CN106588845A - 一种细胞自噬监测探针及其制备方法和用途 - Google Patents
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Abstract
本发明公开了一种细胞自噬监测探针及其制备方法和用途,其细胞自噬监测探针的结构如下:
Description
一、技术领域
本发明涉及一种双光子荧光探针,具体地说是一种细胞自噬监测探针及其制备方法和用途。
二、背景技术
自噬是细胞在受到营养缺乏、氧化应激、高温、损伤等刺激信号时,由一种游离双层膜包裹细胞液或受损的细胞器形成自噬体,并与溶酶体结合形成自噬溶酶体,将内容物降解为氨基酸、核苷酸、游离脂肪酸从而维持细胞自身稳态效益的过程。自噬可分为4个部分,包括:诱导,自噬小体的形成,自噬小体和溶酶体的膜融合以及自噬溶酶体的降解。细胞自噬作为生物体中一种非常重要的代谢途径,调控过氧化物酶体、线粒体和内质网的不断更新,还能清除胞质内受损的细胞器和代谢产物,进行亚细胞水平的重构,保护受损细胞。目前,一些经典的监测自噬的手段大多基于生物学的角度,如扫描电镜(TEM),蛋白免疫印迹(Western Blot)和荧光标记蛋白(GFP-Atg8/LC3)等。这些传统的监测手段都有着其局限性,如扫描电镜和蛋白免疫印迹,它们不能监测活细胞里的自噬状态,同时,这些方法操作都很复杂,花费也很昂贵,对细胞自噬的监测也不是特别的形象。因此,一种能够准确而又经济的自噬监测方法对自噬过程的研究十分必要。
溶酶体是真核细胞中的一种细胞器,内含多种水解酶,专司分解各种外源和内源的大分子物质。在自噬过程中,自噬体的膜会和溶酶体膜发生融合,使自噬体内包裹的物质与溶酶体内的水解酶相接触。在这个膜融合的过程中,溶酶体的微环境势必会发生变化,而这个过程中微环境信号的变化为我们提供了一个监测自噬过程的新思路。
极性是微环境中的一种重要参数,一些特定的无机有机反应均依赖于极性的参与。在生物系统里,尤其在细胞层面上,极性决定了大多数蛋白质和酶的联系并反映了细胞及细胞器膜组分的通透性,甚至极性的反常变化与一些疾病的产生关系密切。荧光探针是在一定体系内,当一种物质或体系中某一物质性质发生变化时,荧光信号能发生相应改变的分子。荧光光谱由于其检测方便,灵敏等优点在痕量检测中展现了优越的性能。近些年来,双光子荧光光谱凭借着自身稳定性高,光漂白性好,细胞穿透性高等优点被广泛使用。因此,能够使用双光子荧光探针来检测细胞溶酶体内极性的变化从而实现监测自噬是非常理想的选择。
三、发明内容
本发明旨在提供一种细胞自噬监测探针及其制备方法和用途。所要解决的技术问题是通过分子设计遴选合适的荧光探针结构,以实现双光子成像定性检测细胞中溶酶体的微环境变化,具有选择性专一、灵敏度高等优点,细胞毒性测试表明本发明对细胞几乎没有毒性作用。
本发明细胞自噬监测探针(Lyso-OSC),是以香豆素为母体,4-甲氧基苯乙烯为供电子基团,2-吗啡-1-乙胺为溶酶体定位基团,简称为荧光探针或荧光探针分子,其结构由下式表示:
Lyso-OSC
本发明细胞自噬监测探针的制备方法,包括如下步骤:
将7-((4-甲氧基苯基)乙烯)-3-羧酸-香豆素(1g,3mmol)、N,N-二异丙基乙胺(1.2g,9mmol)、1-羟基苯并三唑(0.65g,4.5mmol)、EDC·HCl(0.65g,3.3mmol)以及2-吗啡-1-乙胺(1.2g,9mmol)加入史莱克瓶中,在无水无氧条件下加入30mL N,N-二甲基甲酰胺,室温下搅拌反应24h;反应结束后先向反应液中加入50mL二氯甲烷将反应物溶解,再用30mL水萃取(3次),得到有机相,通过柱层析200-300目硅胶(洗脱液由乙酸乙酯和石油醚按体积比1:1混合构成),得到目标产物Lyso-OSC 0.35g,产率40%。
本发明荧光探针分子Lyso-OC的合成过程如下:
本发明荧光探针分子Lyso-OC可在定性检测细胞中溶酶体微环境极性变化时作为检测试剂应用。本发明荧光探针分子Lyso-OC可以通过即时检测溶酶体内极性的变化来监测细胞的自噬过程。
将本发明荧光探针分子溶于DMSO中制得1mM的母液,取50μL的该母液于10mL容量瓶中,再用不同极性(极性参数设置为Δf)的溶剂定容,配制成5μM溶液。荧光探针单光子和双光子的激发波长分别为380nm和760nm,检测390-650nm范围内的荧光光谱变化,可以明显观察到从1,4-二氧六环(极性很小,Δf≈0.09)溶剂极性依次增大到水(极性很大,Δf≈0.32)时,荧光的最大发射峰从470nm红移至525nm(斯托克斯位移约为55nm),荧光强度也随之降低了约550倍。
