CN106220664A - 一种检测自噬流的荧光探针及其制备和应用 - Google Patents
一种检测自噬流的荧光探针及其制备和应用 Download PDFInfo
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Abstract
本发明提供一种检测自噬流的荧光探针，通过N‑苯基‑4‑羟基‑3‑甲氧基苯胺与1，3，5，7‑四甲基‑8‑(3‑醛‑丙基)BODIPY在二氯甲烷与甲醇的混合溶剂中发生还原胺化反应制得。本发明的荧光探针本身在生理环境中无荧光，但可对自噬分子事件中的氧化/硝化自由基及酸性环境做出响应，呈现荧光增强，从而实现对自噬流的检测。本发明涉及的荧光探针具有稳定性好，能够长期保存使用，检测信噪比高，灵敏度好的特点，具有优秀的选择性及生物膜通透性，能在活细胞中中特异性地检测自噬流。荧光探针结构：
Description
技术领域
本发明属生物检测领域，涉及一种特异性检测自噬流的荧光探针及其制备方法和应用。
背景技术
自噬在清理受损细胞器、维持细胞代谢需要的过程中发挥重大作用，是维持细胞稳态的一种生理过程。自噬的过程包括自噬前体的形成，自噬前体延长包裹自噬的底物形成自噬体，自噬体和溶酶体发生融合形成溶酶体，将自噬的底物水解并释放。在许多疾病过程中都有自噬的发生，但是病理条件下自噬对机体损伤发挥保护作用还是加重损伤是难以预测的。因此，发展能对生物体内的自噬流进行实时追踪检测的方法，可极大推动过自噬相关的生理病理学研究。自噬过程中有大量氧化/硝化自由基的产生，包括过氧亚硝基、一氧化氮自由基、超氧阴离子等。而由自噬小体和溶酶体融合产生的自噬溶酶体，则呈明显酸性，因此氧化/硝化自由基及酸性环境的共同存在可作为监测自噬发生的依据。
传统的监测自噬的方法基本基于自噬流过程中相关蛋白的表达水平变化或电镜下观测自噬流中囊泡的形态及数量变化，这种方法存在不能适用于活体检测等方面的问题。荧光探针检测法是近几年来发展起来的新方法。本发明利用自噬发生初期，机体中过量氧化/硝化自由基的产生，及自噬溶酶体形成后环境弱酸性，设计可先后对氧化/硝化自由基及酸性环境做出荧光增强响应的探针，从而实现探针对整个自噬流的监测。由于小分子荧光探针具有体积小、生物膜通透性良好的优点，因而荧光探针法适用于活细胞甚至是动物组织中自噬流的检测。
发明内容
本发明的一个目的是提供一种新型的检测自噬流的荧光探针，具有式I所示的结构：
本发明的又一个目的是提供式I所示化合物的制备方法，通过以下步骤实现：
N-苯基-4-羟基-3-甲氧基苯胺(式II)与1,3,5,7-四甲基-8-(3-醛-丙基)BODIPY(式III)在二氯甲烷与甲醇的混合溶剂中发生还原胺化反应制得。反应式如下：
本发明的再一个目的是提供式I所示的荧光探针在检测细胞自噬中的应用。以活细胞中的应用为例，可通过以下步骤实现：细胞培养基中加入式I所示的荧光探针，使其终浓度为50nM。37℃下孵育20分钟，观察记录细胞荧光强度。
本发明的荧光探针本身在生理环境中无荧光，但可对自噬分子事件中的氧化/硝化自由基及酸性环境做出响应，呈现荧光增强，从而实现对自噬流的检测。本发明涉及的荧光探针具有以下有益效果：1、稳定性好，能够长期保存使用；2、由于探针本身无荧光，只有对自噬分子事件响应后才有荧光，因此，检测信噪比高，灵敏度好；3、具有优秀的选择性及生物膜通透性，能在活细胞中中特异性地检测自噬流。
附图说明
图1.荧光探针分子在pH7.4环境中与过氧化亚硝基反应前后的荧光变化，及反应后酸化溶液至pH4.0后的荧光变化。
图2.荧光探针分子在pH7.4环境中与超氧阴离子反应前后的荧光变化，及反应后酸化溶液至pH4.0后的荧光变化。
图3.荧光探针分子在pH7.4环境中与一氧化氮反应前后的荧光变化，及反应后酸化溶液至pH4.0后的荧光变化。
图4.荧光探针分子AFG-1在饥饿处理的活细胞中的响应。
图5.荧光探针分子AFG-1在活细胞中与LC3的共定位情况。
图6.活细胞检测荧光分子探针AFG-1与溶酶体的共定位情况。
具体实施方式
下面结合附图和实施例对本发明作进一步说明，本实施例并不是限制本发明的范围。
实施例1：荧光探针分子I的制备
N-苯基-4-羟基-3-甲氧基苯胺(式II，0.12g)、1，3，5，7-四甲基-8-(3-醛-丙基)BODIPY(式III,0.20g)分别加入二氯甲烷与甲醇的混合溶剂(5:1，12ml)中，同时加入氰基硼氢化钠(42mg)及一滴乙酸。室温搅拌反应6小时。减压旋干溶剂后，粗产品柱层析纯化，得式I所示化合物，性状：红色固体，产率15％。
1H NMR(400MHz,CDCl3):δ7.18(dd,J＝8.5,7.4Hz,2H),6.90(d,J＝8.4Hz,1H),6.77(t,J＝7.4Hz,1H),6.75(d,J＝7.8,2H),6.67(dd,J＝8.4,2.3Hz,1H),6.63(d,J＝2.3Hz,1H),6.02(s,2H),5.51(s,1H),3.84–3.74(m,5H),3.06–2.95(m,2H),2.50(s,6H),2.32(s,6H),2.04–1.92(m,2H).
