活细胞凋亡死细胞的鉴别方法

线粒体膜电位指示染料线粒体膜电位的下降是细胞凋亡初期的一个标志性事件. 它在凋亡进程中与caspase 活化同时发生并先于磷脂酰丝氨酸（PS）的外翻。

基于以上研究，Biotium 研发了各类的新型的荧光探针用于测量线粒体膜电位。

MitoView ™ 633MitoView ™ 633 是一种新型的用于测量线粒体膜电位的深红染料(激发光/发射光622/648 nm)。

利用NucView ™ 488 和MitoView ™ 633 凋亡检测试剂盒能够在荧光显微镜(图.1)或流式细胞仪(图.2、3)下同时进行线粒体膜电位和caspase-3活性的检测。

图 1. 利用MitoView ™ 633进行活细胞染色:Hela 细胞图 2. 流式细胞仪分析：Jurkat 细胞一组用CCCP 使线粒体去极化，另一组利用staurosporine 作为凋亡诱导剂。

利用MitoView ™633 染色图3. 流式细胞仪分析：对照（A）与经staurosporine 处置(B)的Jurkat 细胞（JC-1 染色）. FL1 (x- 轴) 为绿色荧光; FL2 (y-轴) 为红色荧光。

(A 图) 较高的红绿荧光比例说明线粒体膜电位未下降. （B 图）较低的红绿荧光比例说明：由于staurosporine诱导了凋亡的发生，细胞的线粒体膜电位大幅下降。

JC-1 线粒体膜电位检测试剂盒JC-1通常被用于检测细胞中线粒体膜电位的转变。

在健康细胞中,JC-1以聚合体(J-aggregates)，的形式存在在线粒体基质中，能够产生红色的荧光(激发光/发射光585/590nm)。

相反，在正在凋亡或坏死的细胞中，JC-1不能聚集在基质中，以单体的形式存在，从而发出绿色的荧光( 激发光/ 发射光510/527nm)，如此能够利用流式细胞仪和荧光显微镜、荧光计数仪通过测量荧光颜色的转变来检测线粒体膜电位的转变。

经常使用红绿荧光的相对照例来衡量线粒体去极化的比例。

区别细胞死活的方法细胞是生物体的最小单位，它们构成了人体的所有组织和器官，由此可以看出细胞死活对生物体的关键重要性。

细胞死活的评价是定量化和定性分析生物活动的关键环节。

目前，细胞死活评价仍然是仅限于实验室内容易掌握的技能，且受到许多因素的影响，因此区别细胞死活的方法极其重要。

首先，最常用的方法是通过显微镜观察细胞活力。

要辨识细胞死活，必须通过使用显微镜来确定细胞的形状和质地的变化。

死细胞不易发育，通常会呈现出“静止细胞”的特征，这就是细胞脱水由于内部膜蛋白的变化。

而活着的细胞则会有更多的活力，表面有许多触手，破裂的细胞也很常见。

此外，将细胞从直接观察转变为定量测量也是一种有效的方法。

细胞活力可以用流式细胞术和其他定量技术来测量，最常用的是荧光技术，它可以直接定量测定细胞死亡率。

细胞外基质的变化也可以通过吸收法、荧光比色或其它技术来检测，以区分细胞死活。

此外，细胞死活可以通过凋亡因子、抗凋亡抗体以及蛋白质补体来识别。

凋亡因子有细胞膜活性物质（CAMs）和其他多种因子，可以分子识别细胞外胞质中的死细胞，从而激活死细胞的凋亡过程。

抗凋亡抗体可以识别凋亡过程中的蛋白质进而来识别细胞是否已经死亡。

蛋白质补体可以检测细胞形态上的变化，如染色体、线粒体和其它细胞结构的变化，可以用来识别细胞是否死亡。

最后，通过细胞老化技术也可以识别细胞死活。

细胞老化技术是一种新兴的研究方法，它可以测定细胞的老化情况，也可以检测细胞的死亡率。

主要的方法有DNA电泳、分子探针法和基因表达分析，以及其它从细胞老化到细胞死亡早期检测方法。

总而言之，以上是区别细胞死活的常用方法，我们可以通过显微镜观察细胞活力，定量测量细胞活力，通过凋亡因子和抗凋亡抗体，以及蛋白质补体，来识别细胞死活。

此外，还可以通过细胞老化技术等来识别细胞死活。

在此基础上，精确计算细胞死活率，为生物活动的分析提供重要的依据，成为定量和定性分析生物活动的重要环节。

区别细胞死活的方法细胞死活是生物学家研究细胞活力水平和状态的重要指标，对于评估微生物或细胞繁殖能力、细胞对药物作用与耐药性的反应、细胞功能障碍、细胞毒性研究和诊断实验等都有重要的意义。

近年来，借助现代生物技术的发展，人们发展出了一系列检测细胞死活的方法，有效地区分出细胞的生长和死亡状态。

一、细胞膜活性的检测是区别细胞死活的重要方法。

细胞膜的活性是评估细胞活力的重要指标，可以显示细胞死活。

细胞膜活性可以通过测定细胞膜电位、直接检测细胞膜转运系统活性和监测活细胞特异性指标，如细胞凋亡标志物等来确定。

测定细胞膜电位能直观反映细胞结构的活力水平，是常用的检测细胞死活的方法。

二、一些结构性指标也可以用来区分细胞死活状态，如细胞形态、细胞大小和细胞质组成。

观察活细胞形态，其细胞体形状各异，并具有活动性。

细胞大小可以根据荧光显微镜和流式细胞仪监测，死细胞体积一般小于活细胞体积。

此外，也可以测定细胞质组成，如细胞培养基中活细胞含量远高于死细胞，因此可以通过分析细胞培养基中的活细胞含量来量化评价活细胞的死活。

三、检测细胞代谢特性也是一种常见的检测细胞死活的方法。

细胞代谢特性是衡量细胞活力水平的重要指标，如细胞的糖异构化、蛋白质组合、酶的活性、细胞的ATP水平等均可以用来衡量活细胞的状态。

此外，细胞细胞因子，如细胞因子及表观遗传物质，如mRNA、miRNA等也可以作为调节和检测细胞死活的指标。

四、测定细胞核和其他细胞指标。

细胞死活状态的检测也可以利用细胞核指标来估算，活细胞的细胞核含有较多的染色质和活性核酸，而死细胞核中染色质明显变性，而其他细胞指标如非细胞物质的含量、活性氧的变化、离子通量的变化和细胞内ATP水平的变化也可以用来检测细胞死活。

总之，细胞死活是决定细胞繁殖及功能性状态的关键因素，细胞死活的检测对研究和诊断生物学研究有重要意义。

现代生物技术为探究细胞死活提供了有效的工具，如细胞膜电位检测、细胞结构检测、细胞代谢特性检测和细胞核等技术可以有效地检测细胞死活。

细胞凋亡的几种检测方法Company number：【WTUT-WT88Y-W8BBGB-BWYTT-19998】细胞凋亡的几种检测方法1、形态学观察方法（1）HE（苏木精—伊红染色法）染色、光镜观察：凋亡细胞呈圆形，胞核深染，胞质浓缩，染色质成团块状，细胞表面有“出芽”现象。

（2）丫啶橙（AO)染色，荧光显微镜观察：活细胞核呈黄绿色荧光，胞质呈红色荧光。

凋亡细胞核染色质呈黄绿色浓聚在核膜内侧，可见细胞膜呈泡状膨出及凋亡小体。

（3）台盼蓝染色：如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。

如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。

此方法对反映细胞膜的完整性，区别坏死细胞有一定的帮助。

（4）透射电镜观察：可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。

2、 DNA凝胶电泳细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞内小分子量DNA片断增加，高分子DNA减少，胞质内出现DNA片断。

但凋亡细胞DNA断裂点均有规律的发生在核小体之间，出现180－200bpDNA片断，而坏死细胞的DNA断裂点为无特征的杂乱片断，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。

正常活细胞DNA 电泳出现阶梯状（LADDER)条带；坏死细胞DNA电泳类似血抹片时的连续性条带3、酶联免疫吸附法（ELISA)核小体测定凋亡细胞的DNA断裂使细胞质内出现核小体。

核小体由组蛋白及其伴随的DNA片断组成，可由ELISA法检测。

检测步骤1、将凋亡细胞裂解后高速离心，其上清液中含有核小体；2、在微定量板上吸附组蛋白体’3、加上清夜使抗组蛋白抗体与核小体上的组蛋白结合‘4、加辣过氧化物酶标记的抗DNA抗体使之与核小体上的DNA结合’4、加酶的底物，测光吸收制。

用途该法敏感性高，可检测5*100/ml个凋亡细胞。

细胞凋亡(Apoptosis) 是生物界广泛存在的一种现象,与其它的生命现象一样具有同等重要的生理学或病理学意义。

尽管其最终结果也是导致细胞死亡,但其诱导因素、发生机制和过程及意义均明显不同于一般的病理性细胞死亡(即细胞坏死性死亡,简称细胞死亡) 。

细胞凋亡的形态特征与细胞坏死性死亡的形态特征不同。

细胞坏死性死亡是被动的病理性死亡,其形态特征首先是膜通透性增加,细胞外型发生不规则变化,内质网扩张,核染色质不规则移位,进而线粒体及核肿张,溶酶体破坏,细胞膜破裂,胞浆外溢,这种死亡过程常常引起炎症反应。

细胞凋亡则是在某些因素的诱导下,由细胞内在的有规律的机制引起的,是主动的生理性细胞自杀。

其特征是细胞首先变圆,随即与邻近细胞脱离,失去微绒毛,胞浆浓缩,内质网扩张呈泡状并与细胞膜融合,线粒体无明显变化,核染色质浓缩成块并凝聚在核膜周边,胞膜内陷将细胞自行分割为多个有外膜包裹、内涵物不外溢的凋亡小体,后被吞噬细胞或邻周细胞所识别、吞噬。

由于细胞凋亡过程不导致溶酶体破坏及胞膜破裂,没有细胞内容物外泄,故不引起炎症反应,在生理条件下,生物体内细胞存活与死亡是由自身发育阶段提供的遗传信息,或由邻近细胞和其微环境提供的信号决定的,其中包括细胞相互接触提供的信号以及周围环境中活性物质、激素等。

