CN107205980B - 具有获自虞美人的新型分子的组织和细胞染色剂制剂 - Google Patents
具有获自虞美人的新型分子的组织和细胞染色剂制剂 Download PDFInfo
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Abstract
配制了一种含有新的生物类黄酮的来自虞美人的细胞和组织染色剂,其特异性染色细胞核,用于组织病理学、微生物学和细胞学中的微观评估。它看似是常规使用的苏木精的替代品。此化合物的生化名称为:四氢‑3,4,5‑三羟基‑6‑甲基‑2H‑吡喃‑2‑基氧基)甲基)2H‑吡喃‑2,3,4,5‑四醇)‑4‑甲氧基苯基)‑7‑甲氧基‑4H‑色烯‑4‑酮。NMR分析显示该分子的生化结构是生物类黄酮(图4,5)。虞美人中的分子以及协同分子和其他分子渗入生物和非生物样品中。结合其他协同机制,制备染色剂制剂。媒染剂和pH的量和类型是影响染色结果的品质和时间的参数。制备虞美人制剂特别用于检查实验室和医院的诊断用组织,并且旨在常规使用。全球范围内评估了数百万个用于外科手术准备的生物样品,并对生物样品染色用于诊断。(苏木精组织染料是常规使用的,由被称为苏木精(洋苏木)的树木生产,其是世界热带雨林成员。每年有3000棵洋苏木树木和林场被破坏用于生产,约1200吨粉末和70,000吨液体苏木精染料(150亿美元/年)。来自虞美人的制剂提供突出核的有效和特异性的染色。除了明显的经济贡献之外,虞美人广泛地生长并且可以容易地收获和生产。虞美人制剂也对地球有生态学影响,这将成为重要和有价值的工业产品。
Description
技术领域
虞美人染色剂包含一种新的生物类黄酮以及衍生物和协同分子。用于诊断目的的新的细胞核染色剂由虞美人提取物配制,其为“细胞和组织”的细胞核提供特异性染色。本发明涉及一种染色剂,其是“苏木精”常规染色剂的替代制剂,并且在卫生部门诊断和经济贡献方面具有益处。
概述
虞美人制剂主要用作细胞核染色剂,其含有新的分子,每年可为经济贡献100亿~150亿美元,其是苏木精的替代品。该来源将为保护世界濒危热带雨林平衡区域生态系统作出贡献,并且作为工业产品是有用的(Anatech LTD."histology chemicals,'boilinghematoxylin market"″2008年夏INNOVATOR.)。
虞美人制剂是常规诊断中使用的苏木精常规染色剂的合理替代品。对苏木精需求的增加是由于人口数量的增加和诊断性治疗医疗程序的增加。每克苏木精的销售价格非常高,并且其生产使世界热带雨林面临风险,并对生态系统造成风险。
本文中的虞美人(Papaver Rhoeas)是发现和配制的新的细胞-组织染料来源。虞美人分子含有非常活跃和功能性的生物类黄酮分子,并与曙红结合。分子本身以及衍生物和协同分子在组织和其他来源(如来自微生物样品的真菌)中提供了清晰的细胞核染色,以允许精细的染色细节,并可应用于出于诊断目的的组织病理学、细胞学。颜色的广谱性及诊断中特征的照亮和细化提供了新的诊断参数和发现。
医院、实验室和制造商、医疗设备和材料中的染色方法与传统技术相似,且没有大量的额外投资。所述分子可用作免疫系统调节剂、T细胞活化剂、抗病毒剂、组织保护和再生以及指示剂及用于新维生素复合物的合成。其还可以用作葡萄酒行业中的染料(仍然是用来对食品进行着色的惰性染料),在军装、特别用于婴儿衣服和玩具的对环境友好的纺织品中作为着色剂,作为皮肤恢复和光滑剂,并且可以用作斑点处理和抗皱剂。化妆品产品用于手术假体和外科缝合线的染色。还可用于生化诊断测试中。特别是在工业等离子电视、监控屏幕和纳米技术中用于生产,以及在传感器技术中用于纳米粒子合成,也可用于生产盛放染料的打印机墨盒。同样,免疫组化或与其他数字图像技术相结合将被用于诊断目的。
背景技术
虞美人在自然界中天然生长。其物种名称为Papaver rhoeas L,科学谱系名称为罂粟科(Papaveraceae)。其他名称为红罂粟、玉米罂粟,法兰德斯罂粟、雪莉罂粟、罗比尼罂粟(Papaver Roubini)、细小罂粟(papaver tenuissima)、tumigil罂粟、三裂叶罂粟(Papaver trilobum)、直毛罂粟(Papaver strigosum),为相同的物种(1-6)。虞美人通常在各种尺寸的2个和4个大毛萼片的花瓣上有一个黑点,并具有红色的叶子。子房位于中心,并被黑色区域包围。花粉量取决于果实的大小,每个果实含有大于200个种子。
虞美人生长在阳光充足的气候中,在沙质土壤中生长最好。虞美人的花瓣具有化学发色,并产生涉及罂粟酸的反应。它具有润肤剂的舒缓效果、月经调节、祛痰、催眠、轻微麻醉和镇静作用(7-9)。可用于治疗发热、支气管炎症状和咳嗽、失眠、消化系统疾病和疼痛(10-12)。也可用于神经系统症状,如多动症和失眠。还显示其对儿童是安全的(13-15)。
类黄酮具有酚类结构,其天然存在于水果、蔬菜、谷物,树皮、树根中,并也发现于花、茶和葡萄酒中(16)。已发现超过5000个品种的类黄酮,而其特定效果却显示得很少。生物类黄酮花是果实颜色的原因(17-20)。摄入此类食物可减少心血管疾病,降低死亡率(21,22)。作为一个实例,在蔬菜中也发现这些衍生物,并且仍然显示出其作为维生素(如维生素P)的特征(常规)(23)。20世纪50年代发现,类黄酮增加毛细血管壁(24)的血管通透性。类黄酮主要分为4类(25,26),(27)其又可分为若干亚类。各类类黄酮的分子结构特征均已显示。
黄酮在中心的芳族环上具有双键,具有平面结构。类黄酮包括2-苯基苯并吡喃芳香杂环。生物类黄酮赋予植物颜色,也对类黄酮植物生长和发育起重要作用。用蘑菇生物类黄酮产生UV-B射线的天然屏障有助于防止机会性真菌生长。显示了出抗微生物和抗寄生虫的作用。可以用微生物产生微生物类黄酮分子的遗传变化。但在技术上这些是昂贵的方法(28)。
根据IUPAC 4.5,类黄酮分为异类黄酮和新类黄酮两种。这些是由异黄酮结构衍生出的3-苯基色烯-4-酮(3-苯基-1,4-苯并吡喃)和由生物类黄酮衍生出的苯基香豆满(4-苯基-1,2-苯并吡喃)。
根据是否含有酮及其黄酮和黄酮醇来命名生物类黄酮的化合物。与在非多羟基酮中不同,类黄酮术语也宽泛地用于描述多酚类化合物。类黄酮具有三个环,且杂环结构具有显示出整体三环结构间苯三酚模型的构型。异类黄酮分子可以通过遗传工程产生(28)。
虞美人、花瓣和蒴果产生某些生物碱。当与酸一起被加热时,这些生物碱被转化为称为卟啉的活性络合物。因未知反应,络合物生物碱呈红色。Klayman称其为“红色着色原理”(29-33),并且颜色的发展与反应的pH有关(33-35)。
作为对抗反应性氧自由基的抗氧化剂,生物类黄酮通过防止脂质过氧化(36-38)加速反应来控制氧损伤(39,40),其通过超氧化物歧化酶、过氧化氢酶、和谷胱甘肽过氧化物酶、抗坏血酸和α-生育酚作为还原剂来降低内部氧化活性氧(41)。
某些类黄酮将反应性氧衍生物转化为失活的过氧亚硝酸根(42)。许多类黄酮可以与内皮细胞和巨噬细胞相互作用,在所述细胞中减少一氧化氮、一氧化氮合酶活性(43),从而与极其有害的诱导氧自由基产生的过氧亚硝酸盐反应。
