CN111073947A - 细胞凋亡检测试剂盒及其检测方法和应用 - Google Patents
细胞凋亡检测试剂盒及其检测方法和应用 Download PDFInfo
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Abstract
本发明公开了一种细胞凋亡检测试剂盒及其检测方法和应用,该试剂盒,包括共价结合了荧光素FITC的Annexin V,还包括荧光染料DAPI。所述DAPI的浓度<150nM。本发明还涉及采用所述的试剂盒检测细胞凋亡的方法,以荧光素FITC共价结合的Annexin V特异性结合磷脂酰丝氨酸来识别凋亡细胞,联合DAPI标记死细胞和凋亡晚期细胞;最后利用流式细胞仪或者荧光显微镜检测细胞凋亡的发生并区分凋亡早、晚期的细胞和死细胞。使用Annexin V‑FITC组合DAPI的双染法能够有效避免具有红色到橙色荧光基团的药物对细胞凋亡检测的干扰。
Description
技术领域
本发明涉及生物医药领域,特别是指一种基于AnnexinV-FITC和DAPI双标记的细胞凋亡检测试剂盒及其检测方法和应用。
背景技术
细胞凋亡存在于多细胞生物的整个生命过程当中,可及时清除机体内多余和受损伤的细胞,维持组织器官的稳定性,在维持组织、器官的正常形态和功能方面起重要作用。目前采用的细胞凋亡检测方法包括:利用光学显微镜的细胞形态学检测法、DNA片段化分析法、原位末端缺口标记法(TUNEL)和Annexin V-FITC/PI双染法等。其中Annexin V-FITC/PI双染法由于灵敏度和特异性高,并且操作简便易行,评价指标客观而成为目前应用最为广泛的细胞凋亡检测方法。
其实验原理是:正常细胞中,磷酯酰丝氨酸(PS)位于细胞脂质双层膜的内侧;只有当细胞凋亡发生时,磷酯酰丝氨酸会翻转到细胞膜外侧。Annexin V能在Ca2+存在情况下特异性识别与结合细胞膜外侧的磷酯酰丝氨酸,通过使用共价结合了荧光素FITC的AnnexinV即可标记凋亡细胞。此外,由于正常细胞和早期凋亡细胞的细胞膜相对完整而凋亡晚期及坏死细胞的细胞膜通透性显著增加,使得核酸染料碘化丙啶(propidine iodide,PI)能够选择性进入凋亡中晚期及坏死细胞而标记。由此,通过荧光素FITC(Fluoresceinisothiocyante,异硫氰酸荧光素酯)共价结合的Annexin V特异性识别细胞膜外表面的PS,联合PI检测凋亡晚期和坏死细胞,即可使用流式细胞仪检测细胞凋亡的发生,同时区分凋亡早、晚期的细胞及坏死细胞并确定其比例。
然而,在当前肿瘤细胞凋亡研究中,多种细胞凋亡诱导药物如蒽环类药物盐酸多柔比星和光敏剂如玫瑰红(Rose bengal)等,具有与PI相同的激发光(535nm)和发射光波长(617nm,橙色到红色范围),从而导致上述药物处理后的细胞在使用Annexin V-FITC/PI双染法进行细胞凋亡检测时无法区分坏死细胞与正常、凋亡细胞。
鉴于上述原因,目前仍需提供新的简便易行、具有高灵敏度和特异性的方法用于细胞凋亡的检测。
发明内容
为了解决上述技术问题,本发明提供一种细胞凋亡检测试剂盒及其检测方法和应用,该试剂盒可以可检测细胞凋亡的发生并区分凋亡早、晚期的细胞和死细胞,同时有效避免具有红色到橙色荧光基团的药物对细胞凋亡检测的干扰。
本发明所采用的技术方案是,一种细胞凋亡检测试剂盒,包括共价结合了荧光素FITC的Annexin V,还包括荧光染料DAPI。
进一步地,所述DAPI的浓度<150nM。
本发明还涉及采用所述的试剂盒检测细胞凋亡的方法,以荧光素FITC共价结合的Annexin V特异性结合磷脂酰丝氨酸来识别凋亡细胞,联合DAPI标记死细胞和凋亡晚期细胞;最后利用流式细胞仪或者荧光显微镜检测细胞凋亡的发生并区分凋亡早、晚期的细胞和死细胞。
本发明还涉及所述试剂盒在检测细胞凋亡中的用途。
本发明还涉及所述试剂盒在检测细胞凋亡中的用途,检测过程中使用具有红色到橙色荧光的药物诱导细胞凋亡。