这些刺激信号的增加或减少调节着细胞产生和凋亡,维系着机体细胞的有序状态细胞凋亡的检测细胞凋亡与坏死是两种完全不同的细胞凋亡形式，根据死亡细胞在形态学、生物化学和分子生物学上的差别，可以将二者区别开来。

细胞凋亡的检测方法有很多，下面介绍几种常用的测定方法。

一、细胞凋亡的形态学检测根据凋亡细胞固有的形态特征，人细胞凋亡形态学检测方法。

细胞凋亡( apoptosis)的命名主要是根据某些单个细胞死亡时细胞碎裂如花瓣或树叶散落般的形态学特征。

目前对细胞凋亡的认识不断得到深化，检测凋亡细胞的方法也逐渐增多，但形态改变仍是确定细胞凋亡的最可靠的方法。

死活细胞的鉴定方法The document was prepared on January 2, 2021活细胞的鉴定在生物学和医学上具有很重要的意义.细胞培养过程中要随时记录细胞的生长情况,需要经常测定细胞的存活率；在肿瘤细胞的研究中,为了检验各种药物对肿瘤细胞的杀伤力,也需要测定肿瘤细胞的存活率.在临床医学中死活细胞的鉴定也有很大的应用,例如为了检测某一男子的生育能力,测定精子细胞的存活力是比较常用的办法.死活细胞的鉴定方法有很多种,常用的有染色法和仪器分析法.染色法是常用的细胞死活鉴定方法,简便,易于操作.但是通过直接的形态观察来鉴别细胞死活,实验结果很容易受操作者的主观因素的影响,存在一定的误差,所以,在实际操作中也常采用一些仪器来进行精确的批量检测.不同的死活细胞鉴定方法有各自不同具体的反应机理,但无论采用何种办法,都是利用了死活细胞在生理机能和性质上的差异.常用的方法包括染色法和仪器分析法.1 染色法染色法分化学染色法和荧光染色法,根据染色机理的不同,染料或使死细胞着色,或使活细胞着色.死活细胞在生理机能和性质上的差异主要包括：死活细胞细胞膜通透性的差异：活细胞的细胞膜是一种选择性膜,对细胞起保护和屏障作用,只允许物质选择性的通过；而细胞死亡之后,细胞膜受损,通透性增加.常用的以台盼蓝鉴别细胞死活的方法就是利用了这一性质.台盼蓝,又称锥蓝,是一种阴离子型染料,不能透过完整的细胞膜.所以经台盼蓝染色后只能使死细胞着色,而活细胞不被着色.甲基蓝有类似的染色机理.植物细胞的质壁分离也可鉴定死活.死活细胞在代谢上的差异：是采用美蓝染料鉴定酵母细胞死活的依据.美蓝是一种无毒染料,氧化型为蓝色,还原型为无色.由于活细胞中新陈代谢的作用,使细胞内具有较强的还原能力,能使美蓝从蓝色的氧化性变为无色的还原型,因此美蓝染色后活的酵母细胞无色；而死细胞或代谢缓慢的老细胞,则因它们的无还原能力或还原能力极弱,使美蓝处于氧化态,从而被染成蓝色或淡蓝色.荧光素双醋酸酯FDA是一种常用的培养动植物细胞以及植物细胞原生质体的生活力鉴定染料,其染色机理也利用了死活细胞在代谢上的差异：FDA本身不产生荧光,也无极性,能自由渗透出入完整的细胞膜.当FDA进入活细胞后,被细胞内的脂酶分解,生成有极性的、能产生荧光的物质——荧光素,该物质不能自由透过活的细胞膜,积累在细胞膜内,因而使有活力的细胞产生绿色荧光；而无活力的细胞因不能使FDA分解,而无法产生荧光.除此之外,还有一些细胞器的专有染料.如液泡系的专有染料中性红.中性红是一种低毒性染料,可以使活细胞液泡着红色,而细胞质和细胞核不被着色；死细胞的液泡不被着色或浅染,染料弥散于整个细胞中,细胞核和细胞质被染成红色.有时侯为了增加染色效果可以将两种染料结合使用,如甲基蓝-中性红混合染色法.2 仪器分析法可采用微电极技术利用死活细胞膜两侧电位的差异、全自动分析细胞记数仪和流式细胞仪等,可对细胞的死活进行精确的批量检测.德国INNOVATIS出产的全自动Cedex细胞存活率/细胞计数仪/细胞分析仪是世界上第一台高分辨率、高清晰度扫描专利细胞分析仪,具有全自动台盼蓝染色、显成象、CEDEX工作站软件,可用于制药、医学、发酵、免疫、病理、毒性测试、生物技术等学科的研究开发和工业生产过程中的细胞计数和分析.自动分析结果,检测分析数据输出11项：总细胞数目,总细胞浓度 cells/mL ；活细胞数目,活细胞浓度 cells/mL；死细胞数目,死细胞浓度 cells/mL ；细胞存活率 % ；细胞的平均圆度compactness ；细胞的平均直径 um ；细胞团比例 Aggregate Rate；细胞直径分布图Diameter Histogram；细胞团分布图 Aggregate Histogram；细胞圆度分布图Compactness Histogram ；细胞生长时间曲线Cultivation Time Chart.流式细胞仪法用来判断细胞死活的常用荧光探针有二大类：一类是能透过活的细胞膜进入细胞内而发出荧光的物质,例如FDA可被活细胞持留而发出黄绿色荧光,若细胞有损伤则会从细胞中流失,观察不到荧光.另一类是不能透过活细胞膜,但能对固定的细胞及膜有破损的细胞的核进行染色,例如碘化丙啶 PI,Propidium iodide 和溴化乙锭 EB,Ethidium bromide 就是常用的第二类荧光探针.碘化丙啶PI不能穿透细胞膜,对于具有完整细胞膜的正常细胞或凋亡细胞不能染色.而对于坏死细胞,其细胞膜的完整性丧失,碘化丙啶PI可以染色坏死细胞.目前常用的一种细胞凋亡与坏死检测试剂盒则含有Hoechst 33342和碘化丙啶Propidium Iodide,PI两种荧光染料.细胞发生凋亡时,染色质会固缩,Hoechst 33342可以穿透细胞膜,染色后凋亡细胞荧光会比正常细胞明显增强.上述两种染料双染后,使用流式细胞仪或荧光显微镜检测时,正常细胞为弱红色荧光＋弱蓝色荧光,凋亡细胞为弱红色荧光＋强蓝色荧光,坏死细胞为强红色荧光＋强蓝色荧光.此外,还可以用植物细胞的质壁分离试验来对植物细胞的死活进行见证,还可以通过观察细胞质的流动性进行细胞的死活鉴定.。

