick or aged are or will be present for any extended period of confinement. Do not use where unwrapped food is stored or allow the strip to come into contact with food or cooking utensils. Do not allow children or pets to play or sleep in these areas when treatment is in progress. Do not allow children to handle new or used strips.Attics, Garages and Enclosed Crawl SpacesDO NOT USE IN AREAS WHERE PEOPLE WILL BE PRESENT FOR AN EXTENDED PERIOD OF TIME.NUVAN PROSTRIP kills flies and other insects as listed for up to four (4) months. Wearing gloves, remove the insecticide strip from sealed bag when ready to use and place strip in provided holder if treatment area is routinely accessed by people or pets. Do not allow children to handle new or used strips. Hang or stand, out of reach of children and pets, in an open space of an enclosed area, away from windows. Calculate cubic feet of air space to determine the number of strips required for treatment. Hang strips so as not to impede air circulation. When multiple strips are required, distribute them uniformly within the treated space. Record the date of installation so that the old strip may be replaced with a new, fresh, full-strength strip at the end of the four (4) month period or when effectiveness diminishes.STORAGE AND DISPOSALStorage: Do not contaminate water, food or feed by storage or disposal. Do not open or remove from package before ready to install. Store in a cool, dry place.Foil PouchDo not open or remove from package before ready to use. Disposal:If empty: Place in trash or offer for recycling if available. If partly filled: Call your solid waste agency or 1-800-CLEANUP for disposal instructions. Never place unused product down any indoor or outdoor drain.Outer ContainerNonrefillable container. Do not reuse or refill this container. Offer for recycling if available or dispose of the empty container in trash.The manufacturer warrants (a) that this product conforms to the chemical description on the label; (b) that this product is reasonably fit for the purposes set forth in the directions for use, subject to the inherent risks referred to herein, when it is used in accordance with such directions; and (c) that the directions, warnings, and other statements on this label are based upon responsible experts' evaluations of reasonable tests of effectiveness, of toxicity to laboratory animals and to plants and residues on food crops, and upon reports of field experience. Tests have not been made on all varieties of food crops and plants, or in all states or under all conditions.THERE ARE NO EXPRESS WARRANTIES OTHER THAN THOSE SET FORTH HEREIN. TO THE EXTENT CONSISTENT WITH APPLICABLE LAW THE MANUFACTURER NEITHER MAKES NOR INTENDS, NOR DOES IT AUTHORIZE ANY AGENT OR REPRESENTATIVE, TO MAKE ANY OTHER WARRANTIES, EXPRESS OR IMPLIED, AND IT EXPRESSLY EXCLUDES AND DISCLAIMS ALL IMPLIED WARRANTIES OF MERCHANTABILITY OF FITNESS FOR A PARTICULAR PURPOSE, OR ANY WARRANTY OF QUALITY OR PERFORMANCE. THIS WARRANTY DOES NOT EXTEND TO, AND THE BUYER SHALL BE SOLELY RESPONSIBLE FOR, ANY AND ALL LOSS OR DAMAGEWHICH RESULTS FROM THE USE OF THIS PRODUCT IN ANY MANNER WHICH IS INCONSISTENT WITH THE LABEL DIRECTIONS, WARNINGS OR CAUTIONS.TO THE EXTENT CONSISTENT WITH APPLICABLE LAW BUYER'S EXCLUSIVE REMEDY AND MANUFACTURER'S OR SELLER'S EXCLUSIVE LIABILITY FOR ANY AND ALL CLAIMS, LOSSES, DAMAGES, OR INJURIES RESULTING FROM THE USE OR HANDLING OF THIS PRODUCT, WHETHER OR NOT BASED IN CONTRACT, NEGLIGENCE, STRICT LIABILITY IN TORT OR OTHERWISE, SHALL BE LIMITED, AT THE MANUFACTURER'S OPTION, TO REPLACEMENT OF, OR THE REPAYMENT OF THE PURCHASE PRICE FOR, THE QUANTITY OF PRODUCT WITH RESPECT TO WHICH DAMAGES ARE CLAIMED. TO THE EXTENT CONSISTENT WITH APPLICABLE LAW MANUFACTURER OR SELLER SHALL NOT BE LIABLE FOR SPECIAL, INDIRECT OR CONSEQUENTIAL DAMAGES RESULTING FROM THE USE OR HANDLING OF THIS PRODUCT.AMVAC offers this product, and Buyer accepts it, subject to the foregoing Limited Warranty which may be varied only by agreement in writing signed by an authorized representative of AMVAC.©2014 AMVAC Chemical Corporation. All rights reserved. AMVAC, NUVAN, PROSTRIPS and the BEAKER logo are U.S. registered trademarks of AMVAC Chemical Corporation. Chemtrec is a trademark of the American Chemistry Council, Inc.Proposition 65: Warning: This product contains a chemical known to the State of California to cause cancer.AMVAC Chemical Corporation4100 E. Washington BoulevardLos Angeles, CA 90023 U.S.A.1-888-462-6822。
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Decarburized material
The variations in the carburizing atmosphere, contaminants on the sample or wrong handling can cause low carbon contents on the surface. Example
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Austenitizing temperature defects
If the austenitizing temperature is too low, the microstructure and the hardness do not develop as desired. The Rollscan readings are high. With the Rollscan method the product quality can be improved by optimizing the austenitizing process parameters
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Thickness of nitrided layer
In the calibration sample the Rollscan reading (’s output) correlated well with the thickness of the nitrided layer
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The Rollscan reading indicates the position of the hardening area of the crankshaft main surface.
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Unhardened edge
Example
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Hardness changes
Soft material gives high Rollscan readings
Rollscan readings of partially hardened steel sleeve
VH 2002-09-10
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Missed hardening process
Unhardened parts, e.g. due to the breakdown in the process, can be detected with the Rollscan method
Plot 8 shows the Rollscan reading of an unhardened part
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Location of induction hardening
High Rollscan readings indicate soft material The Rollscan method
can be used to determine the location of the induction hardening coil and the quality of the hardened area. gives feedback of the process in a few seconds suitable for 100 % inspection
100 90 80 70 60 50 40 30 20 10 Lobe angular position 0 0 50 100 150 200 250 300 350 Lobe 1; "Accepted" Barkhausen Noise Amplitude
Lobe 2 Lobe 5
VH 2002-09-10
Effect of soft edge on Rollscan reading (B-Noise) of induction hardened camshaft lobe shown. The edges, which were left soft due to the misalignment of coils in induction hardening, generated about ten times higher signals than the properly hardened valve lobes.
Properly carburized areas give low Rollscan readings (Noise, MP)
VH 2002-09-10
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Microstructure changes
High Rollscan readings on the camshaft lobe show the martensite with Ferrite. Ferrite has soft microstructure so the soft material gives high Rollscan readings (Barkhausen Noise Amplitude).
VH 2002-09-10
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Soft spots
Variations of the carburizing atmosphere, problems with the induction coil or cooling, contaminants on the sample or wrong handling can cause low carbon contents on the surface. Affect structure and cause hardness problems on the sample surface.
VH 2002-09-10
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Low case depth
Example below:
a) Low Rollscan readings correspond to 1 mm case depth b) Increased Barkhausen readings correspond to too low case depth of 0,5 mm The Rollscan method can be used to guarantee the product quality
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Rollscan Method
The Advanced Quality Control Technique based on Barkhausen Noise Analysis
Heat treatment defects
STRESSTECH OY Finland AMERICAN STRESS TECHNOLOGIES, INC. USA STRESSTECH GmbH Germany