为了排除溶剂效应的干扰并且进一步研究本发明荧光探针与溶剂极性的关系,通过调节水-四氢呋喃中水的含量来控制混合溶剂的极性,取50μL的该母液于10mL容量瓶中,再用不同水含量的水-四氢呋喃混合溶剂定容,配置成5μM溶液。单光子和双光子的激发波长和检测范围均同上,可以观察到水含量从1%(Δf≈0.22)增加到80%(Δf≈0.31)时,荧光的最大发射峰从475nm红移至525nm(斯托克斯位移约为50nm),荧光强度随之降低了约75倍,荧光量子产率也随之从55%逐渐降低到了约1%,同时荧光寿命也随着极性的增加线性减少。随后,继续研究本发明荧光探针在其他不同溶剂的混合体系中(如水-1,4-二氧六环、水-甲醇等)随着极性变化而产生的荧光变化,发现其均呈规律性变化。因此本发明荧光探针分子非常有潜力运用于生物检测。
为了排除环境pH值对本发明荧光探针分子荧光变化情况的影响,取50μL的该母液于10mL容量瓶中,再用不同pH的水定容。单光子和双光子的激发波长和检测范围均同上,可以观察到当环境的pH值从4.8变化到8.0(生物系统中常见的pH范围)时,荧光探针的荧光强度基本没有变化,证明pH值的变化对探针的作用影响非常小,并且能够很好地在生物应用中反映极性的变化。
本发明荧光探针检测微环境极性变化的机理是在荧光探针分子吸收光子的过程中,由于整个探针分子各个部分偶极矩的不同而导致“溶剂致变色”现象。当溶剂分子包裹在探针分子周围,“溶剂致变色”现象会导致分子在基态和激发态稳定化能的不同。因此,当溶剂极性增加时,探针分子的激发态需要释放更多地能量来内部转换到一个比之前更稳定的激发态(即比之前激发态更低的能级),甚至可能在一些快速的内部转换之后进一步转换为另一种机理不同却更稳定的激发态(具体为ICT激发态转换为TICT激发态),这也具体表现为荧光强度减弱或荧光光谱红移。
本发明荧光探针分子结构简单,易于合成,以荧光强弱和颜色的变化来检测微环境极性的变化,并且对细胞毒性很小,能够用于检测溶酶体内的极性变化,进而监测细胞自噬过程,操作简单,快速灵敏。
四、附图说明
图1是10μM荧光探针在不同极性的溶剂中的荧光光谱。溶剂包括极性较小的甲苯、1,4-二氧六环等,极性较大的甲醇、水等。
图2是10μM荧光探针在不同极性的溶剂中的紫外吸收光谱。包括甲苯、1,4-二氧六环、甲醇和水等。
图3是10μM荧光探针在不同水含量的水-四氢呋喃混合体系中的荧光光谱。包括含水10%、20%、30%、40%、50%、60%、70%和80%。
图4是10μM荧光探针在不同水含量的水-四氢呋喃混合体系中的紫外吸收光谱。包括含水10%、20%、30%、40%、50%、60%、70%和80%。
图5是在不同含量(10μM、20μM、30μM)的荧光探针分子的作用下的细胞存活率。
图6是荧光探针分子在MCF-7细胞中的溶酶体定位成像图。Lyso-OSC为探针溶酶体定位细胞成像图,Lyso Tracker Red为商品化的溶酶体定位细胞成像图,Merge为Lyso-OSC和Lyso Tracher重叠图。
图7是荧光探针分子在MCF-7细胞中的加入DMSO改变极性后细胞成像图。加入DMSO后极性增大,细胞成像图更明显。
图8是用10μM荧光探针分子培养MCF-7细胞0.5小时后饥饿诱导自噬4小时后的荧光变化情况。
图9是图8中两种状态细胞的荧光数据变化情况。
五、具体实施方式
实施例1:荧光探针分子Lyso-OSC的合成
将7-((4-甲氧基苯基)乙烯)-3-羧酸-香豆素(1g,3mmol)、N,N-二异丙基乙胺(1.2g,9mmol)、1-羟基苯并三唑(0.65g,4.5mmol)、EDC·HCl(0.65g,3.3mmol)以及2-吗啡-1-乙胺(1.2g,9mmol)加入史莱克瓶中,在无水无氧条件下加入30mL N,N-二甲基甲酰胺,室温下搅拌反应24h;反应结束后先向反应液中加入50mL二氯甲烷将反应物溶解,再用30mL水萃取(3次),得到有机相,通过柱层析200-300目硅胶(洗脱液由乙酸乙酯和石油醚按体积比1:1混合构成),得到目标产物0.35g,产率40%。
1H NMR(400MHz,CDCl3)δ9.13(s,1H),8.86(s,1H),7.63(d,J=8.0Hz,1H),7.53(s,1H),7.50(s,1H),7.49(s,1H),7.46(s,1H),6.95(d,J=8.6Hz,1H),6.93(d,J=9.3Hz,1H),6.91(d,J=8.9Hz,2H),3.85(s,3H),3.78(s,4H),3.61(s,2H),2.61(d,J=31.8Hz,6H).
