CN106565596B - 一种萘基衍生物作为线粒体靶向型pH荧光探针的应用 - Google Patents

一种萘基衍生物作为线粒体靶向型pH荧光探针的应用 Download PDF

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CN106565596B
CN106565596B CN201610960390.6A CN201610960390A CN106565596B CN 106565596 B CN106565596 B CN 106565596B CN 201610960390 A CN201610960390 A CN 201610960390A CN 106565596 B CN106565596 B CN 106565596B
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樊丽
林博
葛金印
董川
双少敏
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Abstract

本发明公开了一种萘基衍生物作为线粒体靶向型pH荧光探针的应用，具体为1‑甲基‑4‑[2‑(6‑羟基‑2‑萘)‑乙烯基]‑吡啶碘盐(HNEP+)在活细胞线粒体中的靶向标记及其pH检测的应用。该衍生物以390nm激发，586nm处有最大荧光发射。在磷酸盐缓冲体系中，随着pH由5.00升高至11.50，586nm处的荧光强度逐渐减弱。pKa值为8.8，pH线性范围7.8‑10.0，适宜于线粒体内碱性环境(pH～8.0)的检测。此外，该探针具有超大的Stokes位移(196nm)，良好的选择性，水溶性以及低毒性，有利于细胞内荧光成像研究。细胞内共定位实验及线粒体pH调节实验证明，该探针能够特异性的靶向标记线粒体，并能够高灵敏地检测线粒体内pH的变化。