细胞凋亡检测方法细胞凋亡是一种重要的细胞死亡方式，它在维持机体内稳态和发育过程中起着关键作用。

因此，对细胞凋亡的检测方法研究具有重要意义。

在本文中，我们将介绍几种常用的细胞凋亡检测方法，以及它们的优缺点和适用范围。

1. 形态学观察法。

形态学观察法是最早用于检测细胞凋亡的方法之一。

通过光学显微镜观察细胞形态的变化，如细胞体积缩小、胞质浓缩、核染色质凝聚和核小体形成等，来判断细胞是否发生凋亡。

这种方法简单直观，但不适用于高通量检测和定量分析。

2. DNA断裂检测法。

DNA断裂是细胞凋亡的一个显著特征，因此可以通过检测DNA 的断裂来判断细胞是否发生凋亡。

常用的DNA断裂检测方法包括凝胶电泳、TUNEL法和DNA断裂酶切法。

这些方法可以对凋亡细胞进行定量分析，但需要特殊仪器和试剂，操作较为复杂。

3. 蛋白质检测法。

细胞凋亡过程中，一些特定的蛋白质会发生变化，如Bcl-2家族蛋白、caspase家族蛋白等。

因此，可以通过检测这些蛋白质的表达水平或活性来判断细胞是否发生凋亡。

常用的蛋白质检测方法包括Western blot、免疫荧光染色和流式细胞术。

这些方法对于定量分析和高通量检测具有优势，但需要特异性抗体和专门仪器。

4. 细胞色素C释放检测法。

在细胞凋亡过程中，线粒体膜通透性增加，导致线粒体内膜空间的细胞色素C释放到细胞质中。

因此，可以通过检测细胞色素C的释放来判断细胞是否发生凋亡。

常用的方法包括免疫荧光染色和细胞色素C活性检测。

这些方法对于研究凋亡的机制和药物筛选具有重要意义。

综上所述，不同的细胞凋亡检测方法各有优缺点，研究者可以根据具体的实验目的和条件选择合适的方法。

随着生物技术的发展，新的细胞凋亡检测方法也在不断涌现，相信在不久的将来，会有更多更简便、灵敏的方法问世，为细胞凋亡研究提供更多的选择和可能性。

细胞凋亡检测,细胞凋亡实验步骤,检测方法一、定性和定量研究只定性的研究方法：常规琼脂糖凝胶电泳、脉冲场倒转琼脂糖凝胶电泳、形态学观察普通光学显微镜、透射电镜、荧光显微镜进行定量或半定量的研究方法：各种流式细胞仪方法、原位末端标记法、ELISA定量琼脂糖凝胶电泳;二、区分凋亡和坏死可将二者区分开的方法：琼脂糖凝胶电泳,形态学观察透射电镜是是区分凋亡和坏死最可靠的方法,Hoechst33342/PI双染色法流式细胞仪检测,AnnexinV/PI 双染色法流式细胞仪检测等;不能将二者区分开的方法：原位末端标记法、PI单染色法流式细胞仪检测等;三、样品来源不同选择组织：主要用形态学方法HE染色,透射电镜、石蜡包埋组织切片进行原位末端标记,ELISA或将组织碾碎消化做琼脂糖凝胶电泳;四、细胞凋亡检测1、早期检测:1 PS磷脂酰丝氨酸在细胞外膜上的检测2细胞内氧化还原状态改变的检测3细胞色素C的定位检测4 线粒体膜电位变化的检测2、晚期检测：细胞凋亡晚期中,核酸内切酶在核小体之间剪切核DNA,产生大量长度在180-200 bp 的DNA片段;对于晚期检测通常有以下方法：1 TUNEL末端脱氧核苷酸转移酶介导的dUTP缺口末端标记2 LM-PCR Ladder 连接介导的PCR检测3 Telemerase Detection 端粒酶检测3、生化检测：1典型的生化特征：DNA 片段化2检测方法主要有：琼脂糖凝胶电泳、原位末端标记TUNEL等3TUNEL末端脱氧核苷酸转移酶介导的dUTP缺口末端标记4通过DNA末端转移酶将带标记的dNTP 多为dUTP间接或直接接到DNA片段的3’-OH端,再通过酶联显色或荧光检测定量分析结果;可做细胞悬液、福尔马林固定或石蜡处理的组织、细胞培养物等多种样本的检测;4、LM-PCR Ladder 连接介导的PCR检测当凋亡细胞比例较小以及检测样品量很少如活体组织切片时,直接琼脂糖电泳可能观察不到核DNA的变化;通过LM-PCR,连上特异性接头,专一性地扩增梯度片段,从而灵敏地检测凋亡时产生梯度片段;此外,LM-PCR 检测是半定量的,因此相同凋亡程度的不同样品可进行比较;如果细胞量很少,还可在分离提纯DNA后,用32P-ATP和脱氧核糖核苷酸末端转移酶TdT使DNA标记,然后进行电泳和放射自显影,观察凋亡细胞中DNA ladder的形成;上述两种方法都针对细胞凋亡晚期核DNA断裂这一特征,但细胞受到其它损伤如机械损伤,紫外线等也会产生这一现象,因此它对细胞凋亡的检测会受到其它原因的干扰;需结合其它的方法来检测细胞凋亡;其它方法：1Telemerase Detection 端粒酶检测端粒酶是由RNA和蛋白组成,它可以自身RNA为模板逆转录合成端粒区重复序列,使细胞获得“永生化”;正常体细胞是没有端粒酶活性的,每分裂一次,染色体的端粒会缩短,这可能作为有丝分裂的一种时钟,表明细胞年龄、复制衰老或细胞凋亡的信号;研究发现,90%以上的癌细胞或凋亡细胞都具有端粒酶的活性;2mRNA水平的检测研究者们发现了很多在细胞凋亡时表达异常的基因,检测这些特异基因的表达水平也成为检测细胞凋亡的一种常用方法;据报道,Fas 蛋白结合受体后能诱导癌细胞中的细胞毒性T细胞cytotoxic T cells 等靶细胞;Bcl-2 和bcl-X 长的作为抗凋亡bcl-2 和bcl-X的调节物,它们的表达水平比例决定了细胞是凋亡还是存活;用荧光定量PCR技术来检测基因表达水平无疑比之前者更快更准确;通过检测fas, bax-alpha 和bcl-X 长的基因的mRNA表达水平来进行细胞凋亡的检测; 5、细胞内氧化还原状态改变的检测：正常状态下,谷光苷肽GSH作为细胞的一种重要的氧化还原缓冲剂;细胞内有毒的氧化物通过被GSH还原而定期去除,氧化型的GSH又可被GSH还原酶迅速还原;这一反应在线粒体中尤为重要,许多呼吸作用中副产物的氧化损伤将由此被去除;当细胞内GSH的排除非常活跃时,细胞液就由还原环境转为氧化环境,这可能导致了凋亡早期细胞线粒体膜电位的降低,从而使细胞色素C三羧酸循环中的重要组分从线粒体内转移到细胞液中,启动凋亡效应器caspase的级联反应;6、细胞色素C的定位检测细胞色素C作为一种信号物质,在细胞凋亡中发挥着重要的作用;正常情况下,它存在于线粒体内膜和外膜之间的腔中,凋亡信号刺激使其从线粒体释放至细胞浆,结合Apaf-1 apoptotic protease activating factor-1后启动caspase级联反应：细胞色素C/Apaf-1复合物激活caspase-9,后者再激活caspase-3和其它下游caspase; 7、线粒体膜电位变化的检测：1线粒体跨膜电位的耗散与细胞凋亡有密切关系2近年来陆续有报道说明线粒体跨膜电位的耗散早于核酸酶的激活,也早于磷酯酰丝氨酸暴露于细胞表面;而一旦线粒体跨膜电位耗散,细胞就会进入不可逆的凋亡过程;3在细胞凋亡过程中线粒体跨膜电位的耗散主要是由于线粒体内膜的通透性转变,这是由于生成了动态的由多个蛋白质组成的位于线粒体内膜与外膜接触位点的通透性转变孔道PT孔道能稳定线粒体跨膜电位就能防止细胞凋亡;线粒体在细胞凋亡作用中的进一步证据：1若将纯化的正常的线粒体与纯化的细胞核在一起保温,并不导致细胞核的变化;但若将诱导生成PT 孔道的线粒体与纯化的细胞核一同保温,细胞核即开始凋亡变化;2形态学观察,看到细胞数目有限,统计学上的准确性受影响；3凝胶电泳检测DNA破坏了细胞的完整性也不能测出凋亡细胞占总细胞的比例；4流式细胞术可检测细胞、亚细胞及分子水平的特征性变化;用于流式细胞仪检测的染料：PI、Hoechst、EB、DAPI、丫啶橙等,其中PI、Hoechst 最常用;PI和Hoechst33342双标：PI、Hoechst33342均可与细胞核DNA或RNA结合;但是PI不能通过正常的细胞膜,Hoechst则为膜通透性的荧光染料,故细胞在处于坏死或晚期调亡时细胞膜被破坏,这时可为PI着红色;正常细胞和中早期调亡细胞均可被Hoechst着色,但是正常细胞核的Hoechst着色的形态呈圆形,淡兰色,内有较深的兰色颗粒；而调亡细胞的核由于浓集而呈亮兰色,或核呈分叶,碎片状,边集; 故PI着色为坏死细胞；亮兰色,或核呈分叶状,边集的Hoechst着色的为调亡细胞;PI和Annexin-V双标：磷脂酰丝氨酸PS正常位于细胞膜的内侧,但在细胞凋亡的早期或细胞损伤时PS可从细胞膜的内侧翻转到细胞膜的表面,暴露在细胞外环境中; Annexin-Vgreen可以和磷脂酰丝氨酸PS特异性结合;因此细胞处于调亡或坏死时,Annexin-V可为阳性早期的坏死细胞可能为阴性;但是只有坏死的细胞PI是阳性五、形态学观察1、普通光学显微镜观察2、透射电子显微镜观察3、荧光显微镜观察1、普通光镜下观察：1用苏木素-伊红HE染色：细胞核固缩碎裂、呈蓝黑色、胞浆呈淡红色凋亡细胞,正常细胞核呈均匀淡蓝色或蓝色,坏死细胞核呈很淡的蓝色或蓝色消失2Giemsa染色法、瑞氏染色法等,正常细胞核的色泽均一,凋亡细胞染色变深,坏死细胞染色浅或没染上颜色直接用倒置显微镜观察：1细胞体积变小,全面皱缩；2凋亡小体为数个圆形小体围绕在细胞周围;2、透射电子显微镜观察凋亡细胞体积变小,细胞质浓缩;细胞凋亡过程中细胞核染色质的形态学改变分为三期：Ⅰ期的细胞核呈波纹状或呈折缝样,部分染色质出现浓缩状态；Ⅱa期细胞核的染色质高度凝聚、边缘化；Ⅱb期的细胞核裂解为碎块,产生凋亡小体;3、荧光显微镜常用的荧光染料：丫啶橙、PI 、DAPI、Hoechst33258 和Hoechst33342、EB等Hoechst 33342、Hoechst 33258、DAPI三种染料与DNA的结合是非嵌入式的,主要结合在DNA的A-T碱基区;紫外光激发时发射明亮的蓝色荧光;1PI双染色法基本原理Hoechst是与DNA特异结合的活性染料,能进入正常细胞膜而对细胞没有太大得细胞毒作用;Hoechst 33342在凋亡细胞中的荧光强度要比正常细胞中要高;DAPI为半通透性,用于常规固定细胞的染色;碘化丙啶PI是一种核酸染料,它不能透过完整的细胞膜,但在凋亡中晚期的细胞和死细胞,PI能够透过细胞膜而将细胞核染红;因此将Annexin-V与PI匹配使用,就可以将凋亡早晚期的细胞以及死细胞区分开来;注意事项：细胞凋亡时,其DNA可染性降低被认为是凋亡细胞的标志之一,但这种DNA可染性降低也可能是因为DNA含量的降低,或者是因为DNA结构的改变使其与染料结合的能力发生改变所致;在分析结果时应该注意;六、细胞凋亡的分子生物学检测方法:细胞凋亡中染色体DNA的断裂是个渐进的分阶段的过程,染色体DNA首先在内源性的核酸水解酶的作用下降解为50-300kb的大片段;然后大约30 ﹪的染色体DNA在Ca ²+和Mg²+依赖的核酸内切酶作用下,在核小体单位之间被随机切断,形成180～200bp核小体DNA多聚体;DNA双链断裂或只要一条链上出现缺口而产生的一系列DNA的3’-OH末端可在脱氧核糖核苷酸末端转移酶TdT的作用下,将脱氧核糖核苷酸和荧光素、过氧化物酶、碱性磷酸化酶或生物素形成的衍生物标记到DNA的3’-末端,从而可进行凋亡细胞的检测,这类方法一般称为脱氧核糖核苷酸末端转移酶介导的缺口末端标记法TUNEL;由于正常的或正在增殖的细胞几乎没有DNA的断裂,因而没有3’-OH形成,很少能够被染色;低分子量的DNA分离后,也可使用DNA聚合酶进行缺口翻译nick translation,使低分子量的DNA标记或染色,然后分析凋亡细胞;TUNEL或缺口翻译法实际上是分子生物学与形态学相结合的研究方法,对完整的单个凋亡细胞核或凋亡小体进行原位染色,能准确的反应细胞凋亡最典型的生物化学和形态特征,可用于石蜡包埋组织切片、冰冻组织切片、培养的细胞和从组织中分离的细胞的细胞凋亡测定,并可检测出极少量的凋亡细胞,灵敏度远比一般的组织化学和生物化学测定法要高,因而在细胞凋亡的研究中已被广泛采用;过氧化物酶标记测定法原理：脱氧核糖核苷酸衍生物地高辛digoxigenin-11-dUTP在TdT酶的作用下,可以掺入到凋亡细胞双链或单链DNA的3-OH末端,与dATP形成异多聚体,并可与连接了报告酶过氧化物酶或碱性磷酸酶的抗地高辛抗体结合;在适合底物存在下,过氧化物酶可产生很强的颜色反应,特异准确的定位出正在凋亡的细胞,因而可在普通光学显微镜下进行观察;毛地黄植物是地高辛的唯一来源;在所有动物组织中几乎不存在能与抗地高辛杭体结合的配体,因而非特异性反应很低;抗地高辛的特异性抗体与脊椎动物甾体激素的交叉反应不到1％,若此抗体的Fc部分通过蛋白酶水解的方法除去后,则可完全排除细胞Fc受体非特异性的吸附作用;本方法可以用于福尔马林固定的石蜡包埋的组织切片、冰冻切片和培养的或从组织中分离的细胞凋亡测定;一试剂配制1、磷酸缓冲液PBS：磷酸钠盐50mM,NaCl 200mM;2、蛋白酶K200μg/ml,：蛋白酶K 0.02g；PBS 100ml;3、含2%H2O2的PBS缓冲液：H2O2 ；PBS缓冲液;4、TdT酶缓冲液新鲜配：Trlzma碱3.63g用HCl调节pH至,加ddH2O定容到1000ml；再加入二甲砷酸钠29．96g和氯化钴0.238g;5、TdT酶反应液：TdT酶32μl；TdT酶缓冲液76μl,混匀,置于冰上备用;6、洗涤与终止反应缓冲液：氯化钠17. 