类黄酮作为抗氧化剂起作用,也是由于其消除残留的自由基,因此造成较少的损伤,也可与一氧化氮(44)反应,一氧化氮分子(45)被类黄酮抑制。
类黄酮防止氧化损伤(46,47),导致黄嘌呤氧化酶活性的抑制(48),并能减少白细胞的炎症(49)。某些类黄酮超氧化物的产生(50)抑制中性粒细胞的脱颗粒而无任何影响。肥大细胞脱颗粒导向膜内,Ca2+受体对通道的控制有影响(51,52)。
类黄酮具有铁结合性和铁稳定性质,其也可抑制脂质过氧化(53-56)。某些类黄酮(57-59)减少炎性细胞,因为嗜中性粒细胞粘附会产生最佳的炎症反应。然而,一般来说,可以减少补体激活。类黄酮减少过氧化物酶的释放。通过这种还原,Α1-抗胰蛋白酶活化,抑制反应性氧自由基的产生。蛋白水解酶将显示花生四烯酸对酶系统(60-62)的进行性失活作用,并产生抗炎和抗血栓形成性质(63)。
对类黄酮的平均每日摄入研究有限。例如,维生素C的消耗量是类黄酮摄入量的三倍以上(64)。各国之间类黄酮消耗量差异很大(65,66)。用队列研究来确定类黄酮的食物摄入是困难的。与人体中类黄酮的代谢吸收和排泄相关的研究也是有限的(67-72)。糖基化形式的吸收大于糖苷配基形式。缀合发生在肠和肝脏中(73)。
类黄酮的代谢起始于肠细胞,通过在转运到肝脏后结合白蛋白而形成葡糖苷酸的缀合,硫酸根基团、加入甲基或两者的类黄酮缀合得以完成。活性缀合的生物类黄酮越高,地中海地区的死亡率和心血管疾病的概率越低。
生物类黄酮的所谓毒性作用尚未得到很好的研究(74-79)。有关均匀诱变性的讨论很多(80-82)。然而,对人类的其他长期研究表明,确定的致癌副作用的概率很低。类黄酮对癌细胞或永生化细胞有毒性,但这些分子对正常细胞的毒性较小(83-85)。体外研究显示类黄酮的抗过敏性、抗氧化性、抗微生物性、抗细菌性、抗真菌性以及某些其他生物和药理活性已被证明具有抗病毒作用。
Wang等(87)在单纯性疱疹病毒、HIV以及呼吸道合胞病毒、副流感病毒、和腺病毒的研究中显示了类黄酮的抗病毒活性。在病毒复制周期的不同阶段,类黄酮的作用可能不同(88)。类黄酮糖苷配基(89)的形式对轮状病毒有抑制作用。存在对作为逆转录酶或DNA引导的RNA聚合酶(90)的抗HIV剂的抑制活性的研究。先前确定的类黄酮在治疗感染HIV的患者中的有益显著治疗贡献并不确定(91)。,在对白血病(MLL)基因的体外研究中,认为类黄酮通过抑制拓扑异构酶而在DNA复制突变的发展中起重要作用。通过抗炎作用可见环氧合酶和脂氧合酶。膜酪氨酸激酶(92,93)是类黄酮抑制各种免疫反应的性质所在。
内皮血管生成受多种发展反应的调控。类黄酮的摄入与肺癌和黑色素瘤生长速度呈负相关。但是,类黄酮抗血管生成作用后面的机制尚不清楚。可能的机制是蛋白激酶(94-104)可能被抑制。
在对规律性地从食用食物摄入类黄酮的男性的临床研究中,似乎可降低冠心病死亡的风险。此外,提出摄入类黄酮可防止痴呆(105,106)的发展。据报道,总血浆胆固醇浓度与氧化应激和血管损伤的存在呈负相关。类黄酮的摄入降低了痴呆的风险。改变血管炎症机制,动脉血压和高血压受到高度的调控。血管抑制细胞内氧化应激相关的信号通路,并且通过增加毛细血管内皮功能,降低动脉粥样硬化的风险。通过抑制血小板聚集,抑制了凝块形成和血栓形成。类黄酮具有(a)直接的抗细菌活性,(b)与具有活性的抗生素的协同作用,和(c)具有抑制细菌毒力因子的相同效果。
此外,过程不足和氧化细胞损伤的作用有限,目的在于测量力极限量效果的体内测量值。需要开发分析技术,以确保收集更多关于吸收和排泄的数据。根据现今的信息,推荐从水果、蔬菜和饮料(如茶和红酒)摄入类黄酮(107-112)。
在用于诊断、教学和研究目的的组织病理学和细胞学中,对固定的活组织和细胞样品进行染色,以通过显微镜检查和评估来鉴定特定的病理特征,从而确定组织和细胞中的诊断性特征。首先放置组织或细胞样品的载玻片用于显微镜评估。这些样品在用虞美人制剂染色前是无色、半透明或透明的。新制剂强力渗透组织层,到达细胞膜,然后到达细胞核。由本文的制剂提供的染色是着色方法,并提供适当的显微镜评估,以了解显示结构所需信息之间的关系特征,并且必须确保适当的必要着色来在组织和细胞中再次区分结构。
附图说明
图1:虞美人组织和细胞染色制剂分析方法;
图2:虞美人制剂的制备阶段;
图3:虞美人前体的制备技术;
图4:虞美人生物类黄酮的顺-反化学结构;
图5:分子的名称和生化构型;
图6:虞美人制剂的染色模式;
图7:虞美人制剂染色方法的阶段;
图8:染色变化和差异化脱色(differentiation);
图9:基于染色模式的虞美人细胞和组织染色剂类型;
图10:虞美人制剂制备的实例;
图11:虞美人制剂特别突出肺组织中的细胞核;
图12:虞美人制剂特别突出肺组织中的细胞核;
图13:具有曙红的虞美人制剂在肺组织中显露出细胞核和组织细节;
图14:具有曙红的虞美人制剂在肺组织中显露出细胞核和组织细节;
图15:虞美人制剂染色真菌的结构细节;
图16:虞美人花瓣内的比例螺旋管状结构;
图17:原子力显微镜显示虞美人花瓣内的池(cisternal)结构;
图18:对虞美人分子的NMR分析。
术语和定义
I.本发明涉及生物样品中细胞核的染色剂,描述了新的细胞组织染料(虞美人细胞纹理染料)。发现此染料含有一种新的活性生物类黄酮。
II.术语用于更好地描述本发明的实践并且方便对其进行解释。
III.如果有任何新的特定术语,该新术语的性质将被解释并详细阐述。
IV.定义可以表示为单数或复数。值不限于示例值。
V.“衍生物”涉及分子的种类数,可以是1、2、3种,该种类数可以减少到4以下,或者将其增加到特定的实施方式。
VI.“协同分子”是指1、2、3种分子,并且对于本发明的具体目的该数目可以增加到4以上。
VII.通常,在染色方案中使用自来水作为漂洗溶剂是非常经济的。实际上如本文使用的术语“水”包括诸如蒸馏水或去离子水的应用以及指定的自来水。
VIII.“生物样品”可以是原核生物、古生菌或真核生物(例如昆虫、原生动物、鸟、鱼、爬行动物)。或者为哺乳动物(例如,大鼠、小鼠、牛、狗、驴、豚鼠或兔)或灵长类动物(例如,黑猩猩或人)。可以在体内或体外获得生物组织或液体。所述样品包括体液(如血液、血浆、血清或尿液)、细胞、细胞器、分离的器官、生物样本。但组织和组分可能包含其全部或部分。还从生物样品的一部分提取生物样品。生物样品蛋白质可以包含碳水化合物和核酸。
IX.用于生物样品、组织和生物液体的细胞学不限于取样来评估本发明。体腔清洗、洗眼液、皮肤屏障、颊唾液腺、血液和阴道腺体、骨髓、尿液,预喷乳头抽吸、精液、乳、痰、粘液、胸腔积液、盆腔液、滑液、腹水液、巴氏涂片(pap smear)、直肠拭子、抽吸、针刺活检、手术或尸检液、组织标本、血浆、血清、脊髓液、淋巴液、汗液、眼泪、痰、唾液、肿瘤、液体、器官和体外细胞系和组织培养物,供细胞学采样。生物细胞和组织样品可以在固定包埋在石蜡中后用于染色过程。将该制备物放在保护玻璃上进行检查。样品可用于对真菌、寄生虫和微生物进行着色。生物样品的染色步骤中的制备技术差异不构成对本发明的任何限制。
X.使用诸如水或化学溶剂等液体在溶剂中在室温下储存染料溶液。水(约50体积%以上)含有:包含一种或多种低级链烷醇的液体或多元醇和水。根据修饰而不同。可提及的可使用的溶剂为乙醇、蒸馏水、甲醇、活水、乙二醇、丙二醇、甲醇和甲醛。
