CN109651326B - 一类共价键连接标记细胞的荧光探针和跟踪标记细胞的方法 - Google Patents
一类共价键连接标记细胞的荧光探针和跟踪标记细胞的方法 Download PDFInfo
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Abstract
一类共价键连接标记细胞的荧光探针和跟踪标记细胞的方法，涉及细胞荧光探针。一类共价键连接标记细胞的荧光探针由共价连接部分和荧光基团部分组成；荧光基团可以是任何荧光基团，所述荧光基团可以是任何文献报导过的荧光基团的衍生物，所述荧光基团的衍生物可选自罗丹明、香豆素、荧光素等中的一种，通过在荧光基团上引出一个伯胺基或仲胺基来实现与共价连接部分的键连，由此实现多色的、多功能的共价标记细胞。所述共价标记细胞的特异性为所述荧光探针只能够与细胞内蛋白反应而不与细胞培养基中的蛋白反应。所述共价连接部分中共价标记为探针与细胞内蛋白质形成化学键连接。可应用于长时间、特异性跟踪标记细胞，对细胞毒性小。
Description
技术领域
本发明涉及细胞荧光探针，尤其是涉及一类共价键连接标记细胞的荧光探针和跟踪标记细胞的方法。
背景技术
荧光成像技术已成为一种实时的、高分辨率的、无创的监测生物过程的方法，其中化学小分子探针扮演非常重要的角色。小分子荧光探针易于从生物样品流失，导致荧光保留时间短。通过共价键与生物样品内生物大分子连接的荧光探针在生物样品内保留时间更长，荧光信号更稳定。
羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)是一种广泛应用于生物过程研究的共价连接型的荧光探针，包括CFSE在内的商业化Cell-Trackers不仅会与细胞内的蛋白发生反应，而且容易与全血清细胞培养基中的蛋白质发生反应。
参考文献：
[1]Evans,M.J.；Cravatt,B.F.Chemical Reviews 2006,106,3279.
[2]Lu,C.-P.；Ren,C.-T.；Wu,S.-H.；Chu,C.-Y.；Lo,L.-C.ChemBioChem 2007,8,2187.
[3]Weston,S.A.；Parish,C.R.Journal of Immunological Methods 1990,133,87.
发明内容
本发明的目的在于提供可作为已有共价型细胞探针的补充，具有保留时间长、信号稳定、细胞毒性小等优势，通过引入不同荧光基团，实现不同荧光标记的一类共价键连接标记细胞的荧光探针和跟踪标记细胞的方法。
所述一类共价键连接标记细胞的荧光探针由共价连接部分和荧光基团部分组成，所述共价连接部分的结构式如下：
所述荧光基团可以是任何荧光基团，所述荧光基团可以是任何文献报导过的荧光基团的衍生物，所述荧光基团的衍生物可选自罗丹明、香豆素、荧光素等中的一种，通过在荧光基团上引出一个伯胺基或仲胺基来实现与共价连接部分的键连，由此实现多色的、多功能的共价标记细胞。
所述共价标记细胞的特异性为所述荧光探针只能够与细胞内蛋白反应而不与细胞培养基中的蛋白反应。
所述共价连接部分中共价标记为探针与细胞内蛋白质形成化学键连接。
所述共价连接部分的化学结构式可为：
其中，R基团为CH3-,CH3CH2-,CH3CH2CH2-。
所述一类共价键连接标记细胞的荧光探针可应用于长时间、特异性跟踪标记细胞，对细胞毒性小。
所述一类共价键连接标记细胞的荧光探针的代表之一红色共价标记细胞荧光探针的化学结构式如下：
所述跟踪标记细胞的方法是一类长时间、特异性、对细胞损伤小的跟踪标记细胞的方法，所述细胞为活细胞，跟踪标记的时间为72h以上，且细胞在此期间即使进行细胞分裂，分裂次数达5次以上依旧有荧光信号保留，荧光探针获得的荧光信号可以通过共聚焦光显微镜、激光共聚焦荧光显微镜、转盘共聚焦荧光显微镜、结构分辨荧光显微镜、随机重构荧光显微镜和流式细胞仪检测。
本发明的有益效果如下：
1)该类荧光探针的合成工艺简单，原料廉价，产物稳定性高，易于推广；
2)该种荧光探针在细胞内保留时间长(超过72h)；
3)该种荧光探针在细胞分裂传代之后信号依旧存在，细胞分裂达5次以上仍有信号保留；
4)该荧光探针的共价连接部分可以连接不同的荧光基团，从而制备具有不同荧光激发与荧光发射的荧光探针，进而实现对多种细胞的区分与标记。
5)该荧光探针的细胞毒性小。
附图说明
图1为本发明荧光探针的作用原理图。在图1中，1代表荧光基团，2代表细胞内的酯酶，3代表细胞内蛋白。
图2为本发明荧光探针的合成路线。
图3为本发明红色共价标记荧光探针在细胞中长时间保留的激光共聚焦荧光显微图。在图3中，标尺为10μm。
图4为本发明红色共价标记荧光探针在细胞中长时间保留的流式细胞仪数据。
图5为本发明红色、绿色、蓝色的共价标记荧光探针在多细胞体系中的激光共聚焦荧光显微。在图5中，标尺为10μm。