4g；柠檬酸钠8.82g；ddH2O 1000ml7、％二氨基联苯DAB溶液：DAB 5mg；PBS 10ml,, 临用前过滤后,加过氧化氢至%;8、％甲基绿：甲基绿0.5g；0.1M乙酸钠100ml;9、100％丁醇,100％、95％、90％、80％和70％乙醇,二甲苯,10％中性甲醛溶液,乙酸,松香水等;10、过氧化物酶标记的抗地高辛抗体ONCOR二实验步骤1、标本预处理：1石蜡包埋的组织切片预处理：将组织切片置于染色缸中,用二甲苯洗两次,每次5min;用无水乙醇洗两次,每次3min;用95％和75％乙醇各洗一次,每次3min;用PBS洗5min 加入蛋白酶K溶液20ug／ml,于室温水解15min,去除组织蛋白;用蒸馏水洗4次,每次2min,然后按下述步骤2进行操作;2冰冻组织切片预处理：将冰冻组织切片置10％中性甲醛中,于室温固定10min后,去除多余液体;用PBS洗两次,每次5min;置乙醇：乙酸2：1的溶液中,于-20℃处理5min,去除多余液体;用PBS洗两次,每次5min,然后按下述步骤2进行操作;3培养的或从组织分离的细胞的预处理：将约5 × 107个/ml细胞于4％中性甲醛室温中固定10min;在载玻片上滴加50～100μl细胞悬液并使之干燥;用PBS洗两次,每次5min,然后按下述步骤2进行操作;2、色缸中加入含2％过氧化氢的PBS,于室温反应5min;用PBS洗两次,每次5min;3、用滤纸小心吸去载玻片上组织周围的多余液体,立即在切片上加2滴TdT酶缓冲液,置室温1～5min;4、用滤纸小心吸去切片周围的多余液体,立即在切片上滴加54μl TdT酶反应液,置湿盒中于37C反应1hr注意：阴性染色对照,加不含TdT酶的反应液;5、将切片置于染色缸中,加入已预热到37℃的洗涤与终止反应缓冲液,于37℃保温30min,每10min将载玻片轻轻提起和放下一次,使液体轻微搅动;6、组织切片用PBS洗3次,每次5min后,直接在切片上滴加两滴过氧化物酶标记的抗地高辛抗体,于湿盒中室温反应30min;7、用PBS洗4次,每次5min;8、在组织切片上直接滴加新鲜配制的％DAB溶液,室温显色3～6min;9、用蒸馏水洗4次,前3次每次1min,最后1次5min;10、于室温用甲基绿进行复染10min;用蒸馏水洗3次,前两次将载玻片提起放下10次,最后1次静置30s;依同样方法再用100％正丁醇洗三次;11、用二甲苯脱水3次,每次2min,封片、干燥后,在光学显微镜下观察并记录实验结果;三注意事项一定要设立阳性和阴性细胞对照;阳性对照的切片可使用DNaseI部分降解的标本,阳性细胞对照可使用地塞米松1μM处理3-4hr的大、小鼠胸腺细胞或人外周血淋巴细胞;阴性对照不加TdT酶,其余步骤与实验组相同;一、吖啶橙简称AO荧光染色法原理：吖啶橙Acridine Orange是吖啶的衍生物之一;它是一种荧光染料,激发峰492nm,荧光发射峰530nmDNA,640nmRNA,它与双链DNA的结合方式是嵌入双链之间,而与单链DNA和RNA则由静电吸引堆积在其磷酸根上;在蓝光给502nm激发下,细胞核发亮绿色荧光约530nm,核仁和胞质RNA发桔红色荧光＞580nm;吖啶橙的阳离子也可以结合在蛋白质、多糖和膜上而发荧光,但细胞固定阻抑了这种结合,从而主要显示DNA、RNA两种核酸;1试剂AO贮备液：AO 50mg分析纯蒸馏水 50ml4℃冰箱保存4个月;Tris缓冲液：Tris 50mmol/LMgCl2 LKCl 25mmol/LAO工作液：将AO贮备液10:1用蒸馏水稀释,再用Tris缓冲液将AO稀释到μg/ml的终浓度;2操作步骤①取乙醇固定的细胞悬液,浓度为107/ml,1500r/min,5分钟,弃乙醇;②加入2ml AO工作液,室温染10分钟;③滴在载玻片上,加缓冲甘油封片;④在荧光显微镜下用吸收波长405nm,发射波长530-640nm观察;结果：活细胞核呈黄绿色荧光,胞质呈红色荧光;凋亡细胞核染色质呈黄绿色浓聚在核膜内侧,可见细胞膜呈泡状膨出及凋亡小体;二、Hoechst33258染色Hoechst33258为特异性DNA染料,,与A-T键结合,但在环境下则优先与RNA 结合,染色DNA时应调整染液的pH至,这种染料不溶于磷酸缓冲液；所以配制时必须先以蒸馏水溶解配成储存液在4℃中避光保存;本方法是用于培养细胞、细胞涂片或细胞甩片;试剂及配制1Hoechst33258贮存液：称取Hoechst33258试剂1mg,用20ml蒸馏水溶解后,滤过,4℃避光保存;用时蒸馏水10倍稀释成染色液;2 ,;3封片液：20mmol/L柠檬酸,50mmol/L 磷酸氢二钠,50%甘油;4细胞固定液：甲醇/冰乙酸3:1,现配;操作方法1原代细胞培养、细胞学涂片或细胞甩片机制制备的单细胞片;2细胞固定液4℃固定5min;3蒸馏水稍洗后,点加Hoechst33258染色液,10min;4蒸馏水洗片后,用滤纸沾去多余液体;5封片剂封片后荧光显微镜观察;结果：在荧光显微镜下,活细胞核呈弥散、均匀荧光,坏死细胞不被Hoechst 染色;出现细胞凋亡时,细胞核或细胞质内可见浓染致密的颗粒块状蓝色荧光及明显核形态变化,如果见到3个或3个以上的DNA荧光碎片被认为是凋亡细胞;三、甲基绿-派诺宁染色法一原理细胞凋亡和细胞坏死均可表现为细胞核固缩等细胞死亡形态,但两者发生机制不同;细胞凋亡是一种细胞主动死亡过程,需要有细胞内蛋白酶的激活,细胞质内常有mRNA表达的增强;而细胞坏死是一种被动的细胞死亡过程,细胞质内常有RNA的损失;根据这一特点,可应用试剂甲基绿对DNA染色的特异性和派诺宁对RNA的亲和性,使甲基绿对固缩细胞核内的脱氧核糖核酸着染,如果细胞质内核糖核酸呈派诺宁阳性染色者为凋亡细胞,呈阴性染色者为坏死细胞;二试剂及配制1. 组织固定液无水乙醇 600ml氯仿 300ml冰醋酸 100ml2.甲基绿纯化新购买的甲基绿需用氯仿进行纯化处理以去除甲基紫;方法是将2％甲基绿水溶液20ml倾入洁净分液漏斗,加入氯仿20ml充分,使其内的甲基溶于氯仿中而呈紫红色;旋动分液漏斗下部的砂塞,慢慢把下沉带比红色的氯仿移去,再加入新的氯仿10ml,如此反复更换氯仿,直到无紫红色为止;该液作为贮存液,4℃保存;3.染色液甲基绿贮存液 5ml5%派诺宁水溶液 1ml蒸馏水 12mlL乙酸钠 18ml临用前配制,滤纸过滤;三操作方法1.新鲜取材组织置固定液中4℃固定3-6h或培养细胞、细胞学甩片固定10min;2.直接转入95%乙醇脱水和无水乙醇脱水,二甲苯透明,石蜡包埋;3.切片经二甲苯脱蜡,梯度乙醇水化至蒸馏水;细胞学涂片不用梯度酒精4.置染色液中室温下染色约1h;5.取出切片,不经水洗,用滤纸吸干多余染液;6.插入丙酮中迅速分化;7.转入丙酮二甲苯1:1稍洗;8.二甲苯透明2-3次;9.中性树胶封固;四结果光学显微镜下凋亡细胞固缩,细胞核呈绿色或绿蓝色着染,胞质呈红紫色着染,坏死细胞只有固缩细胞核呈绿色着染;观察时可用凋亡指数进行计数,即随机选择约10-20个视野每张切片约1000-2500个细胞,计数凋亡细胞百分率;四、TdT介导的dUTP缺口末端标记技术TUNEL原理：在机体内部随时都在发生着细胞的死亡;传统上用显微镜来观察细胞的死亡,其特征为核染色质的浓缩及碎片的形成;但是这种现象出现的很晚,时间也很短暂;凋亡的特征是内源性核酸内切酶被激活,细胞自身的染色质或DNA被切割,出现单链或双链缺口,并产生与DNA断点数目相同的3ˉ-OH 末端;末端脱氧核糖核酸转移酶Terminal deoxynucleotidyl Transferase TdT可以将地高辛标记的dUTPDIG-dUTP标记至3’-OH末端,DIG-dUTP核苷酸结合在DNA断点部位,可以通过生物标记的抗地高辛抗体Anti-DIG-Biotin反应后,再结合链酶亲和素-过氧化物酶SABC,然后加入显色底物DAB予以显示;凋亡的细胞核呈棕黄色,从而可以在显微镜下观察到着色的凋亡细胞;试剂1.标记缓冲液Labeling Buffer5×浓度的TdT反应缓冲液,含有以下成分：500mmol/L二甲胂酸钾;10mmol/L CoCl2 氯化钴1mmol/L DTT二硫苏糖醇2.末端脱氧核糖核酸转移酶TdT,×20×204.封闭液Blocking Reagent5.生物素化抗地高辛抗体×100×100×2008.抗体稀释液9. 多聚赖氨酸或APES;10. 0.01M TBS,配法：1L双蒸馏水中加入8.5克氯化钠,1.2克Tris和纯乙酸;11. DAB显色试剂;操作步骤1.样品处理1玻片预先用多聚赖氨酸或APES进行处理;2细胞涂片和冰冻切片：最重要的是及时固定;用4%多聚甲醛/0.01M PBS室温下固定30-60分钟;0.01M PBS洗2分钟×2次;蒸馏水洗涤2分钟×2次; 3组织：有条件时应及时固定;常规4%多聚甲醛/0.01M PBS或10%中性缓冲福尔马林固定4小时以上,石蜡包埋;切片常规脱蜡入水脱蜡务必干净;2.新鲜配制3%H2O2,室温处理10分钟;蒸馏水洗涤2分钟×3次;3.标本片加0.01M TBS1:200新鲜稀释Proteinase K37℃消化5-15分钟,0.01M TBS洗2分钟×3次;细胞涂片和冰冻切片一般不消化或消化10-60秒钟;新鲜石蜡切片消化5-10分钟;陈旧石蜡切片消化10-30分钟;4.标本片加标记缓冲液Labeling Buffer20μl/片,以保持切片湿润;按每张切片取TdT和DIG-d-UTP各1μl,加入18μl标记缓冲液中,混匀;甩去切片上多余液体后加标记液,20μl/片;置样品于湿盒中,37℃标记2小时;5.0.01M TBS洗2分钟×3次;6.加封闭液50μl/片,室温30分钟,甩掉封闭液,不洗;7.用抗体稀释液1:100稀释生物素化抗地高辛抗体：取1ml抗体稀释液加生物素化抗地高辛抗体10μl,混匀后50μl/片加至标本片上;置样品于湿盒中,37℃反应30分钟;0.01M TBS洗2分钟×3次;8.用抗体稀释液1:100稀释SABC：取1ml抗体稀释液加SABC10μl,混匀后50μl/片加至切片;37℃反应30分钟;0.01M TBS洗5分钟×4次;显色;10.苏木素轻度复染;脱水,透明,封片;显微镜观察;结果判定细胞核固缩有的呈碎片状,不规则,大小不一致,呈棕黄色颗粒者为阳性细胞; 即凋亡的细胞TUNEL改良方法在细胞凋亡检测中的应用.0M枸橼酸盐缓冲液、通透液%Triton-100、枸橼酸钠、标记缓冲液：二甲胂酸钠100mmol/L标记、二巯基苏糖醇L,氯化钴coci;TdT反应液；标记缓冲液中加TdT酶100U/ml,biotin-dUTP；链霉菌素标记的辣根过氧化物酶,蛋白酶K20μg/ml;显色剂氨乙基咔唑amino ethyl carbazole,AEC的配制AEC 20mg二甲酰胺DMF0.05M醋酸缓冲液 50ml%H2O2实验步骤1切片用冷风吹干后为防止冰冻切片脱片用1%火棉胶封片1分钟,PBS漂洗3×5分钟；23%H2O2阻断10分钟后PBS洗3×5分钟；3组织切片浸泡于盛有L枸橼酸盐缓冲液的烧杯中,置于微波炉内,在650W,辐射时间15min的条件下进行组织处理;冷却至室温;4放在通透液%Triton -100溶于%枸橼酸钠溶液中室温20分钟,PBS漂洗3×5分钟；5滴加50μl TdT反应液37℃1h,PBS漂洗3×5分钟；6滴加50μl biotin-dUTP,反应液37℃1h,PBS漂洗3×5分钟；7滴加50μl链霉菌标记辣根过氧化物酶液37℃孵育30分钟,8PBS漂洗3×5分钟, AEC-H2O2显色10-15分钟,苏木素复染用1%的酸性水分化,水洗、水溶性封片;阳性对照dTd 反应前用20μg/ml蛋白酶K处理切片;阴性对照用PBS代替dTd反应液; 结果判断及标准改良的方法：凋亡细胞核呈红色固缩,有白边集或碎列,细胞膜皱褶,有的卷曲和出泡,在计数时避开坏死区域,以避免假阳性;计数500个细胞中的凋亡细胞数目,得出凋亡百分率;凋亡染色分度如下：每个高倍视野平均1-3个为Ⅰ级；3-6个为Ⅱ级；6-10个为Ⅲ级；>10个者为IV级;阳性对照：经在dTd反应认前用20μg/ml蛋白酶,处理切片30分钟的切片同改良方法基本相同,但红色较浅;阴性对照：切片无棕色反应;评价正常的或正在增殖的细胞没有DNA的断裂,没有3’-OH末端被标记,因此镜下无显色的物质;这种分子生物学与形态学相结合的方法,可以对完整的单个凋亡细胞核或凋亡小体进行标记,准确反应细胞凋亡最典型的生物化学与形态学特征,灵敏度远远高于一般的组织化学和生物化学方法;在检测过程中,可设阴性对照不加TdT阳性对照已知的阳性标本;注意:AEC孵育切片,反应产物为红色;可用苏木精对衬染色,反应产物是纯酒精溶性的,因此;可用酸性水溶液分化,然后用水溶性封固剂封片;五、凋亡细胞：凝胶电泳检测前已提及凋亡细胞的突出特征是由于内源性核酸内切酶的激活而导致细胞核DNA的断裂;典型的细胞凋亡,在核内形成大量200bp大小及其倍体的核苷酸片段,而非典型的凋亡细胞,由于核内的DNA降解不完全,仅形成300~50kb的DNA大片;利用这一特性,可通过DNA凝胶电泳来判定凋亡的发生和发展情况;试剂1. PBS缓冲液2. 消化液的配制：100mmol/L NaCl10mmol/L Tris-HCL25mmol/L EDTA%SDSml蛋白酶K3. 酚：氯仿：异戊醇混合液按25:24:1比例配制;4. 氯仿：异戊醇混合液按24:1比例配制;5. 醋酸铵,L;6. 100%和70%的乙醇;7. TE 缓冲液：100mmol/L Tris-HCL,5mmol/L EDTA;8. TBE缓冲液;9. 电泳仪;。