实施例2：荧光探针分子I与过氧化亚硝基反应前后的荧光变化
将探针分子以少量DMSO溶解，分别加入PBS缓冲液或过氧化亚硝基的PBS溶液，使探针分子的终浓度为5μM,过氧化亚硝基终浓度为10μM。反应10min后使用荧光分光光计以485nm激发，记录溶液在最大发射波长(约505nm)下的荧光强度，进而确定探针分子与过氧化亚硝基反应后荧光强度增强。反应后的溶液加少量1N盐酸调节pH至4.0，继续以荧光分光光计485nm激发，记录溶液在最大发射波长(约505nm)下的荧光强度，确定探针与过氧化亚硝基反应后，体系酸化对荧光强度的影响。如图1所示。
实施例3：荧光探针分子I与超氧阴离子反应前后的荧光变化
将探针分子以少量DMSO溶解，分别加入PBS缓冲液或超氧阴离子的DMSO溶液，使探针分子的终浓度为5μM,超氧阴离子终浓度为10μM。反应10min后使用荧光分光光计以485nm激发，记录溶液在最大发射波长(约505nm)下的荧光强度，进而确定探针分子与超氧阴离子反应后荧光强度增强。反应后的溶液加少量1N盐酸调节pH至4.0，继续以荧光分光光计485nm激发，记录溶液在最大发射波长(约505nm)下的荧光强度，确定探针与超氧阴离子反应后，体系酸化对荧光强度的影响。如图2所示。
实施例4：荧光探针分子I与一氧化氮反应前后的荧光变化
将探针分子以少量DMSO溶解，分别加入PBS缓冲液或一氧化氮的PBS溶液，使探针分子的终浓度为5μM,一氧化氮终浓度为10μM。反应10min后使用荧光分光光计以485nm激发，记录溶液在最大发射波长(约505nm)下的荧光强度，进而确定探针分子与一氧化氮反应后荧光强度增强。反应后的溶液加少量1N盐酸调节pH至4.0，继续以荧光分光光计485nm激发，记录溶液在最大发射波长(约505nm)下的荧光强度，确定探针与一氧化氮反应后，体系酸化对荧光强度的影响。如图3所示。
实施例5：荧光探针分子AFG-1在饥饿处理的活细胞中的响应
将探针以DMSO溶解为10mM作为储存浓度，储存于-40℃，再用DMSO稀释为0.05mM的浓度，储存于4℃。EA.hy296细胞生长到80％左右的密度时，用HBSS溶液替换含10％胎牛血清的DMEM高糖培养基，分别孵育不同时间。吸去多聚甲醛，用PBS清洗3次，每次10min；DAPI以1:500比例溶于PBS染色10min，再用用PBS清洗3次，每次10min。最后封片，保存于4℃，用Zeiss共聚焦显微镜采集图像，结果如图4所示。
实验结果显示随着HBSS孵育时间的增加，细胞内探针的荧光强度增加，且逐渐出现点状聚集，提示该探针在饥饿条件下有响应。
实施例6：荧光探针分子AFG-1在活细胞中与LC3的共定位情况
血管内皮细胞EA.hy296细胞接种于6孔板中，1.2μg RFP-LC3转染。约24h后用胰酶将细胞消化并接种于放置用D-多聚赖氨酸包被的小玻片的24孔板内，待EA.hy296细胞生长到80％左右的密度时，设置Ctrl、HBSS、Ctrl+Bafilomycin A1(100nM)、HBSS+BafilomycinA1(100nM)组，造模4h并固定封片，用Nikon A1R共聚焦显微镜采集图像，结果如图5所示。
实验结果显示，和Ctrl组相比，Ctrl+Bafilomycin A1(100nM)组探针荧光强度略有增强，LC3荧光强度也略有增强，HBSS组AFG-1探针荧光强度明显增强，有点状聚集，LC3荧光强度明显增加，有点状，两者有部分共定位，而HBSS+Bafilomycin A1(100nM)组探针荧光强度较HBSS组略弱，LC3荧光也有点状，两者有部分共定位。这说明AFG-1探针和自噬体有部分共定位。
实施例7：活细胞检测荧光分子探针AFG-1与溶酶体的共定位情况
血管内皮细胞EA.hy296细胞接种于Time lapse的小皿中，48h内细胞密度约为80％，在培养液中加入lysotracker RED，37℃孵育45min，将培养液更换为HBSS溶液，在转盘倒置荧光显微镜下以每分钟一帧的频率连续记录4h，结果如附图6所示。
结果显示HBSS组的AFG-1荧光探针强度随记录时间逐渐增强，且出现点状聚集的数量也逐渐增多，和lysotracker RED所标记的点状有部分共定位，说明AFG-1探针和溶酶体(包括自噬溶酶体)有部分共定位。
Claims (3)
1.一种检测自噬流的荧光探针，其特征在于，如式I所示结构：
2.权利要求1所述的一种检测自噬流的荧光探针的制备方法，其特征在于，通过以下步骤实现：
N-苯基-4-羟基-3-甲氧基苯胺与1，3，5，7-四甲基-8-(3-醛-丙基)BODIPY在二氯甲烷与甲醇的混合溶剂中发生还原胺化反应制得，反应式如下：
3.根据权利要求1所述的一种检测自噬流的荧光探针在检测细胞自噬中的应用。
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