XI.该术语描述了具有高级氧化物的分子的抗氧化能力,并且该分子处于防止其他分子氧化的相同环境中。
XII.可以添加:OH基、烷基(甲基、乙基,含有n为1~5个碳原子的烷基)、-丙基、(异丙基、正丁基、丁基、叔丁基、正-基、异戊基、新戊基或戊基)和低级链烷醇分子(链烷醇是甲醇、乙醇和异丙醇)。
XIII.该术语是指具有比其他分子更强的还原电位的次级氧化剂分子。不同的化学氧化剂可以用于不同的制剂。氧化实例有碘盐和碘酸钾、碘酸钠、氧化锌、高锰酸盐或高锰酸钾、高碘酸钠、高碘酸钾盐和过氧化物、过氧化氢、高碘酸盐、次氯酸钙、氯醛甲酰胺、氯石灰、碘酸钠、氧化锌、碘、次氯酸钠、盐酸盐和氢氧化钡。
XIV.媒染剂(mordant)是能够结合染料分子以形成阳离子络合的金属。新分子可以检测细胞、髓鞘、弹性蛋白和肌肉条纹的胶原纤维和线粒体内的DNA,也与分子的协同作用相关。如下列出媒染剂的实例:硫酸铝、硫酸铝钾、硫酸铝铵、氯化铝、铁、钨、锆、铋、钼磷钼酸或钼酸、钒(钒酸盐)铝或明矾铵、硫酸铝、明矾钾、乙酸铝、氯化钙、硝酸铝、铁、硫酸铵、硫酸亚铁、亚铁氰化钾、铁氰化钾、氯化铁、乙酸铜、明矾铁、铝、硝酸铋、钼酸和磷钼酸。不同的金属媒染剂会产生不同的有色部分。例如;铝:紫-蓝,铁:蓝-黑,铬:蓝-黑,铜:蓝-绿,镍:紫,锡:红,铅:深棕;锇:绿棕。
XV.通过向生物样品的细胞中加入酸来制备染色剂制剂混合物,其细胞核染色特异性增加。出于此目的,可以使用乙酸、水杨酸、柠檬酸、盐酸、硫酸、饱和乙酸甲酸、抗坏血酸等。
XVI.术语抗氧化剂用于感官稳定化,其有助于保持染料氧化的最佳持续时间并延长保质期。要求的稳定剂为甘油、水合氯醛、二乙二醇、碘化钾、乙基二醇。
XVII.“分子”是可以通过各种附着点连接到其他分子并形成复合结构的有机分子。新组合物和分子结构可以与染料分子组合物组合而特异性地形成多样性。附着于这些分子的不同分子结构和合成结构和不同的性质可以形成衍生物。所考虑的这些多糖的某些实例是:直链淀粉或环糊精,和许多醛糖环、单糖、葡萄糖、果糖和半乳糖、二糖(蔗糖)、带有穴醚的其他结合性分子(例如麦芽糖和乳糖)、树状聚合物、纳米管、缬氨霉素和尼日利亚菌素。
XVIII.在本发明公开的整个说明书和权利要求中,该术语用于特定语言表达。术语“约”指指定的值不限于被该术语修饰的确切值。除非另有说明,如本文所用的分子量、反应条件和成分,例如权利要求中的所有表示特征的量的数字,应当理解的是,在每种情况下都可以用该术语修饰。因此,除非有相反的指示,可以在以下描述中具体化,其中记载了至少由所需性质及其数值参数所指定的每个数字参数的有效数字的数量。
XIX.术语“烷基”是指支化的或直链的烷基(例如甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基等),是指饱和的脂肪族基团,其包括链烷基(异丙基、叔丁基、异丁基等),直链或支化链烷基,主干具有6个以下碳原子(例如,C1-C6,C3-C6支链、直链),主干具有4个以下碳原子(例如,C1-C4直链或C3-C4支链)。“C1-C6”烷基是指含有1-6个碳原子的烷基。如本文所用,术语“C1-C4”烷基是指含有1-4个碳原子的烷基。此外,术语烷基包括“未经取代的烷基”和“经取代的烷基”,后者的经取代的烃主干是指具有一个或多个碳上的氢被取代基替换的烷基。这些取代基例如包括(C1-C4)烷基、(C1-C4)烷氧基、氨基(包括C1-C4烷基氨基和(C1-C4)二烷基氨基)、环烷基和芳基(包括苯基、萘基)、羟基、氰基、卤素或硝基。芳基烷基和环烷基取代基也如上所述。
XX.术语“烷氧基”是指共价连接有氧原子的经取代和未经取代的烷基、烯基和炔基。烷氧基的实例包括甲氧基、乙氧基、异丙氧基、丙氧基、丁氧基、戊氧基,但不限于此。在某些实施方式中,其是具有四个以下碳原子的直链或支链烷氧基,支链骨架(例如,C1-C4直链,C3-C4支链)。如本文使用的,“C1-C4”烷基是指含有1-4个碳原子的烷基。
XXI.如本文使用的,“胺”或“氨基”是指其中至少一个碳改变或氮原子与杂原子共价结合的结构。烷基可以在主干中具有4个以下碳原子(例如,C1-C4直链,C3-C4支链),(C1-C4)烷基氨基,该基团或化合物意味着氮至少键合于一个另外的C1-C4烷基。(C1-C4)二烷基氨基,该基团或化合物意味着氮至少键合于两个另外的C1-C4烷基。
XXII.术语“芳基”例如包括苯、苯基、吡咯、呋喃、噻吩、噻唑、含有的0~4个杂原子的基团(例如,指5元和6元单环芳基)、异噻唑、咪唑、三唑、四唑、吡唑、噁唑、异噁唑、吡啶、吡嗪、哒嗪和嘧啶等。此外,“芳基”指多环芳基(例如三环、双环),例如萘、苯并噁唑、苯并二噁唑、苯并噻唑、苯并咪唑、苯并噻吩、亚甲二氧基苯基、喹啉、异喹啉,这些基团还包括吲哚、苯并呋喃、嘌呤、苯并呋喃,包括脱氮嘌呤结构或在吲嗪中具有杂原子。这些芳基,“芳基杂环”,“杂芳基”或“杂芳香基”也可以表示如下。如上所述的芳族环,例如在一个或多个环位置具有这种取代基:(C1-C4)烷基、(C1-C4)烷氧基、氨基(包括(C1-C4)烷基氨基和(C1-C4)二烷基氨基)、羟基、氰基、卤素或硝基。芳基也可以稠合,多环(例如,四氢化萘)可产生足够的非芳族脂环族或杂芳族。例如包括Lazarus杂环,其包括环氧乙烷、二硫杂环丁烷、吡咯啉、吡咯、呋喃、二氢呋喃、二氢噻吩、噻吩、吡唑、咪唑、噁唑、噻唑、异噻唑,12,2,3-三唑、1,2,4三唑、二噻唑、四唑、吡啶、吡喃、嘧啶、吡喃、噻喃、二嗪、噻嗪、二噁英、三嗪和并四苯,包括不饱和环状化合物。
XXIII.术语“抗体”特异性结合特定的空间和极性,其进一步指定义为与构造互补的免疫球蛋白分子。抗体可以是单克隆或多克隆的,并且通过收集分泌蛋白(单克隆)的连续杂交细胞或通过收集宿主血清(多克隆)形成获得。抗体是完整的免疫球蛋白,或者可以包含其片段,例如IgA、IgD、IgE、IgG1、IgG2a、IgG2b和IgG3,IgM类别和同种型,功能性抗体片段包括,可以包含能够以与全长相似的亲合力保持结合的部分(例如,Fab、Fv和F(ab')2或Fab')。
XXIV.术语结合物是指能够结合生物样品中的一种或多种靶标的分子。结合物可以以独特的方式连接到目标。适合的结合物包括天然或修饰的肽、蛋白质(例如,抗体,其本身或适体)、核酸(例如,多核苷酸、DNA、RNA或适体)、多糖(例如凝集素、糖)、脂质、酶、酶底物或抑制剂、配体、受体、抗原或半抗原。可根据当前靶标进行选择和确定用于待分析样品的适合的结合物。
XXV.可以根据当前靶标来选择和确定用于待分析样品的适合的结合物。例如,靶标可以包括样品中的配体且结合物可以包括受体,或者靶标可以包括受体且结合物可以包括配体。类似地,靶标可以包括抗原和抗体或抗体结合片段,或者反之,或可以包括抗体。在某些实施方式中,靶标可以包括核酸,并且结合物可以包括互补核酸。在某些实施方式中,靶标和结合物均可以包括能够彼此结合的蛋白质。
XXVI.新的功能和分子产物的任何改变将不会限制本发明,并且被认为是本发明的可能内容。
具体实施方式