一、实验目的1. 掌握动物细胞培养的无菌操作技术。

2. 了解并掌握用组织块贴壁培养法和消化细胞培养法进行动物细胞原代培养的实验方法。

3. 熟练掌握动物细胞传代培养的实验方法。

4. 学习细胞生死状态鉴别的方法，掌握细胞生死状态鉴别的原理。

5. 熟悉和掌握各种鉴别细胞生死状态方法的判定特征。

6. 掌握细胞技术方法，计算细胞存活率。

二、实验原理细胞培养是在无菌条件下，将动、植物细胞从组织中取出，在体外模拟体内的生理环境，使离体的细胞在体外生长和繁殖，并维持其结构和功能的一种培养技术。

细胞生死状态的鉴别方法主要包括化学染色法和荧光染色法。

活细胞和死亡细胞在生理功能和性质上存在以下差异：1. 细胞膜通透性的差异：活细胞的细胞膜是一种选择性膜，对细胞起保护和屏障作用，只允许物质选择性地通过；而细胞死后，细胞膜受损，其通透性增加。

基于此，发展出了以台盼蓝、伊红、苯胺黑、赤藓红、甲基蓝以及荧光染料碘化丙啶或溴化乙啶等为染料鉴别细胞生死状态的方法。

上述染料能使死亡细胞着色，而活细胞不被着色。

2. 代谢上的差异：活细胞能够进行正常的代谢活动，如糖酵解、蛋白质合成等；而死亡细胞代谢活动减弱或停止。

三、实验材料1. 实验动物：小鼠2. 实验试剂：台盼蓝染液、生理盐水、显微镜、盖玻片、载玻片、吸管、剪刀、镊子等3. 实验仪器：恒温培养箱、离心机、细胞培养瓶、移液器等四、实验步骤1. 组织块贴壁培养法（1）取小鼠肝脏组织，用生理盐水清洗后，剪成1mm×1mm×1mm大小的组织块。