DAPI即4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole),分子式:C16H15N5分子量:277.32,化学结构式:
DAPI是一种能够识别DNA双链并结合于DNA双链大沟的荧光染料。当DAPI与DNA双链结合时,DAPI的最大激发光波长为358nm,最大发射波长为461nm,呈现为蓝色。因其发射光的波长范围含盖了蓝色至青绿色,因此在荧光显微技术中DAPI可被紫外线激发而被蓝或青色滤镜检出。DAPI也可以结合RNA,但在358nm激发光波长激发下其最大发射光波长范围约在400nm,且其产生的荧光强度显著低于与DNA结合的荧光强度,因此能够有效标记DNA双链。重要的是,在相当宽泛的较低浓度范围内(<150nM)DAPI不能自由通过完整的细胞膜,因而和PI一样可以选择性通过凋亡晚期及坏死细胞的细胞膜,从而可用于标记凋亡晚期及坏死细胞。
鉴于DAPI的激发光和发射光波长远离前述药物的激发和发射光波长,使用Annexin V-FITC组合DAPI的双染法能够有效避免具有荧光基团的药物(红色到橙色)对细胞凋亡检测的干扰。
附图说明
图1是不同浓度DAPI对4T-1活细胞的标记后的荧光显微图片。
图2是不同浓度DAPI对4T-1死细胞的标记后的荧光显微图片。
图3是AnnexinV-FITC/PI检测不同浓度Rose bengal经光激发诱导的4T-1细胞凋亡实验结果图。
图4是AnnexinV-FITC/DAPI检测不同浓度Rose bengal经光激发诱导的4T-1细胞凋亡实验结果图。
图5是AnnexinV-FITC/DAPI检测不同浓度Rose bengal经光激发诱导的MDA-MB-231细胞凋亡实验结果图。
图6是AnnexinV-FITC/DAPI检测不同浓度Rose bengal经光激发诱导的MDA-MB-468细胞凋亡实验结果图。
图7是AnnexinV-FITC/DAPI检测不同浓度盐酸多柔比星诱导的MDA-MB-231细胞凋亡实验结果图。
图8是AnnexinV-FITC/DAPI检测不同浓度盐酸多柔比星诱导的MDA-MB-468细胞凋亡实验结果图。
具体实施方式
下面结合具体实施,进一步阐明本发明。
1)将DAPI溶解在50%DMSO中,配置成浓度300μM的母液;
2)传代培养小鼠乳腺癌4T-1细胞1×105/孔于两个六孔细胞培养板中,用含10%胎牛血清的DMEM培养基于细胞培养箱(95%湿度,37℃,5%CO2)内孵育24小时。然后将其中一块六孔板弃去DMEM,加入70%乙醇处理15分钟杀死细胞,此为死亡细胞组;正常培养的细胞为活细胞组。再于两组细胞分别加入不同浓度的DAPI(0、18.75、37.5、75、150、300nM),继续避光孵育15分钟后用荧光显微镜(350nM波长激发光)观察不同浓度DAPI对细胞的标记情况,具体如图1和图2所示。可发现低浓度DAPI(18.75nM)即可将死细胞核标记为蓝色,细胞核蓝色标记随DAPI浓度增加而增加,呈线性关系。但150nM浓度以下的DAPI均不可标记活细胞,当DAPI浓度达到150nM及以上时方可对活细胞核进行标记。这一结果表明较低浓度的DAPI可有效区分活细胞与死亡细胞。
3)传代培养小鼠乳腺癌4T-1细胞1×105/孔于六孔细胞培养板中,用含10%胎牛血清的DMEM培养基于细胞培养箱(95%湿度,37℃,5%CO2)内孵育24小时。然后各孔分别加入不同浓度的光敏剂Rose bengal。Rose bengal浓度依次为0μM、0.5μM、1μM、2.5μM、5μM、10μM。24小时后,分别以510nM波长激发光照射90秒。然后以胰蛋白酶消化液消化、收集各孔的细胞于1.5mL EP管中,离心机1200转/分钟离心3分钟,弃去培养基后重悬细胞于500μL PBS溶液中,再次离心收集细胞。然后用200μL Binding buffer重悬后分为两组:第一组为Annexin V-FITC/PI检测组;第二组为Annexin V-FITC/DAPI检测组。Annexin V-FITC/PI检测组各孔细胞加入AnnexinV-FITC 5μL和PI 5μL;Annexin V-FITC/DAPI检测组加入AnnexinV-FITC 5μL和DAPI(终浓度150nM),然后避光室温孵育十五分钟,再向各管中加入400 L Binding buffer。以流式细胞术检测细胞凋亡,对比Annexin V-FITC/PI双染法和Annexin V-FITC/DAPI双染法对细胞凋亡的检测作用,具体检测结果见图3和图4。