本发明描述了获自虞美人提取物的新的细胞核染色剂。这种新的组织学染料含有非常活跃和功能性的生物类黄酮。虞美人制剂作为新的核染色剂显示出细胞和组织样品的特征,表明了在组织病理学、细胞学和微生物学中应用的独特模式的生物学特征(图11,12)。
评估细胞核特征的目的在于病理学家的诊断,特别是检测恶性或转移性细胞,然而,诊断有助于从正常发现中分离出病理学发现,特别是术后手术样本和活检、细针活检、涂片、洗涤液,以及侵入性或非侵入性方法样品,并将用于检查尸检制备物。
虞美人在自然界中自发生长,并且易于产生来维持染色,以产生组织细胞核制剂。染色剂制剂包含突显细胞核的分子。分析该分子并用于其他实验室研究,如此处示出和阐明的。
掺入媒染剂(金属)以提供足够和稳定的染色,并加入酸。虞美人制剂显示具有清晰细胞形态和简洁的永久染色性。特别是当其与曙红染色剂结合时,虞美人制剂对组织和细胞、组织层、膜、肌肉组织、核内或胞质内结构进行染色,并以精细的细节着色(图13,14)。
长时间储存的染料溶液被自然氧化或化学氧化(113,114)。氧化剂的最佳效率和用量将防止产物形成过度氧化(115,116)。
染料浓度的波动干扰染色的稳定性(117)。此时应该应用必须被校正的染色剂来代替。目视评估染色剂的强度。训练有素的专家对染色进行评估并调节染色强度,直到达到理想的染色品质。
一般来说,类黄酮含有两个芳香环。这些环中的每一个形成至少一个羟基和具有三碳桥的6-杂环构造。根据特征或官能团的氧化态以及杂环结构和结合部分,由杂环与结合的芳族环形成类黄酮亚类。
该分子使用结合物以连接到不同基团和原子,所述不同基团和原子为天然或修饰的肽、蛋白质(例如抗体或适体)、核酸(例如多核苷酸、DNA、RNA或适体)、多糖(例如凝集素、糖)、脂质、酶、酶底物或抑制剂、配体、受体、抗原或半抗原。类似地,连接的靶标基团可以是抗原和抗体或抗体片段。此制剂中的虞美人靶向核酸。
对虞美人进行了微观、分子和原子力显微镜检查(图1),已针对过滤的提取物溶液的分子分析进行了调整,其通过在热乙醇中半速温育来干燥。将干燥样品研磨三次,然后放入甲醇浴中1小时。再次,将液体部分在30℃~60℃的培养箱中蒸发并干燥过夜。将1g所得粉末溶解在100cc乙醇中并吸附在纤维板上。具有1%铵溶液的粉红色板上特异性观察到淡蓝色。将该蓝染料溶于乙酸。除去液体部分。然后处理剩余的干燥粉末进行分析(图2,3)。
分子分析显示了生物类黄酮的特征和(图18)结构生化名称:5-羟基-2-(3-(四氢-6-((四氢-3,4,5-三羟基-6-甲基-2H-吡喃-2-基氧基)甲基)-2H-吡喃-2,3,4,5-四醇)-4-甲氧基苯基)-7-甲氧基-4H-色烯-4-酮。
它具有生物类黄酮的特征性生化构造(图4,5)。
虞美人制剂和曙红(118)可以组合使用。分子及其衍生物协同生物类黄酮和新分子染色组织层、膜、肌肉细胞,详细显示了胞质内和核内结构以及粘蛋白和神经胶质纤维。
染料制剂可以被修饰以利用掺有媒染剂、溶剂、酸、氧化剂或氧化剂和防腐剂的制剂(119)。
酸通常用于调节溶液的pH值,并能保持更耐用的染色剂制剂。并且更有选择地使细胞着色并防止过量的氧化物。因此酸防止沉淀物的形成。可以选择自动或手动染色方法。
稳定添加剂防止超快氧化,基本上通过优化提供更长的保质期。出于此目的,使用直链淀粉、环糊精、穴醚、大环内酯(cryptophycin)、穴状配体(Kavitand)、冠醚、杯芳烃、缬氨霉素、环糊精或尼日利亚菌素。
液态溶剂可溶于水中,加入其他抗氧化剂。例如,加入没食子酸正丙酯、没食子酸正辛酯和没食子酸正十二烷基酯、没食子酸正烷基酯;诸如山梨糖醇和甘露醇等可还原糖;苯甲酸酯和羟基苯甲酸酯;亚硫酸盐和偏亚硫酸氢盐;柠檬酸、酒石酸、乳酸、异抗坏血酸、抗坏血酸、尿酸、单宁酸、及碱性盐(Mg2+、NH4+、Na+、K+和Ca2+盐)、EDTA和水合氯醛。
一种或多种溶剂可用于虞美人制剂。可以包含水、诸如乙醇等低级链烷醇、多元醇。可以应用的多元醇的实例包括甘油、乙二醇、丙二醇、聚乙二醇和聚丙二醇。
福尔马林是组织和细胞样品适合的固定剂。石蜡组织盒用于制备和填充并包埋样品,然后进行冷冻。用切片机将石蜡固定的组织切成薄片并置于玻璃显微镜载玻片上。然后将制备物置于烘箱中以熔化多余的石蜡并在剩余的蜡上熔化。必须完全建立用二甲苯和甲苯脱蜡的载玻片。
可以取冷冻的组织样品作为染色方法的切片。将冷冻的组织切片短时间保存于10%的福尔马林中。染色方法、时机和顺序可以根据制剂方法和其他条件而变化。还可以使用对曙红Y、橙1G、浅绿SF、黄色苯胺棕、牢固绿FCF、O-6、EA25、EA36、EA50和EA65进行反细胞质染色(122)。
所述染色剂制剂可以在以下染料制剂中进行修饰:Gills氏、Anderson氏、DeGroot氏、Baker氏、Bennett Bohmer氏、Bosma氏、Bullard氏、Coca Cola的Carazzi、Debi氏、Delafield氏、Duval氏、Ehrlich氏、Friedlander氏、Gadsdo氏、Gage氏、Galigh氏、Garvey氏、Graham氏、Mitchell氏、Mayer氏、Masson氏、Martinotti氏、Mann氏、Mallory氏、McLachlan碘、Lillie氏、Lee氏、Launoy氏、Langeron氏、Krutsay氏、Kleinenberg氏、Horneyold氏、Haug氏、Hamilton氏、Harris氏、Harris&Power氏、Haugen氏、Molnar氏、Papamiltiades氏、Pusey氏、Rawitz氏、Reddy氏、Sass氏、Schmorl氏、Sliders氏、Unna氏、Watson氏、以及Weigert和Wright氏染料、铁媒染的苏木精、Anderson氏、Cretin氏、Faure氏、Goldman氏、Hansen氏、Heidenhain氏、Janssen氏、Kefalas氏、包括Krajina氏、Krutsay氏、Manna氏、Lillie氏、Lillie和Earle氏、Masson氏、More&Bassal氏、Murray氏、Paquin和Goddard氏、agaud氏、Rozas氏、Seidel氏、Thomas氏、Weigert和Yasvoyn氏、或铋媒染的苏木精、Roach&Smith氏、铜媒染的苏木精、Bensley氏、Cook氏和Faure氏、钼媒染的苏木精、Held氏、可考虑钒媒染的苏木精、Hedenham氏、Smith氏、锆媒染的苏木精和McNulty&Smith氏(123)。