（2）将组织块接种于培养瓶中，加入适量DMEM培养液，放入恒温培养箱中培养。

（3）待细胞贴壁生长后，进行细胞传代培养。

2. 细胞传代培养（1）将培养瓶中的细胞用胰酶消化后，收集细胞悬液。

（2）将细胞悬液离心，弃去上清液，加入适量DMEM培养液重悬细胞。

（3）将重悬后的细胞按1:2比例接种于新的培养瓶中，放入恒温培养箱中培养。

3. 死活细胞鉴别（1）取一定量的细胞悬液，加入适量台盼蓝染液，混匀。

This article was originally published in a journal published by Elsevier,and the attached copy is provided by Elsevier for the author’s benefit and for the benefit of the author’s institution,for non-commercial research and educational use including without limitation use in instruction at your institution,sending it to specific colleagues that you know,and providing a copy to your institution’sadministrator.All other uses,reproduction and distribution,including without limitation commercial reprints,selling or licensing copies or access, or posting on open internet sites,your personal or institution’s website or repository,are prohibited.For exceptions,permission may be sought for such use through Elsevier’s permissions site at: /locate/permissionusematerialA u th or 'spe rs on alc op yAcenaphtho[1,2-b ]pyrrole derivatives as new family of intercalators:Various DNA binding geometry andinteresting antitumor capacityZhichao Zhang,a Yuanyuan Yang,b Danni Zhang,a Yuanyuan Wang,aXuhong Qian a,c,*and Fengyu Liu d,*aState Key Laboratory of Fine Chemicals,Dalian University of Technology,Dalian 116012,ChinabSchool of Environmental and Biological science and Technology,Dalian University of Technology,Dalian 116024,China cShanghai Key Laboratory of Chemical Biology,East China University of Science and Technology,Shanghai 200237,ChinadChemistry department of Dalian University of Technology,Dalian 116012,ChinaReceived 12May 2006;revised 15June 2006;accepted 16June 2006Available online 7July 2006Abstract—A series of acenaphtho[1,2-b ]pyrrole derivatives were synthesized and their intercalation geometries with DNA and anti-tumor activities were investigated in detail.From combination of SYBR Green–DNA melt curve,fluorescence titration,absorption titration,and circular dichroism (CD)studies,it was identified that to different extent,all the compounds behaved as DNA inter-calators and transformed B form DNA to A-like conformation.The different intercalation modes for the compounds were revealed.The compounds containing a methylpiperazine substitution (series I )intercalated in a fashion that the long axis of the molecule par-alleled to the base-pair long axis,while the alkylamine-substituted compounds (series II and III )located vertically to the long axis of DNA base pairs.Consequently,the DNA binding affinity of these compounds was obtained with the order of II >III >I ,which attributed to the role of the substitution in binding geometry.Further,cell-based studies showed all the compounds exhibited out-standing antitumor activities against two human tumor cell lines with IC 50ranging from 10À7to 10À6M.Interestingly,compound 1a (a compound in series I ),whose binding affinity was one of the lowest but altered DNA conformation most significantly,showed much lower IC 50value than other compounds.Moreover,it could induce tumor cells apoptosis,while the compounds 2a and 3a (in series II and III ,respectively)could only necrotize tumor cells.Their different mechanism of killing tumor cells might lie in their different DNA binding geometry.It could be concluded that the geometry of intercalator–DNA complex contributed much more to the antitumor property than binding affinity.Ó2006Elsevier Ltd.All rights reserved.1.IntroductionThere is substantial and continuing interest in artificial molecules that bind and interact with DNA.Most of these molecules have been studied with the purpose of developing novel antitumor lead compound.1–3Studies on the binding geometry of various ligands to DNA have revealed that the biological activity correlated with not only their DNA binding affinity,but also the bind-ing mode.4,5For example,Viola G.found the more effi-ciency of DNA cleavage of externally bound dye compared with the intercalated one.6Cholody W.M.demonstrated that the best model to account for the excellent antitumor property of BIAs involved the inter-calation of one chromophore while the other resided in the minor groove.7,8So,understanding the nature of binding geometry of ligand–DNA complex may contrib-ute to the design of more efficient antitumor leads.The heterocyclic molecules containing a flat,generally p -deficient aromatic system form a famous class of anti-tumor agents.9–12They could bind to DNA by intercala-tion between base pairs of the double helix.Recently,we reported a series of electron-deficient acenaphtho-het-erocyclic compounds as novel antitumor leads,13,148-oxo-8H -acenaphtho[1,2-b ]pyrrole-9-carbonitrile and its bining the results of gel-shift studies and binding mode studies,it was illustrated that the dif-ferent binding mode of these compound to DNAowingBioorganic &Medicinal Chemistry 14(2006)6962–6970Keywords :Antitumor agents;DNA;Intercalation;Geometry.*Corresponding authors.E-mail addresses:xhqian@ ;annyuer_liu@h or 'speo p yto their different side chain rendered them different anti-tumor potency.These results offered us a promising platform to design novel antitumor lead compound.In addition,it promoted us to gain insight into the key structural features that account for binding geometry and consequently the antitumor potency,and the possi-ble relationship between the binding mode of ligand–DNA and its antitumor property.To this end,we further synthesized nine derivatives characterized by various substitutions with different length,flexibility,and p -deficiency.The binding geome-try of these compounds to DNA was investigated by means of molecular spectra,and cell-based assay was performed to evaluate their antitumor properties.Through the SAR (structure–activity relationship)stud-ies,we expected to find the constituent for more efficient biological and medical application.2.Results and discussion2.1.Synthesis and spectraThe structures of the new compounds are shown in Scheme 1.They were divided into three series I ,II ,and III according to methylpiperazine,dimethylethane,and dimethylpropane side chain.Spectral data of them were measured and are summarized in Table 1.2.2.Binding affinityThe intercalation of the nine compounds to CT DNA was evaluated by SYBR Green–DNA melt curve.SYBR Green could intercalate all double-stranded DNA.Its self-fluorescence intensity is very weak but increases greatly when intercalates into DNA.15,16Figure 1(curve j )shows as the temperature increased from 45–95°C and CT DNA became single-stranded,the fluorescence signal decreased due to the SYBR Green molecules re-leased.When the nine compounds were added to the SYBR Green–DNA complexes,different fluorescence curves were exhibited due to their different binding affin-ity with DNA.The obtained flat curves strongly indicat-ed the seven compounds (1a ,2a ,2b ,2c ,3a ,3b ,and 3c )competed with all of the SYBR Green molecules for the intercalation sites.Obviously,their DNA-intercalat-ing properties were stronger than the typical intercala-tor–SYBR Green.The much weaker and the weakest competition were observed for 1b and 1c ,respectively.The results proved the binding strength variations among these compounds.To identify the binding affinity to DNA,the Scatchard binding constants of the nine compounds were deter-mined through fluorescence titration.The fluorescenceTable 1.The spectral characteristics of the nine compounds in Tris–HCl buffer (pH 7.0)CompoundLog e Absorption k max (nm)Emission k max (nm)Stock shift (nm)1a 3.417590603132a 3.0736********a 2.857602613111b 3.546570587172b 3.220593608153b 3.167592608161c 3.302572589152c 3.141594610163c3.08259461117Z.Zhang et al./Bioorg.Med.Chem.14(2006)6962–69706963A uon alc op yof these compounds was quenched upon addition of CT DNA.The binding constants K b were calculated (Fig.2)and are summarized in Table 2with the order of series II >III >I .The compounds of series I containing meth-ylpiperazine substitution exhibited weaker affinity to DNA,while the much higher affinity was observed for alkylamine-substituted compounds of series I and III .These results were in good agreement with the SYBR Green–DNA melt curve experiment.In addition,the DNA affinity decreased in the order,CN >COOCH 3>COOCH 2CH 2Br-introduced compounds.The stacking of p -bond interactions between the elec-tron-deficient chromophore and the electron-rich pur-ine–pyrimidine base plays an important role on DNA binding affinity.10–12,17As anticipated,the higher affinity was found for CN-introduced compounds,because the strongly electron-withdrawing moiety of the CN group led to higher electron-deficiency of planar ring system.Although COOCH 2CH 2Br group was characteristic of stronger electron-withdrawing than COOCH 3owing to the bromine atom,the lower DNA affinity was found for the former one.The more steric hindrance of larger moiety-COOCH 2CH 2Br appeared to be responsible for it.Another crucial structural feature for DNA binding affinity of intercalators is the properties of side chainon naphthalene moiety,such as the length and rigidi-ty.17–19The data in Table 2clearly indicated the weaker affinity for series III than II ,which was much likely ow-ing to the longer length of the substitution that sterically hindered the intercalation of the chromophore into the DNA base pairs.Similarly,the steric hindrance of semi-rigid methylpiperazine substitution resulted in the weak-est affinity of series I .The n (apparent number of independent binding sites of DNA)values were also calculated by Scatchard analysis,as shown in