Description

一种萘基衍生物作为线粒体靶向型pH荧光探针的应用
技术领域
本发明涉及pH荧光探针，具体为1-甲基-4-[2-(6-羟基-2-萘)-乙烯基]-吡啶碘盐(HNEP+)作为线粒体靶向型pH荧光探针的应用。
背景技术
细胞内pH在许多细胞事件中发挥着关键作用，例如细胞的生长和凋亡、内吞作用、受体介导的信号转导、离子转运以及钙调控等。在不同的原核生物和真核细胞的不同亚细胞结构间存在各自的酸碱平衡。pH异常往往会细胞功能紊乱。因此，监测活细胞内pH变化有助于更好的了解细胞的生理和病理过程。
线粒体是真核细胞中一类至关重要的细胞器，它是双层膜包裹的囊状结构，内膜上含有由四个蛋白质复合物构成的电子传递链，在传递电子过程中释放的能量不断将质子从基质泵至膜间隙，使得膜间隙(pHIMS～7.0)的质子浓度高于基质(pHmito～8.0)，由此在内膜两侧形成pH梯度。这种pH梯度在细胞凋亡、神经传递以及胰岛素分泌的过程中发挥着重要的作用，尤其是线粒体酸化已成为线粒体自噬异常的一个特异性事件，而线粒体自噬异常被证实与心血管疾病、神经退行性疾病和雷氏症候群密切相关。因此，对线粒体基质内pH变化进行灵敏、准确的监测，将会对探索线粒体病变以及相关疾病的早期诊断与病理研究产生极大的推动作用。
荧光法由于具有非破坏性、高灵敏性、特异性响应等优点备受关注。尤其是结合激光共聚焦显微成像技术，在细胞内pH检测时显示出其独特的时间和空间分辨率高的性质，成为分子水平上实时检测细胞内pH的重要手段。
目前，文献报道了众多性能优异的pH荧光探针，但是这些探针大多适用于近中性细胞质(pH 6.8-7.4)和弱酸性溶酶体(pH 4.0-5.5)pH变化的检测。遗憾的是，对于线粒体基质内的碱性环境(pH～8.0)缺乏关注，导致这方面荧光探针的种类十分有限。因此，非常有必要开发碱性pH探针，兼具大的Stokes位移和低毒性，并能够靶向应用于线粒体基质内pH变化的检测。
1-甲基-4-[2-(6-羟基-2-萘)-乙烯基]-吡啶碘盐(HNEP+)作为一种有机吡啶盐衍生物，由于分子中存在典型的D(给体)-π-A(受体)结构特点，具有良好的光学活性，目前主要用于非线性光敏材料，也有人用其作为中间体合成荧光探针。但是将该衍生物用于pH荧光探针检测细胞内线粒体pH变化和细胞成像还未见报道。
发明内容
本发明的目的是提供的一种萘基衍生物作为线粒体靶向型pH荧光探针在检测细胞中线粒体pH变化中的应用；所述的萘基衍生物，是1-甲基-4-[2-(6-羟基-2-萘)-乙烯基]-吡啶碘盐(HNEP+)，其结构式为：
该萘基衍生物作为线粒体靶向型pH荧光探针能够特异性的靶向标记线粒体，并能够高灵敏检测线粒体内pH变化。
与现有技术相比，本发明提供的线粒体靶向型pH荧光探针具有如下优点：(1)基于分子内电荷转移原理(ICT)设计，吡啶盐既是电子受体(A)，也是线粒体靶向基团；萘酚为电子给体(D)，同时酚羟基为pH敏感位点，在碱性条件下酚羟基去质子化，使得分子极性增大，与水分子的相互作用增强，从而阻碍了整个体系的ICT效应，导致荧光发射减弱。(2)该探针具有超大的Stokes位移(196nm)，有利于降低成像过程中激发光的干扰。(3)对pH响应具有较高的灵敏性和选择性，不受其他常见金属离子及氨基酸的干扰。(4)pKa值为8.8，pH响应线性范围7.8-10.0，适宜于线粒体内碱性环境(pH～8.0)pH变化的检测。(5)该探针具有良好的细胞膜通透性，能够特异性的靶向标记线粒体，并能够高灵敏检测线粒体内pH变化。
附图说明
图1.实施例1中探针HNEP+随pH变化的紫外吸收光谱图。
图2.实施例1中探针HNEP+在自然光下识别H+前后颜色变化，由黄色变为无色。
图3.实施例1中探针HNEP+随pH变化的荧光发射光谱图。
图4.实施例1中探针HNEP+在紫外光下识别H+前后颜色变化，由蓝色变为无色。
图5.实施例1中探针HNEP+的荧光强度I586随pH值变化的Sigmoidal拟合曲线；插图：pKa值为8.8，pH响应范围7.8-10.0。
图6.实施例1中探针HNEP+在pH 7.0时，在常见金属离子及氨基酸存在下对H+的选择性。
图7.实施例1中探针HNEP+在人肝癌细胞(HepG2)中与市售线粒体特异选择性染料MitoTracker Deep Red FM的共定位激光共聚焦成像图。
图8.实施例1中探针HNEP+分别在pH 7.0，pH 8.5和pH10.0时，与HepG2细胞共同孵育10min的激光共聚焦成像图。
具体实施方式
实施例1