可以结合使用其他染料,诸如吖啶染料、蒽醌染料、芳基甲烷染料、偶氮染料、重氮染料、诸如硝基染料等染料,可结合(特别是自动使用的方法)酞菁染料、奎宁亚胺染料、四唑鎓染料、氧杂蒽染料和噻唑染料。转化染料样品用于组织学染色,乙酰黄、酸性黑l、酸性蓝22、酸性蓝93、酸性品红、酸性绿、酸性绿1、酸性绿5、酸性红、酸性橙10、酸性红4、酸性红26、酸性红29、酸性红44、酸性红51、酸性红66、酸性红73、酸性红87、酸性红91、酸性红92、酸性红94、酸性红101、酸性红103、酸性玫瑰红、酸性复红、酸性紫19、酸性黄l、酸性黄9、酸性黄23、酸性黄24、酸性黄36、酸性黄73、酸性黄S、酸性黄T、吖啶、吖啶黄、阿尔新蓝、阿尔新黄、醇溶性曙红、茜素、茜素蓝、茜素蓝2RC、茜素胭脂红、茜素花青BBS、茜素花青R、茜素红S、茜素红紫素、氧化铝、酰胺黑10B、酰胺萘酚红、酰胺黑、苯胺蓝WS、苯胺紫、蒽G偶氮蓝SWR、蒽蓝SWX、金胺0、偶氮曙红、偶氮胭脂红B、偶氮胭脂红G、偶氮重氮5、偶氮重氮48、偶氮焰红、伊文斯蓝、深蓝、天蓝B、天蓝C、碱性蓝8、碱性蓝9、碱性蓝12、碱性蓝15、碱性蓝17、碱性蓝20、碱性蓝26、碱性棕1、碱性品红、碱性红4、碱性红5、碱性绿2、碱性橙14、碱性绿5、碱性红9、碱性紫2、碱性紫4、碱性紫10、碱性紫14、碱性黄1、碱性黄2、猩红R、苯胺棕Y、巴西、巴西、亮番红、亮结晶猩红6R、猩红、钙红、胭脂红、胭脂红酸淡红6、天青石蓝B、中国蓝、染料牢固红5B、红、天青石蓝、芝加哥蓝4B、铬紫CG、铬变素2、铬花青R、刚果紫、刚果红、棉蓝、棉红、克罗克红3D、克罗克红Moon、sketch染料、结晶丽春红6R、结晶红、结晶紫、大丽紫染料、钻石绿B、直接蓝14、直接蓝58、直接番红、直接红10、直接红28、直接红80、直接红81、直接黄7、耐尔晒蓝4、耐尔晒蓝8G、黄光曙红、曙红Y、曙红B、蓝光曙红、耐尔晒曙红、伊利石榴红B、羊毛铬青R、赤藓红B、乙基曙红、乙基绿、乙基紫、伊文思蓝、牢固蓝B、牢固绿FCF、牢固红B、牢固黄、超牢固黄G、油性黑HB、荧光素、食品绿3、帆船色(galleon)、没食子胺蓝、樟花青(gallocy)、龙胆紫、黄、丽丝安牢固黄1、INT、胭脂、胭脂酸、Kemechtrot、紫胶(Lac)、紫胶酸、劳氏紫、牢固亮绿、牢固蓝I、牢固复红BBL、拿破伦蓝、霍夫曼紫、联氨黄、帝国红、丽丝安绿SF、Luxol牢固蓝、品红0、品红II、品红II、品红III、孔雀石绿、曼彻斯特棕、马修黄、丁香紫(lilac)、苯胺紫、汞溴红、红汞、甲基黄、亚甲基蓝、亚甲基天蓝B、亚甲基蓝C、亚甲基蓝、亚甲基绿、甲基蓝、甲基绿、甲基紫、甲基紫2B、甲基紫10B、研磨黄3G、媒介蓝3、媒介蓝10、媒介蓝14、媒介蓝23、媒介蓝32、媒介红4、天然媒介蓝紫45、媒介红紫11、媒介紫325、紫39、萘蓝黑、萘酚蓝黑、萘酚绿B、萘酚黄S、自然黑1、自然红、自然红3、自然红28、自然红25、自然红24、自然红16、自然红8、黄6、NBT、中性红、新品红、尼亚加拉蓝3B、夜蓝、耐尔蓝、耐尔蓝A、硫酸耐尔蓝、耐尔红、硝基BT、四唑鎓硝基蓝、核牢固红、油红O、橙G、地衣红、副玫瑰苯胺、帕金斯紫、焰红B、苦味酸、丽春红2R、丽春红6R、丽春红B、二甲苯胺丽春红、丽春红S、滂胺天蓝5B、四季樱草、报春花、红紫素、焦宁B、焦宁G、焦宁Y、罗丹明B、玫瑰苯胺、焦宁OR、红中红R、猩红R、虫漆、天狼红F3B、天狼红4B、番红花红、孟加拉、番红花玫瑰、天狼蓝F3、单铬青R、可溶蓝、溶剂黑3、溶剂蓝38、溶剂红23、溶剂红24、溶剂红27、溶剂红45、溶剂黄94、醇溶性曙红、苏丹III、苏丹IV、苏丹黑B、苏丹红BK、硫黄S、瑞士蓝、酒石黄、硫代黄素S、硫代黄素T、硫堇(tion)、甲苯胺蓝、甲苯胺红、金莲橙G、吖啶黄、硫代黄素、台盼蓝、荧光素钠、维多利亚蓝4R、维多利亚蓝B、维多利亚蓝R、维多利亚绿B、水溶曙红、木曙红、二甲苯胺丽春红和黄曙红、及它们的组合(123-128)。
组织学中作为协同分子的两种最常见的金属媒染剂是铝和三价铁,媒染剂和染料以共价或配位络合物形式形成螯合。媒染剂的多价金属离子和染料分子产生配位络合物。螯合物的形成构成“色淀”,其定义为染料媒染剂多价络合物。该分子具有多于一个连接点,并且所形成的络合物在多样性和功能上是不同的。
改变制剂的pH值显示出非常突然和急剧的颜色变化。突然的颜色变化可解释为离域电子的位置变化和金属电子的较低能级。与络合物分子结构相比,金属具有相对较低的能级,即,溶液能量水平因金属的添加而突然下降。
在碱基的DNA结构中,其在脱氧核糖磷酸酯后面,胸腺嘧啶、胞嘧啶、鸟嘌呤和腺嘌呤以匹配互补的方式形成螺旋结构。磷酸酯基团是组织学染色的基础。向染料中掺混媒染剂,DNA中的磷酸酯会产生螯合物的形成。
形成配位键集合体所必需的电子来自与磷酸氧相连的媒染剂染料。DNA和蛋白质含有羟基和羧基,核染色质也是如此,其包括蛋白质组分和DNA复合物。因此,即使从DNA中去除染色剂后,核组分仍然可见。
固定不充分的组织不能被最佳地染色。快速加工技术影响染色品质。施加10%中性缓冲福尔马林几个小时,蛋白质的固定会不足。乙醇是稳定剂和脱水剂。出于此目的,用福尔马林溶液固定就足够。当单独使用乙醇时,脆性和扭曲部分留下组织的假象。
与退行性(regressive)染色方法相比,进行性(progressive)染色法不需要进行差异化脱色。细胞核染色外的背景染色也可以是在不同程度上所需的。进行性染色溶液显示无粘蛋白的很少或没有背景染色。
然而,酸添加中较高的染料浓度(1.5g/l~2.0g/l)也提供了清晰的背景染色。退行性染料(特别是上述制剂中5g/l的染料浓度)看起来着色更强。在退行性染色后,差异化脱色提供了清晰和干净的核形态外观。退行性制剂可以是大规模实验室和医院优选的。
单独的铵离子和钾离子的铝离子或铝离子与溶剂显示出较低pH水平,然而,如果缓冲能力非常有限,pH最终会增加。然而,将染料制剂调整为酸性的pH,碱溶液在短时间内与该液体的接触不会影响该液体的pH。染料溶液的寿命涉及返回到碱性液体的携带量上的媒染剂或染料。以此方式,染料开始降低活性。降解的第一个迹象是染色剂溶液从红亮紫色到红色不透明樱桃色的变化。通过将少量的酸性染料添加到染料溶液中来激活。出于此目的,按照pH控制所要求的量,应当用1升溶液定容25%的乙酸、0.1%柠檬酸或0.5%盐酸酒石酸、乳酸、异抗坏血酸、抗坏血酸、尿酸、丹宁酸。也可以选择其他酸来控制pH。溶液的pH应在2.5左右。然而,根据该制剂可以在pH 1~4之间调节修改(图6)。
添加强酸会溶解组织中的钙储存,染色的组织中钙的沉积对于诊断(例如乳腺癌)是否可能误导诊断是重要的。因此,应该避免非常低的pH值,即强酸。诸如乙酸和柠檬酸,应该预期会提供含酸清洁剂和显著的核染色的制剂。换句话说,可以实现含有弱酸的进行性理想制剂的细胞核染色。与此相反,制剂不含酸显示更好的背景,观察内核会不太清楚。制剂中退行性添加酸将有助于染色,但为了提供清晰度,再次增加染料溶液的酸性pH值水平,并会延长染色剂的寿命。
染色方案的步骤包括:1.脱蜡;2.再水化(使用醇);3.用水冲洗;4.虞美人制剂实施;5.用水冲洗;6.差异化脱色;7.用水冲洗;8.蓝化;9.用水冲洗;10.用醇冲洗;11.曙红;12.脱水(使用醇);13.用水(或二甲苯)清洗(图7)。
所施加的染色时间与其他染料一样,可根据制剂比例在30分钟~1或2分钟的范围内进行调节(131~133)。非常短的温育期导致染色不完整和不一致。若无法做好充分的准备工作,可以通过搅动来纠正。酸可以清除载玻片上的矫作物。然而,这可能降低媒染剂染料络合物的有效性。对染色剂进行15秒的搅拌(摇动)会减少总染色时间。如果没有搅动,持续时间应该更长。
染色5分钟是足够的。通过调整时间长度来调整进行性染色的品质。理想的染色时间为5分钟~10分钟。进行性染色首先快速开始且在时机上更加独立,随后因动态平衡点而自身受限。对于退行性染色技术,5分钟~10分钟是足够的时间。用酸性醇除去过量的染料。