Table 2.The n varied remarkably among the three series,much likely due to the difference in their chemical structure.The very small n of series I are consistent with their poor binding affinities.2.3.Absorption titrationTo further investigate the DNA binding mode of the three series of compounds to DNA and understand if the two kinds of substitution,methylpiperazine and alkylamine,played a different role in the process of binding to DNA,the compounds 1a ,2a ,and 3a were selected to perform the titration absorption spectra by CT DNA due to their highest binding affinity in each series (Fig.3).In the case of compound 1a ,addi-tion of DNA induced hypochromicity (32%)and a small red shift of the absorption maximum (2nm)in the UV–vis spectra.A significantly difference and unusual phenomenon,however,was obtained for com-pounds 2a and 3a .Two bands were observed for the complexes.At low DNA/compound ratio,hypochrom-icity was found for the two bands.As the DNA con-centration was increased,the spectra for the longer band showed pronounced hyperchromicity and batho-chromic shift.The maximum bathochromic shifted about 12and 16nm as compared with that of the free 2a and 3a ,respectively.The hypochromicity of the shorter band continued.The distinguishable results of the spectrophotometric titration for series I and II or III could be interpreted as the existence of the different binding mode between the compounds containing methylpiperazine and alkyl-amine substitution.Significant hypochromicity and a slight red shift phenomenon exhibited in 1a –DNA com-plex revealed a classical intercalation of 1a –DNA.20,21While the absorption spectra of 2a and 3a recorded during the increase of DNA concentration could infer that two-step binding process occurred.13The first step occurred at low DNA/compound ratio,which spectra might attribute to the complexation and stacking of the compounds along DNA surface.This cooperative binding originated from intercalation and electrostatic attraction of the positively charged substitution and the negatively charged phosphate groups of DNA.22The second step occurred at high DNA/compound ra-tio,which spectra reflected the formation of a complete intercalation mode.In summary,their DNA intercala-tion geometry was very different from that of 1a .It is reasonable to infer the stacking of series II and III was driven by the flexibility of the alkylamine substitution.Table 2.The DNA binding affinities and site densities for the nine compounds Compound K b ·106(M À1)n (Sites per base)R 21a 0.813±0.0350.084±0.0020.9971b 0.458±0.0110.010±0.0020.9991c 0.035±0.0030.050±0.0010.9912a 2.753±0.2920.171±0.0110.9832b 1.930±0.3490.302±0.0250.9843b 1.782±0.2210.285±0.0140.9783a 1.803±0.0430.216±0.0020.9993b 1.690±0.1950.286±0.0160.9933c1.555±0.1630.305±0.0130.9896964Z.Zhang et al./Bioorg.Med.Chem.14(2006)6962–6970n alc op y2.4.Induced circular dichroismTo further distinguish the intercalation geometry of ser-ies I and II or III ,circular dichroism spectra were per-formed and the role of the two kinds of substitutions in binding geometry to DNA was investigated.To illus-trate how the compound located in the intercalation pocket,the induced circular dichroism (ICD)of 1a ,2a ,and 3a was performed at DNA/compound ratio of 10,where all the compounds showed intercalative mode.According to experiments and theoretical studies,an intercalated chromophore centered near the helix axis of DNA should exhibit negative ICD for all long-wave-length transitions polarized parallel to the long axis of the base-pair pocket,while transitions perpendicular to this direction,but still in the plane of the nucleobases (i.e.,parallel to the pseudo-dyad axis),should give posi-tive ICD.23–26As shown in Figure 4,when CT DNA was added,a negative ICD signal was observed for 1a which contained a methylpiperazine substitution,suggesting that 1a intercalated into DNA with its long axis parallel to the base-pair long axis.On the contrary,the two alkylamine-substituted compounds 2a and 3a exhibited positive ICD signals.It indicated a vertical orientation in the intercalation pocket (Fig.5).The different ICD signals additionally supported the notion that the com-pound 1a and 2a or 3a intercalated into DNA in different fashion.So far,the binding affinities of the three series of compounds could be explained further.In the case of series II and III ,because chromophore intercalated into DNA in the way that long axis of the molecule perpendicularly oriented to the base-pair long axis,the alkylamine group exposed on DNA surface.By con-trast,due to the parallel orientation of series I in DNA base-pair pocket,the semirigid methylpiperazineFigure 5.Molecular modeling of 1a –DNA complex ((A)long axis of 1a was oriented parallel to the long axis of DNA base pairs)and 2a –DNA complex ((B)long axis of 2a was vertical to the long axis of DNA base pairs).Z.Zhang et al./Bioorg.Med.Chem.14(2006)6962–69706965A u thn alc op ymoiety involved much in the binding progress.So the binding affinities of series I were lower than those of ser-ies II and III .In addition,the 2a –DNA complex showed larger ICD signal than 3a –DNA,suggesting that the length of the substitution did affect the intercalation into DNA and short side chain on naphthalene be helpful for intercalation,which was in consistent with the binding affinity results.2.5.DNA conformational changesThe intrinsic CD spectra of DNA were applied to under-stand the potential of the present compounds changing the DNA conformation.As shown in Figure 6,the CD spectrum of free CT DNA exhibited a negative band at 244nm due to the helicity and a positive band at 275nm due to the base stacking,which was the charac-teristic of DNA in the right-hand B form.27,28When the compounds were incubated with CT DNA,the CD spec-trum of DNA underwent changes in both the positive and negative bands (Table 3).The increase of the posi-tive bands and decrease of the negative bands with no significant red shift was observed for all the compoundsof the three series,which was consistent with the B to A-like conformational change.29,30The DNA helical band at 244nm corresponding to the DNA unwound extent exhibited decrease for all the compounds,with the same orders as that of binding affinity,1a >1b >1c ,2a >2b >2c ,and 3a >3b >3c .Consequently,in each of the three series,the CN-introduced compounds caused the B form DNA to the most unwound form regardless of the substitution.In particular,it is worth noting that 1a ,whose binding affinity was one of the lowest,led to the most significant base pair stacking at 275nm.This may attribute to its intercalation geometry,which was different from that of compounds 2a and 3a .It was exciting to evaluate 1a and other compounds on living species to explore the features that contributed to the biological activity,bind-ing affinity,binding mode,or both.2.6.CytotoxicityThe nine compounds were tested for in vitro cytotoxicity against human cervical carcinoma (HeLa)cell line and Human caucasian breast adenocarcinoma (MCF-7)cell line utilizing the MTT assay.All the compounds exhibited outstanding cytotoxic activity with IC 50rang-ing from 10À7to 10À6M 31,32(Table 4).Compounds 1c ,2c ,and 3c ,which contain a COOCH 2CH 2Br group in the chromophore,were the strongest growth inhibitors against both the two cell lines.It is acceptable because halogenated hydrocarbons are well-known toxicants.33,34Interestingly,no obvious correlation was found between their DNA binding affinities and antitumor activities.The higher cell killing ability was astoundingly found in the series I whose binding affinity was one order of magnitude lower than that of series II and III .The possible model to account for this behavior involved the different DNA binding geometry between the com-pounds containing methylpiperazine and alkylamine substitution.One used to believe that for DNA interca-lating agents,the main mode of action was potent inhib-it of nucleic acid synthesis,which enhanced linearly upon their DNA binding affinity.10,17Recently,more and more research revealed that the formation of a ter-nary complex of DNA,intercalator,and a critical DNA binding protein was much important for the antitumor property,which is related with the binding geometry of ligand–DNA complex.7,8So we hypothesized that 1a could change the DNA conformation so significantly that the 1a –DNA complex might interfere with some critical DNA binding protein.The consequent event of 1a intercalating into DNA was,at least partly,the mech-anism for its cell killing ability.To this point,the antitu-mor molecular mechanism of series I should be different from that of the series II and III .pound 1a induced apoptosis in vitroThe mechanism of compounds 1a ,2a ,and 3a causing cell death was investigated.Under fluorescence microscope,the live,dead,and apoptotic cells were distinguished clearly through color and morphology by means ofTable 3.CD parameters for the interaction of CT DNA with the compoundsCompoundMolecular ellipticities [É]·10À5(deg cm 2dmol À1)Positive band (276nm)Negative band (244nm)DNA 50l M 71628644DNA+1a 10l M 130605540DNA+1b 10l M 107086420DNA+1c 10l M 72118755DNA+2a 10l M 122424454DNA+2b 10l M 120945966DNA+2c 10l M 118086791DNA+3a 10l M 110214389DNA+3b 10l M 109435847DNA+3c 10l M1147472196966Z.Zhang et al./Bioorg.Med.Chem.14(2006)6962–6970A u th or 'spe rs on alc op ytwo-color test.Figure 7shows 1a induced MCF-7cell apoptosis in a dose-dependent manner.After 24h expo-sure of 1l M 1a ,25.6%cells exhibited apoptosis,most of which were in early phase of