图6为本发明红色共价标记荧光探针的MTT比色法测细胞毒性实验数据图。在图6中，a为二甲亚砜，b为1μM，c为2μM，d为4μM，e为8μM。
具体实施方式
以下实施例将结合附图对本发明作进一步的说明。
本发明的原理如下：
原理图参见图1，该荧光探针通过乙酰化扁桃酸的两个羟基，得化合物Ⅱ(图2)，再利用化合物Ⅱ中的羧基与荧光基团露出的伯胺基或仲胺基发生酰胺缩合反应，从而制备得到。该种探针在细胞内能与酯酶发生反应进而形成醌甲基的结构，由于醌甲基的结构十分不稳定，易与蛋白质中的亲核基团发生反应，因此能够与细胞内的蛋白质发生共价键连接，从而达到长时间标记跟踪细胞的作用。
以下给出具体实施例。
实施例1：本发明红色、绿色、蓝色共价标记荧光探针的制备
1)取50ml圆底烧瓶，加入3.4g、20mmol 2-羟基-2-(4-羟基苯基)乙酸(既扁桃酸，图2化合物Ⅰ)，溶于10ml吡啶中。逐滴加入100mmol乙酰氯，在室温下搅拌1h。随后旋干溶剂并过硅胶柱提纯，得图2化合物Ⅱ，产率86％。
2)取504mg，2.0mmol化合物Ⅱ溶于5ml二氯甲烷中。加入500mg，2.6mmol1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和606mg，6.0mmol三乙胺。随后加入914mg，2.0mmol化合物罗丹明衍生物(记图2，化合物Ⅲ)。反应2h结束后用1M稀盐酸处理，二氯甲烷萃取。有机相旋干溶剂过柱提纯，得红色共价标记荧光探针，记图2化合物Ⅳ。
使用类似的合成方法，将化合物Ⅱ与其他荧光基团的衍生物发生反应，可以分别得到绿色共价标记荧光探针(图2，化合物Ⅴ)，和蓝色共价标记荧光探针(图2，化合物Ⅵ)
实施例2：本发明红色共价标记荧光探针在细胞中长时间保留的细胞实验。
1)称取2.5mg，约4μmol的红色共价标记荧光探针溶于1mL的二甲亚砜。即得4mM的红色共价标记荧光探针的标准溶液。
2)取10μL红色共价标记荧光探针的标准溶液与10mL细胞培养溶液混合，得到含有4μmol/L(4μM)红色共价标记荧光探针的细胞培养液。
3)将含有4μM红色共价标记荧光探针的细胞培养液与HeLa细胞在35mm玻璃底培养皿共同孵育1h，用新鲜的细胞培养液洗三遍，经过0h、24h、48h、72h用共聚焦荧光显微镜对细胞内荧光信号进行采集。实验结果见图3，说明探针在经过72h，细胞分裂多代后仍有信号保留。
4)将含有4μM红色共价标记荧光探针的细胞培养液与HeLa细胞在35mm玻璃底培养皿共同孵育1h，用新鲜的细胞培养液洗3遍，经过0h、24h、48h后用流式细胞仪对荧光信号进行采集。使用结果见图4，说明探针在经过长时间，细胞分裂多代后仍能用流式细胞仪检测信号。
实施例3：本发明红色、绿色、蓝色共价标记荧光探针联用，运用于多细胞体系跟踪标记细胞的实验
1)与实施例2步骤1,2相同，得浓度为4μM的红色、绿色、蓝色共价标记荧光探针的标准细胞培养液。
2)将分别含有4μM红色、绿色、蓝色共价标记荧光探针的细胞培养液与HeLa细胞孵育在24孔板的孔中，孵育1h。用新鲜的细胞培养液洗3次。将3种不用处理的细胞进行混合传代播种于同一个35mm玻璃底培养皿中。经过24h之后用共聚焦荧光显微镜对细胞内荧光信号进行采集。实验结果见图5，说明本发明共价标记荧光探针可以运用于多细胞体系的荧光跟踪与标记。
本发明红色共价标记荧光探针的MTT比色法测细胞毒性实验数据图参见图6。
Claims (3)
1.一类共价键连接标记细胞的荧光探针，其特征在于由共价连接部分和荧光基团部分组成，所述共价连接部分的结构式如下：
所述荧光基团是任何文献报导过的荧光基团的衍生物，所述荧光基团的衍生物选自罗丹明、香豆素、荧光素中的一种，通过在荧光基团上引出一个伯胺基或仲胺基来实现与共价连接部分的键连，由此实现多色的、多功能的共价标记细胞；所述共价标记细胞的特异性为所述荧光探针与细胞内蛋白反应而不与细胞培养基中的蛋白反应；所述共价连接部分中共价标记为探针与细胞内蛋白质形成化学键连接；
其应用于长时间、特异性跟踪标记细胞，对细胞毒性小；所述跟踪标记细胞的方法是一类长时间、特异性、对细胞损伤小的跟踪标记细胞的方法，所述细胞为活细胞；跟踪标记的时间为72h以上，且细胞进行细胞分裂，且分裂次数达5次以上依旧有荧光信号保留，荧光探针获得的荧光信号通过共聚焦光显微镜、激光共聚焦荧光显微镜、转盘共聚焦荧光显微镜、结构分辨荧光显微镜、随机重构荧光显微镜和流式细胞仪检测。
2.如权利要求1所述一类共价键连接标记细胞的荧光探针，其特征在于所述共价连接部分的化学结构式为：
其中，R基团为CH3-,CH3CH2-,CH3CH2CH2-。
3.如权利要求1所述一类共价键连接标记细胞的荧光探针，其特征在于其红色共价标记细胞荧光探针的化学结构式如下：
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