出于此目的用乙醇进行差异化脱色,且改变酸性乙醇的浓度。样品;70%乙醇(标准酸性乙醇),使用5%盐酸混合物30-45秒。需要稀释强酸性染料才能完全删除剪贴板。本文中,可以使用70%蒸馏水代替乙醇。水可以稀释酸,差异化脱色可以导致最佳染色。然而,使用水代替醇会遇到染色不规则物。可以选择正异丙醇。
组织载玻片上的酸性介质的初始颜色是亮红紫色。这不是永久的颜色,应该转换为蓝色持久色。颜色的红色相在玻片下方浸出。蓝色相是耐溶剂的、不溶的,向其提供水和温和溶剂,通过调节pH值来提供永久转化。自来水或其他碱性溶液提供蓝化。水以及其他碱溶液可能足以使核变成蓝色,例如0.1%~1%的碳酸锂溶液、0.5%的乙酸钠、2%的碳酸氢钠和Scott氏自来水替代品(图8)。
水中的氯可导致载玻片上的染料颜色褪色,并可完全消除染色并漂白。在使用含氯水的情况下,必须避免使用自来水冲洗或应当使用非含氯水。如果强碱用于蓝化,高pH可以抑制后续的曙红染色。可以通过pH控制、固定剂、氧化剂和其他染料提供并用溶剂来维持差异化脱色。
通过使用媒染剂可以减少过量的染色剂。溶液中包含的更多媒染剂染料进入,竞争与组织的结合,并使染料与组织分离。通过这种方法可以在染色剂制备上完全去除。熟化(ripening)将提高染色剂溶液的效率。熟化可以通过天然氧化工艺实现,其更加可靠和耐用。可以使用从顶部进入空气的用棉花松散闭口的超大型烧瓶。烧瓶必须放置在温暖、黑暗和通风的地方,以允许缓慢氧化。
用于化学熟化的化学氧化剂,提供更快更有效的成熟。沸腾加速此操作,在煮沸碘酸钠、次氯酸钙、过氧化氢、USP、高锰酸钾、铁氰化钾、碘化钠、氧化锌后,可以用作除高碘酸钾和次氯酸钠以外的化学氧化剂。
当与如硫酸铝钾等铝盐结合时,溶液是淡色(未成熟)不透明的透明紫,当氧化时,其变成明亮的深紫色(熟化)。它们也可以与铁盐组合。这些是颜色通常较深的非常暗的紫色。根据所使用的化合物,草酸、铁氰化硼砂、高锰酸钾可用作氧化剂。
用于染色溶液的主要媒染剂是硫酸铝或硫酸铝钾、硫酸铝铵、硫酸铝钠。乙酸铝和硝酸铝也可以。5克染料粉末/升用作水溶剂,应使用蒸馏水。媒染剂染料比约为10倍,或者稍低水平。过度和持续的氧化会累积沉淀物。该沉淀物还含有媒染剂,其通常为通过氧化所得的不活泼的衍生物。定期过滤染色剂制剂。
从提取物获得的虞美人液体制剂为暗红色,并以不同的方法处理。产品本身的研磨制剂适合于商业销售(特别是在易于运输方面),并适于由技术人员、医院和实验室制备。经过测试的溶液作为即用型包装易于运输,或者可以可以在黑暗的容器内运送到适当的医院。
通过与乙酸反应,核染色质核给出了更一致和清晰的细胞核图像。媒染剂铝染料制剂为酸性pH值(pH在3.3~2.5之间)。如果染色时染料容器在多次洗涤期间移动,以长度染色的结构的有效性可能会降低。添加新的溶液将加强涂覆。在氧化结束后,在溶液中形成的衍生物的有效性较差,导致某些沉淀的形成。液体制剂可以储存在桶或容器中,其上覆盖有薄层油,以防止有氧氧化。
曙红Y或其他染料可用作细胞质的复染色剂。这里适用一般原则,但结果是主观评估的。对比染料是理想的,不仅使细胞核之外的蓝色核组分看起来良好,而且应该允许组织彼此清楚地区分(图14),还必须能够从胶原蛋白样品中区分新的肌肉组织。曙红Y通常可溶于95%乙醇,所以一旦封闭式薄层制剂CA或乙醇放置时间太长,染色剂将变得无色。如果在95%乙醇中留下太长时间,可用染色的EO无色组织制成,其小于100%乙醇对Eon的分辨率,但这里也从组织中除去染料。为了避免此问题,在嗜曙红制剂7次~10次的快速浸泡后,应该用95%乙醇洗涤来减去或形成。曙红色与蓝色核形成适合的对比。曙红Y通常具有微黄橙色,但是乙酸使其变得更红。
红色的强度对于区分核外的结构是重要的。染色不足会导致微弱的染色,另一方面,细胞核上的染料太多,会难以区分组织结构。曙红染料通过结合胶原蛋白来复染;淡粉色,肌肉;暗粉色,嗜酸性细胞质;红色,嗜碱性细胞质;紫色,核;暗紫色蓝,用红色染色的红血细胞。染色虞美人制剂后,必须将曙红施加于载玻片上约1分钟。
然后再洗涤更多的曙红染料(129,130)。出于此目的,优选选择50%、75%和95%浓度的一系列乙醇溶液。
染色过程的终止是一个重要的标准,但何时终止是对颜色强度和对比度的视觉评估的最佳方法。制备标准物和染色品质控制的经验导致染色效率的改善(134-138)。
虞美人制剂用于对非人组织生物样品进行染色。例如来自酵母菌的样品中的酵母真菌的细节被染色(图15)。
通过检查光学显微镜再次观察到,光学和原子力显微花瓣中的花瓣显示出以完美队列排列的池结构,其中填充有由优异的比例螺旋管产生的液体(图16,17)。
实施例
1.实施例1.前体的制备:冷冻、新鲜或干燥的虞美人花瓣(花瓣)用于制备提取的染料(图9)。将叶片破碎。然后添加蒸馏水静置过夜,随后进行挤压来提取液体,得到染料前体。第一染料是紫色、红色、紫红色、红紫色和红蓝色。过滤之后,以1/10的比例提供福尔马林、乙醇、甲醇等溶液,溶剂作为其他醚加入,并在30℃~60℃的培养箱中加热干燥。用甲醇再次处理干燥的粗材料,分离沉淀物。该沉淀物用于制备粉末粗产物。将该沉淀物加入冷的或沸腾的乙醇(1g染料/100g乙醇)中并溶解。将该液体混合物(冷的或沸腾温度)加入1000ml去离子水中。
2.实施例2:对于分子分析,将提取液过滤并用乙醇处理,并在培养箱内干燥。粉碎干燥的样品,然后通过甲醇浴3次。并在放入30℃~60℃的培养箱中过夜,并蒸发液体部分。将1g所得粉末溶解在100cc的乙醇中并吸附在板的纤维上。观察到粉红色板在1%氨溶液下的蓝化。将该蓝色染料溶于乙酸。除去液体部分。并对剩余的干粉进行分析。
3.实施例3:在该新分子的组合物中部分或完全使用化学氧化剂。作为氧化的结果将形成分子的衍生物。然而,还可以形成活性较低的复合物。氧化剂和分子的摩尔平衡为4:1~1:1。溶剂中的染料具有多种媒染剂(铝、铁、铋、铜、钼、铬、钒和锆媒染剂)(120,121)。分子和媒染剂的摩尔比为2:1~1:100,该比例根据制剂而不同,例如在1:20~1:5之间变化。制剂还可以含有诸如乙酸等酸。最终媒染剂染料和溶剂的溶液(如AlCl3 80%,10克/升~50克/升)具有酸性pH,向制剂中加入酸调节核染色的特异性,并增加保质期。蓝化突出显示了细胞核的形状。向染料制剂中加入乙酸将颜色变成明亮的棕紫色(图10)。
4.实施例4:虽然制剂的理想pH为2.5,但其变化范围为1~4。在某些具体实施方式中,组合物中的氧化剂为碘酸钠,媒染剂为硫酸铝,氯化铝抗氧化剂为β-环糊精,以60%~90%的水作为溶剂,可以以10%~40%比例的乙二醇混合物使用。可以使用没食子酸正丙酯、氢醌和一种或多种水溶性抗氧化剂。
5.实施例5:以进行性或退行性染色的方式建立着色。生物样品由显微镜载玻片支撑。该方法也可用于安装在显微镜载玻片上的细胞学样品。选择曙红进行复染。
6.实施例6:稳定剂防止过度氧化、蒸发和最终沉淀。染料制剂以设定为1克/升~20克/升的溶剂量和染料/溶剂比制备。进行性染色溶液包含最少1克染料/升,退行性染料包含至少5克染料/升。当媒染剂的量保持不变时,溶液中更低浓度的染料显示出对细胞核的更具选择性的染色。例如,加入5mL的10%染料溶液和逐渐增量直至超过10%的醇染料溶液,以达到最佳选择性。