13C NMR(101MHz,CDCl3)δ161.65,161.09,160.49,154.31,147.46,133.52,133.05,129.87,129.53,128.24,127.4,118.81,118.13,118.00,114.24,114.14,95.16,87.03,66.49,56.74,55.32,53.26,36.30.
实施例2:荧光探针分子Lyso-OSCC的双光子测试
利用双光子测量技术,测试荧光探针分子的双光子吸收截面,从图8可以看出,荧光探针分子的最大有效吸收截面是93GM,双光子激发波长在760nm。对荧光探针分子的双光子吸收进行验证,在760nm的双光子激发波长下,将电压从200w逐渐调至800w,双光子有效吸收截面成线性增长,线性斜率为1.98,证明此荧光探针的确可以双光子激发。
实施例3:细胞毒性测试
MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐)实验是根据已报道的文献,做细胞毒性测试。分别在同一批MCF-7细胞中加入0,10,20,30μM的荧光探针分子,此条件是在37℃、含5%CO2的细胞培养箱中孵育24小时,根据细胞存活度的公式:细胞存活率%=OD570(样品)/OD570(对照组)×100,可算得细胞存活率(图5)。从图5中我们可以看出,浓度为5μM时,细胞存活率为98%左右,当探针浓度达到15μM时,细胞存活率仍然有约82%,说明了本发明荧光探针分子对细胞基本没有毒性作用,因此可以用来做细胞检测及监测溶酶体中的极性变化。
实施例4:细胞定位测试
MCF-7细胞由DEME(invitrogen)培养液培养,成像前一天,HeLa细胞放于激光共聚焦皿中,成像时MCF-7细胞和10μM的荧光探针的DMSO溶液于37℃、含5%CO2的细胞培养箱中孵育0.5小时,用中性的PBS缓冲溶液洗涤3次后,再往培养皿中加入1μM商品化溶酶体染色剂Lyso Tracker Red溶液继续孵育0.5小时,用中性的PBS缓冲溶液洗涤3次。用双光子荧光共聚焦成像,设置绿色通道tracker1,激发波长为760nm,发射波段为495-545nm,这个通道用来接受探针分子Lyso-OC发射的荧光。设置红色通道tracker2,激发波长为577nm,发射波长为580-600nm,这个通道用来接收商品化溶酶体染色剂Lyso Tracker Red发射的荧光。
实施例5:细胞成像及细胞自噬过程测试
MCF-7细胞由DEME(invitrogen)培养液培养,成像前24小时,MCF-7细胞放于激光共聚焦皿中贴壁培养,成像时MCF-7细胞和10μM的荧光探针的DMSO溶液于37℃、含5%CO2的细胞培养箱中孵育0.5小时,用中性的PBS缓冲溶液洗涤3次后进行细胞成像测试,设置通道tracker1,激发波长为760nm,发射波段为495-545nm,此时记录下正常状态下的细胞形态和荧光情况。将培养皿里面的培养基换成HBSS缓冲溶液来饥饿诱导细胞发生自噬,此时溶酶体的形态及微环境发生变化,随着时间的变化,分别记录下各个时间点的细胞形态及荧光变化情况,得图8。
Claims (5)
1.一种细胞自噬监测探针,是以香豆素为母体,4-甲氧基苯乙烯为供电子基团,2-吗啡-1-乙胺为溶酶体定位基团,其结构由下式表示:
2.一种权利要求1所述的细胞自噬监测探针的制备方法,包括如下步骤:
将7-((4-甲氧基苯基)乙烯)-3-羧酸-香豆素3mmol、N,N-二异丙基乙胺9mmol、1-羟基苯并三唑4.5mmol、EDC·HCl 3.3mmol以及2-吗啡-1-乙胺9mmol加入史莱克瓶中,在无水无氧条件下加入30mL N,N-二甲基甲酰胺,室温下搅拌反应24h;反应结束后先向反应液中加入50mL二氯甲烷将反应物溶解,再用30mL水萃取,收集有机相,通过柱层析200-300目硅胶,得到目标产物。
3.根据权利要求2所述的制备方法,其特征在于:
柱层析时洗脱液由乙酸乙酯和石油醚按体积比1:1混合构成。
4.一种权利要求1所述的细胞自噬监测探针的用途,其特征在于:在定性检测细胞中溶酶体微环境极性变化时作为检测试剂应用。
5.一种通过权利要求1所述的细胞自噬监测探针监测细胞自噬过程的方法,其特征在于:以所述细胞自噬监测探针作为检测试剂,通过定性检测细胞中溶酶体微环境的极性变化以实现对细胞自噬过程的监测。
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