然后再进行两次独立实验,将三次实验的结果进行统计分析,确定该方法的重复性和准确性。
从图3的结果可以看出:AnnexinV-FITC/PI双染法检测时,随着Rose bengal处理浓度增加细胞坏死信号持续增加,二者呈线性相关;而且不同浓度Rose bengal处理的细胞均显示为晚期凋亡,且最大浓度Rose bengal处理的细胞几乎没有存活。这一结果与PI和Rose bengal激发光/发射光波长重叠对细胞凋亡检测的干扰作用相符合。
从图4的结果可以看出:AnnexinV-FITC/DAPI双染法检测时,随着Rose bengal处理浓度增加细胞坏死信号也增加,但二者不呈线性相关;而且随着Rose bengal处理浓度增加细胞坏死、早期凋亡和晚期凋亡信号均持续增加。这一结果表明该方法可有效避免与Rose bengal激发光/发射光波长重叠所导致的检测干扰。
进一步地将该方法用于人三阴性乳腺癌MDA-MB-231细胞和人三阴性乳腺癌MDA-MB-468细胞凋亡的检测。实验中分别采用临床化疗药物盐酸多柔比星和光敏剂Rosebengal诱导细胞凋亡,然后以Annexin V-FITC/DAPI双染法检测。从图5和图6的结果可以看出:Annexin V-FITC/DAPI双染法能够有效检测光敏剂Rose bengal诱导的MDA-MB-231细胞和MDA-MB-468细胞凋亡,该两种细胞的凋亡发生率随着光敏剂的浓度增加而显著增加。从图7和图8的结果可以看出:Annexin V-FITC/DAPI双染法能够有效检测盐酸多柔比星诱导的MDA-MB-231细胞和MDA-MB-468细胞凋亡,该两种细胞的凋亡发生率在溶剂对照组和盐酸多柔比星低浓度组(0.5μM)没有显著差别(MDA-MB-231)或2.42倍度增加,在盐酸多柔比星浓度从0.5μM提高到2.5μM时,细胞总体凋亡发生率分别提高了5.44倍(MDA-MB-231)和5.46倍(MDA-MB-468)。其中,细胞晚期凋亡率也随药物浓度增加而增加,但两种细胞的坏死率均无显著增加,表明尽管两种凋亡诱导药物均为红色荧光诱导药物,但对Annexin V-FITC/DAPI双染法检测细胞凋亡没有显著影响。
由上述描述可知,本发明提供的基于AnnexinV和DAPI双标记的细胞凋亡检测方法能够用于细胞凋亡检测,特别是可有效用于检测某些与PI激发光/发射光波长重叠的药物诱导的细胞凋亡。
上述的实施例仅为本发明的优选技术方案,而不应视为对于本发明的限制,本发明的保护范围应以权利要求记载的技术方案,包括权利要求记载的技术方案中技术特征的等同替换方案为保护范围。即在此范围内的等同替换改进,也在本发明的保护范围之内。
Claims (5)
1. 一种细胞凋亡检测试剂盒,其特征在于:包括共价结合了荧光素FITC的Annexin V,还包括荧光染料DAPI。
2.根据权利要求1所述的试剂盒,其特征在于:所述DAPI的浓度<150nM。
3.采用权利要求1或2所述的试剂盒检测细胞凋亡的方法,其特征在于:以荧光素FITC共价结合的Annexin V特异性结合磷脂酰丝氨酸来识别凋亡细胞,联合DAPI标记死细胞和凋亡晚期细胞;最后利用流式细胞仪或者荧光显微镜检测细胞凋亡的发生并区分凋亡早、晚期的细胞和死细胞。
4.权利要求1或2所述试剂盒在检测细胞凋亡中的用途。
5.权利要求1或2所述试剂盒在检测细胞凋亡中的用途,其特征在于,检测过程中使用具有红色到橙色荧光的药物诱导细胞凋亡。
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