7.实施例7:如果浓度较低且溶液中染料的媒染剂染料/染料比较高,则其残留在溶液中,少量的染料粘附于组织。加入诸如0.1%或2%乙酸、柠檬酸等酸可延长保质期。
8.实施例8:非常低pH水平的溶液对于诊断而言是重要的,因为组织钙化也可以溶解。因此,用用强酸溶液进行的诊断可能导致错误和遗漏。增加0.2ml冰醋酸的浓度,或者可以通过加入0.1ml HCl来测试。通过使用5g染料和50g酸性媒染剂(硫酸铝)(比例为1/10)获得退行性染色制剂,通过使用1g染料和50g酸性媒染剂(硫酸铝)(比例为1/50)获得进行性染色制剂(图9)。
9.实施例9:将1000ml沸腾的去离子水置于大烧瓶中。加入50g硫酸铵并使混合物冷却。将4g干燥粉末置于烧瓶中并与0.4g碘化钠一起搅拌,溶于50ml冷水中。加入100cc乙二醇并与之混合,加入10cc乙酸。(pH单位在2.7~3.1之间)。将储备液相互混合。染料即可使用。染色的施加时间应当通过试用来优化。
10.实施例10:还可以进行修饰。将酸加入100g无酸的退行性染料中,其中该制剂具有5g染料媒染剂染料。此时比例为1/20。(5g染料+100g硫酸铝)。进行性染料制剂含染色剂1克/升~2克/升,退行性染料制剂含染色剂5克/升~6克/升,中间制剂含染色剂3克/升~4克/升。技术人员应完全了解染料的制备和应用。改变染料的含量可以调节所得染料的品量。
11.实施例11:所生产的粉末是砖红紫棕色,可溶于乙醇,较少溶于水(100cc的水,1g染料溶于100g乙醇,35g~50g溶解除去染料)。可以掺入甘油作为抗氧化剂,并防止制剂过度氧化,还防止真菌的发生。加入任何抗微生物剂以在2.0~5的pH单位内抑制微生物生长,抗微生物剂例如有Proclin叠氮化钠、ProclinProclin和Proclin(例如)向溶液中加入约0.04%量的Proclin(Sigma Aldrich,St Louis,MO)(图10)。
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Claims (28)
1.一种用于细胞核染色的化合物,其为5-羟基-2-(3-(四氢-6-((四氢-3,4,5-三羟基-6-甲基-2H-吡喃-2-基氧基)甲基)-2H-吡喃-2,3,4,5-四醇)-4-甲氧基苯基)-7-甲氧基-4H-色烯-4-酮,所述化合物获自虞美人(Papaver Rhoeas)提取物。
3.一种细胞核特异性的组织-细胞染色剂制剂,其包含权利要求1或2所述的化合物。
4.如权利要求3所述的染色剂制剂,所述染色剂制剂是虞美人提取物。
5.一种体外细胞核染色方法,包括使用权利要求1或2所述的化合物或权利要求3或4所述的染色剂制剂作为染料对生物样品进行染色。
6.如权利要求5所述的方法,其中,所述生物样品的来源是原核生物、古生菌或真核生物。
7.如权利要求5所述的方法,其中,所述生物样品的来源是哺乳动物。
8.如权利要求5所述的方法,其中,所述生物样品的来源是灵长类动物。
9.如权利要求5至8中任一项所述的方法,其中,所述生物样品包括体液、分离的器官、组织、细胞或细胞器。
10.如权利要求5所述的方法,所述方法包括使用权利要求4所述的染色剂制剂作为染料对生物样品进行染色,其中,染料前体是通过压制和过滤获自花瓣的液体组分来制备的。
11.如权利要求10所述的方法,其中,使用新鲜、干燥或冷冻的花瓣制备染料液体组分的前体。
12.如权利要求10所述的方法,其中,0.1体积%~10体积%的液体前体转化为干粉染料。
13.如权利要求5所述的方法,其中,所述染色剂制剂与作为复染剂的曙红组合,其区别性地染色组织层、膜、肌肉条纹、胞质内核内结构。
14.如权利要求13所述的方法,其中,所述染色剂制剂染色神经胶质结构。
15.如权利要求5所述的方法,其中,所述生物样品用福尔马林和醇固定。
16.如权利要求5所述的方法,其中,使用冷冻细胞和组织样品用于染色。
17.如权利要求5所述的方法,所述方法包括使用权利要求4所述的染色剂制剂作为染料对生物样品进行染色,所述染色包括(1)使用5 g染料和50 g媒染剂的退行性染色剂或(2)使用1g染料和50g媒染剂的进行性染色剂。
18.如权利要求5所述的方法,所述方法包括使用权利要求4所述的染色剂制剂作为染料对生物样品进行染色,其中,向5 g染料和100 g媒染剂的制剂中加入酸使pH为2.3~4,以提供退行性染色。
19.如权利要求17或18所述的方法,其中,所述媒染剂选自硫酸铝、硫酸铝铵、乙酸铝、硝酸铝或硫酸铝钾。
20.如权利要求5所述的方法,其中,所述染料是溶液,所述方法还包括对所述溶液进行化学熟化,所述化学熟化使用碘酸钠、次氯酸钙、过氧化氢、USP、高锰酸钾、铁氰化钾、碘化钠、氧化锌、高碘酸钾或次氯酸钠进行氧化。
21.如权利要求5所述的方法,其中,在所述染料中掺入媒染剂、溶剂或酸。
22.如权利要求21所述的方法,其中,所述溶剂包含水、甲醇、乙醇、异丙醇或多元醇。
23.如权利要求21所述的方法,其中,所述溶剂选自乙醇、甘油、乙二醇、丙二醇、聚乙二醇和聚丙二醇。
24.如权利要求5所述的方法,所述方法包括在所述染料中添加抗微生物剂以抑制微生物生长。
25.如权利要求24所述的方法,其中,所述抗微生物剂包括Proclin 300®叠氮化钠、Proclin 150®、Proclin 200®或Proclin 950®。
26.如权利要求5所述的方法,所述方法还包括将如下染料与所述化合物或染色剂制剂组合使用:吖啶染料、蒽醌染料、芳基甲烷染料、偶氮染料、重氮染料、硝基染料、酞菁染料、奎宁亚胺染料、四唑鎓染料、噻唑染料或氧杂蒽染料。
27.如权利要求5所述的方法,所述方法还包括将如下染料与所述化合物或染色剂制剂组合使用:乙酸、酸性黄、酸性黑l、酸性蓝22、酸性蓝93、酸性品红、酸性绿、酸性绿1、酸性绿5、酸性红、酸性橙10、酸性红4、酸性红26、酸性红29、酸性红44、酸性红51、酸性红66、酸性红73、酸性红87、酸性红91、酸性红92、酸性红94、酸性红101、酸性红103、酸性玫瑰红、酸性复红、酸性紫19、酸性黄l、酸性黄9、酸性黄23、酸性黄24、酸性黄36、酸性黄73、酸性黄S、酸性黄T、吖啶、吖啶黄、阿尔新蓝、阿尔新黄、醇溶性曙红、茜素、茜素蓝、茜素蓝2RC、茜素胭脂红、茜素花青BBS、茜素花青R、茜素红S、茜素红紫素、氧化铝、酰胺黑10B、酰胺萘酚红、酰胺黑、苯胺蓝WS、苯胺紫、蒽G偶氮蓝SWR、蒽蓝SWX、金胺0、偶氮曙红、偶氮胭脂红B、偶氮胭脂红G、偶氮重氮5、偶氮重氮48、偶氮焰红、伊文斯蓝、深蓝、天蓝B、天蓝C、碱性蓝8、碱性蓝9、碱性蓝12、碱性蓝15、碱性蓝17、碱性蓝20、碱性蓝26、碱性棕1、碱性品红、碱性红4、碱性红5、碱性绿2、碱性橙14、碱性绿5、碱性红9、碱性紫2、碱性紫4、碱性紫10、碱性紫14、碱性黄1、碱性黄2、猩红R、苯胺棕Y、亮番红、亮结晶猩红6R、猩红、钙红、胭脂红、胭脂红酸淡红6、天青石蓝B、中国蓝、染料牢固红5B、天青石蓝、芝加哥蓝4B、铬紫CG、铬变素2、铬花青R、刚果紫、刚果红、棉蓝、棉红、克罗克红3D、克罗克红Moon、sketch、结晶丽春红6R、结晶红、结晶紫、大丽紫染料、钻石绿 