将实施例1中的探针HNEP+用二次水(电导率为18.2)配制成浓度为1mM的储备液保存。实验中用不同pH值的磷酸盐缓冲液(PBS)将探针稀释为终浓度10μM，记录HNEP+在PBS体系中随pH变化的紫外吸收光谱(图1)。随着pH值由6.89升高到11.65，390nm处的吸收峰依次下降，445nm处的吸收峰逐渐增强，并且在413nm附近出现一个明显的等吸收点。同时溶液颜色由黄色变为无色(图2)。
实施例2
同样用不同pH值的磷酸盐缓冲液将探针HNEP+稀释为终浓度10μM，固定激发波长为317nm，记录HNEP+在PBS体系中随pH变化的荧光发射光谱变化(图3)。随着pH值由5.00升高至11.50，586nm处的荧光强度逐渐降低，有适宜于线粒体内碱性环境(pH～8.0)pH变化的检测。在紫外灯照射下，溶液的颜色由黄色变为无色(图4)。根据HNEP+在586nm处的荧光强度值随pH变化的Singmoidal拟合曲线(图5)，计算pKa值为8.8，pH响应线性范围为7.8-10.0。线性回归方程为I＝-9151.11×pH+94210.98，相关系数R2＝0.9948。
实施例3
将实施例1中的探针HNEP+浓度保持在10μM，分别考察该探针在常见离子及氨基酸存在下，对H+的选择性。如图6所示，在PBS体系中，pH 7.0时，探针对上述物质几乎没有响应，证明HNEP+对H+具有很高的选择性。图6中物质的顺序和浓度依次为：1,空白；2,F-(1mM)；3,Cl-(10mM)；4,Br-(1mM)；5,I-(1mM)；6,SO4 2-(1mM)；7,S2O3 2-(1mM)；8,SO3 2-(1mM)；9,HS-(1mM)；10,NO3 -(1mM)；11,NO2 -(1mM)；12,Ac-(1mM)；13,HCO3 -(1mM)；14,ClO4 -(1mM)；15,K+(140mM)；16,Cd2+(1mM)；17,Mg2+(1mM)；18,Li+(1mM)；19,Co2+(1mM)；20,Hg2+(1mM)；21,Ba2+(1mM)；22,Ni2+(1mM)；23,H2O2(1mM)；24,L-GSH(1mM)；25,Hcy(1mM)；26,Cys(1mM).
实施例4
为了观察实施例1中的探针HNEP+是否具有线粒体靶向性，我们首先进行探针与线粒体特异选择性染料MitoTracker Deep Red FM的共定位激光共聚焦成像实验。将贴壁的HepG2细胞与HNEP+(终浓度10μM)在pH 7.0的条件下，于37℃、5％CO2的孵育箱中共同孵育10min，然后用磷酸盐缓冲液(pH 7.0)轻轻洗涤3次，除去多余的探针，再加入MitoTrackerDeep Red FM(终浓度30nM)继续孵育20min后，在激光共聚焦显微镜下观察二者的共定位情况。其中，HNEP+固定激发波长为405nm，收集绿色发射范围545-600nm(由于HNEP+本身发黄色荧光，为更好地观察HNEP+与红色市售线粒体探针的共定位图像，此处将HNEP+设置为绿色(假色)荧光)；MitoTracker Deep Red FM固定激发波长为635nm，收集红色发射范围650-700nm。由图7a可知，HNEP+呈绿色(假色)棒状荧光分布于细胞质区域，说明探针具有良好的细胞膜通透性。此外，HNEP+的绿色荧光与MitoTracker Deep Red FM的红色荧光(图7b)能够很好地重叠，经软件处理得到黄色荧光(图7c)，表明HNEP+与MitoTracker Deep Red FM具有显著的共定位成像，能够靶向定位于线粒体中。明场成像进一步证实了经HNEP+孵育后细胞的存活性(图7d)，说明HNEP+对细胞具有低毒性。
实施例5
将贴壁的HepG2细胞与实施例1中的探针HNEP+在pH 7.0的条件下，于37℃、5％CO2的孵育箱中共同孵育20min，然后用磷酸盐缓冲液(pH 7.0)轻轻洗涤3次，除去多余的HNEP+，在激光共聚焦显微镜下观察。固定激发波长为405nm收集黄色发射范围545-600nm。pH7.0时细胞在黄色通道呈现明亮的黄色(图8a)；当pH降至10.0时，细胞的黄色荧光几乎猝灭(图8b,c)。明场成像进一步证实了经HNEP+孵育后细胞的存活性(图8d-f)。这些结果说明HNEP+能够高灵敏检测细胞内线粒体碱性环境pH的变化。

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1.一种萘基衍生物作为线粒体靶向型pH荧光探针在检测细胞中线粒体pH变化中的应用；所述的萘基衍生物是1-甲基-4-[2-(6-羟基-2-萘)-乙烯基]-吡啶碘盐，其结构式为：
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