apoptosis (green cells whose nuclei present pyknosis),and 12.3%necrotic cell stained as brown cells with nuclear morphology resem-bling that of viable cells.At this time point,5l M 1a caused 48.3%cell apoptosis,among which cells under-went later cell apoptosis were available (moon-like brown cells whose nuclei present pyknosis),and 23.7%necrotic cells.By contrast,1l M 2a necrotized 22.5%MCF-7cells,without any significant apoptosis.The similar phenomenon was found for 3a .As a tightly con-trolled network of protein–protein interaction,apopto-sis was so complicated that multiple protein families were involved.Now it was difficult to assess which pro-tein or factors were interfered with 1a or 1a –DNA com-plex,but it was clear that except for the cytotoxic function which was the same as compounds 2a and 3a ,1a did lead to cell spontaneous programmed death.In summary,a detailed description of the DNA binding affinity,geometry,and antitumor potential of acenaph-tho[1,2-b ]pyrrole derivatives was obtained.These series of compounds presented a novel promising platform for the development of antitumor lead compound.In partic-ular,the role of methylpiperazine and alkylamine substi-tution in both the DNA binding affinity and antitumor property was analyzed.It could be concluded that the binding geometry contributed not only to the binding affinity,but also to the antitumor capacity.In our case,binding geometry contributed much more than binding affinity to the antitumor property of the compounds.3.Experimental3.1.SynthesisAll the solvents were of analytical grade.8-oxo-8H -ace-naphtho[1,2-b ]pyrrole-9-carbonitrile and its derivatives were prepared according to our previous report.35Thesynthesis route of 8-oxo-8H -acenaphtho[1,2-b ]pyrrole-9-carboxylic acid methyl ester,8-oxo-8H -acenaph-tho[1,2-b ]pyrrole-9-carboxylic acid-2-bromo-ethyl ester,and their derivatives referred to another previous publi-cation of us.36Melting points were determined by an X-6micro-melting point apparatus (China)and are uncorrected.The 1H NMR spectra were obtained with Bruker AV-400spectrometer (USA)with chemical shifts reported as ppm (in CDCl 3/DMSO-d 6,TMS as internal standard).The IR spectra were measured using a Perkin-Elmer 2000FTIR instrument (USA).High-reso-lution mass spectra were obtained on HPLC-Q-Tof MS (Micro)spectrometer (USA).Column chromatography was performed using silica gel 200–300mesh.3.1.1.3-(4-Methyl-piperazin)-8-oxo-8H -acenaphtho[1,2-b ]pyrrole-9-carbonitrile (1a).Yield 28%;dark purple sol-id;mp:201–202°C;1H NMR (400MHz,CDCl 3),d =8.68(d,J =7.6Hz,1H),8.50(d,J =8.4Hz,1H),8.12(d,J =8.4Hz,1H),7.82(dd,J =8.4,7.6Hz,1H),7.06(d,J =8.4Hz,1H), 3.67(t,J =4.6Hz,N(C H *2CH 2)2NCH 3,4H), 2.77(t,J =4.6Hz,N(CH 2CH *2)2NCH 3,4H), 2.45ppm (s,NCH 3,3H).IR (KBr):m =3423,2941,2214,1623,1572cm À1.HRMS (ESI)m /z :329.1415[M+H]+(m /z calcd for [C 20H 17N 4O]+:329.1402).3.1.2.3-(2-Dimethylamino-ethylamino)-8-oxo-8H -ace-naphtho[1,2-b ]pyrrole-9-carbonitrile (1b).Yield 38%;dark purple solid;mp:>300°C;1H NMR (400MHz,DMSO-d 6)d =8.99(d,J =7.6Hz,1H),8.64(d,J =7.6Hz,1H),8.02(d,J =9.2Hz,1H),7.93(dd,J =8.0,8.0Hz,1H),7.06(d,J =9.2Hz,1H),3.79(br s,NHC H *2CH 2,2H),2.93(br s,NHCH 2C H *2,2H),2.44ppm (s,N(CH 3)2,6H).IR (KBr):m =2949,2212,1631,1577,1526cm À1.HRMS (ESI)m /z :343.1557[M+H]+(m /z calcd for [C 19H 17N 4O]+:343.1559).3.1.3.3-(3-Dimethylamino-propylamino)-8-oxo-8H -ace-naphtho[1,2-b ]pyrrole-9-carbonitrile (1c).Yield 38%;dark purple solid;mp:>300°C;1H NMR (400MHz,DMSO-d 6)d =8.86(d,J =8.0Hz,1H),8.58(d,Table 4.Cytotoxicity evaluation of the nine compounds against HeLa and MCF-7cell line in vitro (IC 50,l M)Compound 1a1b1c2a2b2c3a3b3cHeLa 1.76 2.330.797.368.80.72 4.028.56 1.38MCF-72.1 1.9 1.18.88.60.33 1.79.10.44Figure 7.LIVE/DEAD/APOPTOSIS two-color cell viability experiment:(A)untreated MCF-7cells (live,negative control),(B)cells exposed to 1l M 1a for 24h (cells indicated by arrows are early apoptotic cells),(C)cells exposed to 5l M 1a for 24h (later apoptotic cells),and (D)cells exposed to 5l M 2a for 24h (dead cells).Z.Zhang et al./Bioorg.Med.Chem.14(2006)6962–69706967A u th or 'spe rs on alc op yJ =7.6Hz,1H),7.95(d,J =9.2Hz,1H),7.90(dd,J =8.0,7.6Hz,1H),7.02(d,J =9.2Hz,1H),3.65(t,J =6.7,7.1Hz,NHC H *2CH 2,2H), 3.28(m,C H *2N(CH 3)2,2H),2.30(s,N(CH 3)2,6H),1.92ppm (m,CH 2,2H).IR (KBr):m =2927,2821,2202,1625,1572,1535cm À1.HRMS (ESI)m /z :329.1409[M ÀH]À(m /z calcd for [C 20H 17N 4O]À:329.1402).3.1.4.3-(4-Methyl-piperazin)-8-oxo-8H -acenaphtho[1,2-b ]pyrrole-9-carboxylic acid methyl ester (2a).Yield 32%;dark purple solid;mp:210°C dec;1H NMR (400MHz,CDCl 3):d =9.01(d,J =8.0Hz,1H),8.77(d,J =7.6Hz,1H),8.46(d,J =8.4Hz,1H),7.78(dd,J =8.0,7.6Hz,1H),7.17(d,J =8.4Hz,1H),3.55(br s,N(C H *2CH 2)2NCH 3,4H),3.17(s,COOC H *3,3H),3.46(br s,N(CH 2C H *2)2NCH 3,4H),2.48ppm (br s,NCH 3,3H).IR (KBr):m =2929,1765,1702,1629,1572,1509cm À1.HRMS (ESI)m /z :362.1449[M+H]+(m /z calcd for [C 21H 20N 3O 3]+:362.1505).3.1.5.3-(Dimethylamino-ethylamino)-8-oxo-8H -acenaph-tho[1,2-b ]pyrrole-9-carboxylic acid methyl ester (2b).Yield 38%;dark purple solid ;mp:220°C dec;1H NMR (400MHz,DMSO-d 6):d =8.84(d,J =8.8Hz,2H),8.62(d,J =7.2Hz,1H),7.85(dd,J =7.2,8.0Hz,1H),7.02(d,J =8.8Hz,1H), 3.71(br s,NHC H *2CH 2,2H),2.98(s,COOCH 3,3H),2.84(br s,2H,C H *2N(CH 3)2),2.41ppm (s,CH 2N(C H *3)2,6H).IR (KBr):m =2923,1765,1703,1685,1654,1626,1563cm À1;HRMS (ESI)m /z :350.1505[M+H]+(m /z calcd for [C 20H 20N 3O 3]+:350.1505).3.1.6.3-(Dimethylamino-propylamino)-8-oxo-8H -ace-naphtho[1,2-b ]pyrrole-9-carboxylic acid methyl ester (2c).Yield 35%;dark purple solid;mp:186–187°C;1H NMR (400MHz,DMSO-d 6):d =8.79(d,J =8.8Hz,2H),8.59(d,J =7.6Hz,1H),7.84(dd,J =7.6,8.0Hz,1H),6.99(d,J =8.8Hz,1H),3.61(br s,NHC H *2CH 2,2H),3.17(br s,(CH 3)2NC H *2CH 2,2H),2.98(s,COOCH 3,3H),2.38(s,N(CH 3)2,6H), 1.95ppm (m,NHCH 2C H *2,2H).IR (KBr):m =3047,2912,1749,1702,1624,1567,1545,1511cm À1.HRMS (ESI)m /z :364.1665[M+H]+(m /z calcd for [C 21H 22N 3O 3]+:364.1661).3.1.7.3-(4-Methyl-piperazin-1-yl)-8-oxo-8H -acenaph-tho[1,2-b ]pyrrole-9-carboxylic acid 2-bromo-ethyl ester (3a).Yield 26%;dark purple solid;mp:220°C dec;1H NMR (500MHz,DMSO-d 6):d =8.97(d,J =8.0Hz,1H),8.65(d,J =7.6Hz,1H),8.61(d,J =8.4Hz,1H),7.94(dd,J =8.0,7.6Hz,1H),7.47(d,J =8.4Hz,1H),4.13(t,J =6.4Hz,OCH 2,2H), 3.72(t,J =6.4Hz,CH 2Br,2H), 3.55(br s,N(C H *2CH 2)2NCH 3,4H),3.46(br s,N(CH 2C H *2)2NCH 3,4H),2.49ppm (br s,NCH 3,3H).IR (KBr):m =2929,1765,1702,1629,1572,1509cm À1.HRMS (EI)m /z :453.0692[M Å](m /z calcd for [C 22H 20BrN 3O 3Å]453.0688).3.1.8.3-(Dimethylamino-ethylamino)-8-oxo-8H -acenaph-tho[1,2-b ]pyrrole-9-carboxylic acid 2-bromo-ethyl ester (3b).Yield 35%;dark purple solid;mp:>300°C;1H NMR (400MHz,DMSO-d 6):d =8.85(d,J =8.0Hz,1H),8.78(d,J =8.8Hz,1H),8.60(d,J =7.2Hz,1H),7.85(dd,J =7.2,8.0Hz,1H),7.03(d,J =8.8Hz,1H),3.92(t,J =6.4Hz,OC H *2,2H), 3.71(t,J =6.4Hz,CH 2Br,2H),3.69(t,J =6.4Hz,NHC H *2,2H),2.69(t,J =6.4Hz,C H *2N(CH 3)2,2H), 2.29ppm (s,N(CH 3)2,6H).IR (KBr):m =2928,1750,1698,1625,1572,1546cm À1.HRMS (ESI)m /z :442.0753[M+H]+(m /z calcd for [C 21H 21BrN 3O 3]+:442.0766).3.1.9.3-(Dimethylamino-propylamino)-8-oxo-8H -ace-naphtho[1,2-b ]pyrrole-9-carboxylic acid 2-bromo-ethyl ester (3c).Yield 32%;dark purple solid;mp:220°C dec;1H NMR (400MHz,DMSO-d 6):d =9.52(br s,NH),8.84(d,J =8.0Hz,1H),8.83(d,J =8.8Hz,1H),8.63(d,J =7.6Hz,1H),7.88(dd,J =7.6,8.0Hz,1H),7.05(d,J =8.8Hz,1H), 3.93(t,J =6.4Hz,OCH 2,2H),3.71(t,J =6.4Hz,CH 2Br,2H),3.63(br s,2H,NHC H *2),2.67(br s,C H *2N(CH 3)2,2H),2.40(s,N(CH 3)2,6H),1.96ppm (m,CH 2C H *2CH 2,2H).IR (KBr):m =2938,2776,1748,1698,1626,1571,1546,1507cm À1.HRMS (ESI)m /z :456.0930[M+H]+(m /z calcd for [C 22H 24BrN 3O 3]+:456.0923).3.2.DNA binding studies3.2.1.Materials.SYBR Green was purchased from The Molecular Probes Company (USA).Tris Base was from Promega Company (USA).Calf thymus DNA (CT DNA)was obtained from Sigma Chemical Company (USA).Stock of CT DNA was prepared by dissolving commercial nucleic acids in Tris–HCl buffer at pH 7.0and stored at 4°C for more than 24h to get homogene-ity.The concentration of CT DNA was determined spectrophotometrically from the molar absorption coef-ficient (6600M À1cm À1)at 260nm as well as its purity checked by the absorbance ratio A 260/A 280that should not be less than 1.8.37Doubly purified water used in all experiment was from MILLI-Q system.All the chem-icals and solvents were of reagent grade and used with-out further purification.3.2.2.DNA binding affinity.SYBR Green–DNA melt curve was recorded on SmartCycler II (TaKaRa,USA).The contrast was obtained by incubating 1:5000dilution SYBR Green with CT DNA (50l M)for 15min.Each compound (10l M)was added into the former mixture then samples were transferred to SmartCycler tube and heated from 45to 95°C.Fluorescence intensities were recorded as SYBR Green–DNA melt curve.The fluorescence spectra were scanned with a FP-6500spectrophotometer (Jasco,Japan)at room temperature in a 1-cm quartz cuvette.Titrations were performed by keeping the DNA concentration as 50l M while varying the compound concentration from 1to 10l M.Fluores-cence intensity was recorded after each addition of DNA.The difference in fluorescence intensity of the compounds in the absence and presence of DNA (fluo-rescence quenching)was assumed to be proportional to the amount of DNA-bound compound.The binding affinity (K b )and binding intensity n were calculated.383.2.3.Absorption titration.The absorption titrations were performed using a HP 8453spectrophotometer (HP,USA).A 1-cm path quartz cell was used for the6968Z.Zhang et al./Bioorg.Med.Chem.14(2006)6962–6970。