B、直接蓝14、直接蓝58、直接番红、直接红10、直接红28、直接红80、直接红81、直接黄7、耐尔晒蓝4、耐尔晒蓝8G、黄光曙红、曙红Y、曙红B、蓝光曙红、耐尔晒曙红、伊利石榴红B、羊毛铬青R、赤藓红B、乙基曙红、乙基绿、乙基紫、牢固蓝B、牢固绿 FCF、牢固红B、牢固黄、超牢固黄G、油性黑HB、荧光素、食品绿3、帆船色、没食子胺蓝、樟花青、龙胆紫、丽丝安牢固黄1、INT、胭脂、胭脂酸、Kernechtrot、紫胶、紫胶酸、劳氏紫、牢固亮绿、牢固蓝I、牢固复红BBL、拿破伦蓝、霍夫曼紫、联氨黄、帝国红、丽丝安绿SF、Luxol牢固蓝、品红0、品红II、品红II、品红III、孔雀石绿、曼彻斯特棕、马修黄、丁香紫、苯胺紫、汞溴红、红汞、甲基黄、亚甲基蓝、亚甲基天蓝B、亚甲基蓝C、亚甲基蓝、亚甲基绿、甲基蓝、甲基绿、甲基紫、甲基紫2B、甲基紫10B、研磨黄3G、媒介蓝3、媒介蓝10、媒介蓝14、媒介蓝23、媒介蓝32、媒介红4、天然媒介蓝紫45、媒介红紫11、媒介紫325、紫39、萘蓝黑、萘酚蓝黑、萘酚绿B、萘酚黄S、自然黑1、自然红、自然红3、自然红28、自然红25、自然红24、自然红16、自然红8、黄6、NBT、中性红、新品红、尼亚加拉蓝3B、夜蓝、耐尔蓝、耐尔蓝A、硫酸耐尔蓝、耐尔红、硝基BT、四唑鎓硝基蓝、核牢固红、油红O、橙G、地衣红、副玫瑰苯胺、帕金斯紫、焰红B、苦味酸、丽春红2R、丽春红6R、丽春红B、二甲苯胺丽春红、丽春红S、滂胺天蓝5B、四季樱草、报春花、红紫素、焦宁B、焦宁G、焦宁Y、罗丹明B、玫瑰苯胺、焦宁OR、红中红R、猩红R、虫漆、天狼红F3B、天狼红4B、番红花红、孟加拉、番红花玫瑰、天狼蓝F3、单铬青R、可溶蓝、溶剂黑3、溶剂蓝38、溶剂红23、溶剂红24、溶剂红27、溶剂红45、溶剂黄94、醇溶性曙红、苏丹III、苏丹IV、苏丹黑B、苏丹红BK、硫黄黄S、瑞士蓝、酒石黄、硫代黄素S、硫代黄素T、硫堇、甲苯胺蓝、甲苯红、金莲橙G、吖啶黄、硫代黄素、台盼蓝、维多利亚蓝4、维多利亚蓝B、维多利亚蓝R、维多利亚绿B、水溶曙红、木曙红、二甲苯胺丽春红或黄曙红,或它们的组合。
28.如权利要求5所述的方法,所述方法包括:使用不同的复染剂与所述染料组合,所述复染剂选自曙红Y、橙G、浅绿SF、黄色苯胺棕、牢固绿FCF、O-6、EA25、EA36、EA50和EA65。
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AU2018267059B2 (en) * | 2017-05-10 | 2021-08-12 | Ventana Medical Systems, Inc. | Stabilized two-part hematoxylin solution utilizing pH adjustment |
CN107246988A (zh) * | 2017-06-06 | 2017-10-13 | 珠海贝索生物技术有限公司 | 一种抗酸性菌染色方法 |
KR101982521B1 (ko) | 2018-04-04 | 2019-05-27 | 부산대학교병원 | 염료 및 고차가지구조 고분자의 합성 복합체를 유효성분으로 포함하는 조직 염색 화합물 및 그의 제조방법 |
CN108479427B (zh) * | 2018-05-25 | 2020-05-22 | 南京佳乐净膜科技有限公司 | 抗菌除重金属共混改性的聚合物膜的制备方法 |
CN108918236B (zh) * | 2018-08-01 | 2020-12-01 | 中国农业科学院烟草研究所 | 一种烟草主茎徒手切片快速双重染色液 |
CN111829858A (zh) * | 2019-04-18 | 2020-10-27 | 湖北文理学院 | 实验发现两种简单、低廉、来源丰富的新型组织切片染色剂 |
RU2725291C1 (ru) * | 2019-05-27 | 2020-06-30 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Тихоокеанский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Способ донозологической неинвазивной диагностики состояния здоровья работников |
WO2020261299A1 (en) * | 2019-06-28 | 2020-12-30 | Rajiv Gandhi University Of Health Sciences | Method for preparing biodegradable staining composition |
CN111018893B (zh) * | 2019-11-25 | 2020-09-25 | 桂林医学院 | 一种含硫氨基醇席夫碱铜配合物及其制备和应用 |
KR102332433B1 (ko) * | 2020-01-13 | 2021-11-30 | 사회복지법인 삼성생명공익재단 | 조직 염색용 조성물 및 이를 이용한 조직 염색 방법 |
CN111337332A (zh) * | 2020-03-17 | 2020-06-26 | 宁波江丰生物信息技术有限公司 | 一种巴氏染色液ea50及其制备方法 |
WO2021194436A1 (en) * | 2020-03-26 | 2021-09-30 | Nanobiomed Sağlik Ve Yaşam Bilimleri A. Ş. | The natural antiviral and anti-inflammatory compound consist of bioflavonoids which extracted from papaver rhoeas red petals |
CN111624065B (zh) * | 2020-05-22 | 2023-10-27 | 嘉兴优瑞生物科技有限公司 | 一种动物专用Diff-Quik染液及制备方法 |
CN111982626B (zh) * | 2020-08-20 | 2023-07-25 | 江苏瑞思坦生物科技有限公司 | 一种软骨球快速石蜡切片鉴定方法 |
CN112649595B (zh) * | 2020-11-11 | 2022-02-22 | 西安交通大学 | 一种基于单脉冲激光诱导光致击穿可控射流的系统和方法 |
KR102635263B1 (ko) * | 2021-11-18 | 2024-02-08 | 경희대학교 산학협력단 | 양귀비속 식물의 추출물을 포함하는 항산화 및 항염증용 조성물 |
CN115200963A (zh) * | 2022-07-15 | 2022-10-18 | 重庆医科大学附属儿童医院 | 一种油红o染色液及油红o染色方法 |
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