CN110387226A - 一种用于检测肿瘤的荧光探针及用途 - Google Patents

一种用于检测肿瘤的荧光探针及用途 Download PDF

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CN110387226A
CN110387226A CN201810359911.1A CN201810359911A CN110387226A CN 110387226 A CN110387226 A CN 110387226A CN 201810359911 A CN201810359911 A CN 201810359911A CN 110387226 A CN110387226 A CN 110387226A
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高清志
刘胜男
石蕴丽
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Tianjin University
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Abstract

本发明公开了一种用于检测肿瘤的荧光探针及用途，一种用于检测肿瘤的荧光探针，其结构如式(I)所示：

Description

一种用于检测肿瘤的荧光探针及用途
技术领域
本发明属于生物分析检测领域，特别涉及一种靶向核苷转运蛋白通道的荧光探针及其在制备检测肿瘤试剂中的用途。
技术背景
分布于细胞膜上的核苷转运蛋白是负责核苷类药物转运和传递的主要通道。其分为两类，即平衡型核苷转运体(equilibrative nucleoside transporter,ENT)和浓度型核苷转运体(concentrative nucleoside transporter,CNT)。其中，ENT在转运和传递核苷类药物过程中起到十分重要的作用。ENT在肿瘤研究中的重要性主要体现在它在多种人类恶性肿瘤细胞均有广泛的表达。例如，研究表明，hENT1在人宫颈癌(Hela)，人乳腺癌(MCF-7)，人肺癌(A549)，人黑色素瘤(G361)，人白血病(K562)等均有中等及高水平的表达(Neuropharmacology 36(1997)1167-1179).目前，人类的ENT(hENT)被证实由四种亚型(hENT1、2、3、4)，其中对hENT1和hENT2完成了较多的科学研究。结果发现，hENT1能够有效转运核苷类抗肿瘤药物，例如：克拉屈滨(Cladribine)、阿糖胞苷(Ara-C)、氟尿嘧啶(5-FU)，氟达拉滨(Fludarabine)、卡培他滨(Capecitabine)、吉西他滨(Gemcitabine)等等。进一步研究发现，某些核苷类抗肿瘤药物在对肿瘤细胞发挥药效的同时，会同时上调hENT的表达和转运活性，相反，如果肿瘤细胞对某些抗癌药物产生耐药，则hENT会表现为下调(BiochemJ 136(2004)733-740)。此外，蛋白激酶对细胞膜表面的ENT1具有灵敏的调控作用，例如蛋白激酶C(PKC)的某些亚型可增强hENT1的活性和功能(FEBS Letters 517(2002)201-205)。而且，蛋白激酶抑制剂类药物对PKC的抑制作用也直接影响hENT1的表达和转运活性(Pharmacol Exp Ther304(2003)753-760)。
综上所述，如果能够实现对核苷转运蛋白hENT1的针对性荧光识别，通过测试和掌握肿瘤细胞的hENT表达水平，不仅可用于发现对肿瘤有效的核苷类或者蛋白激酶类抗癌药物，还能够通过检测肿瘤患者癌细胞的hENT1表达水平对化疗药物的耐药性进行监控和评价。18F-脱氧胸腺嘧啶核苷(18F-FLT)是目前基于hENT核苷转运蛋白而开发并广泛用于前临床和部分临床研究的肿瘤示踪和显像试剂。18F-FLT的肿瘤检测原理基于放射性F18原子和PET成像技术，由于其在临床研究中被试者出现显著的贫血、粒细胞降低和外周神经病变以及肝肾毒性等，并造成受试者意外死亡(J Infect Dis 170(1994)1394-1403；MolImaging Biol 11(2009)343-355)。结合上述临床应用中出现的问题，该肿瘤示踪剂需要放射性同位素在分子中的植入以及合成效率低等因素极大地限制了它的应用。
发明内容
本发明的目的是克服现有技术的不足，提供一种用于检测肿瘤的荧光探针。
本发明的第二个目的是提供一种与ENT蛋白特异性结合或细胞内转运的用于检测肿瘤的荧光探针在检测肿瘤中的用途。
本发明的技术方案概述如下：
一种用于检测肿瘤的荧光探针，其结构如式(I)所示：
其中，
Z选自氢原子，乙酰基，C1-C8的直链烷基，F，Cl，Br，I，NH2，CN，NO2；
R选自(A-1)、(A-2)、(A-3)、(A-4)、(A-5)、(A-6)、(A-7)或(A-8)；
n＝0-4。
较好地，Z选自氢原子，甲基，F；R选自(A-1)、(A-2)、(A-3)、(A-4)或(A-5)；n＝1-4。
更好地，Z选自氢原子，甲基，F；R选自(A-1)、(A-4)或(A-5)；n＝1-4。
最好是，Z选自氢原子，甲基，F；R选自(A-1)。
上述荧光探针在制备肿瘤检测试剂中的用途。
本发明具有以下的有益效果：
本发明用于检测肿瘤的荧光探针能够直接用在细胞体系中分析和检测肿瘤细胞ENT蛋白的表达程度，从而完成对肿瘤细胞的筛选，肿瘤细胞对化疗药物的耐药预测，以及筛选不同种类抗肿瘤药物的目的。为肿瘤早期筛选和诊断，发现和开发新的抗肿瘤药物，预测和评估肿瘤对化疗药物的耐药性等提供了有效的手段和有用的工具。
附图说明
图1为实施例1、2和3制备的用于检测肿瘤的荧光探针在正常乳腺细胞与乳腺癌细胞中荧光强度差别；
图2为实施例4、5和6制备的用于检测肿瘤的荧光探针在正常乳腺细胞与乳腺癌细胞中荧光强度差别；
图3为实施例7、8和9制备的用于检测肿瘤的荧光探针在正常乳腺细胞与乳腺癌细胞中荧光强度差别；
图4为实施例10、11和12制备的用于检测肿瘤的荧光探针在正常乳腺细胞与乳腺癌细胞中荧光强度差别；
图5为实施例13、14和15制备的用于检测肿瘤的荧光探针在正常乳腺细胞与乳腺癌细胞中荧光强度差别.
具体实施方式
本发明的设计理念是创造一种带有荧光平衡型核苷转运体ENT底物(荧光探针)，荧光探针含有尿嘧啶结构，能够被平衡型核苷转运体ENT有效识别，当荧光探针与ENT相互作用时依据ENT表达量的不同而产生荧光强度的变化，从而通过测试荧光探针在细胞体系中产生的荧光信号变化，达到诊断肿瘤，检测肿瘤耐药以及筛选肿瘤候选药物的目的。
本发明的用于肿瘤检测和诊断的荧光探针可经过下述合成路线制备获得：
方法A：用于肿瘤检测和诊断的荧光探针(含有荧光团A-1和A-2)的制备：
方法A中，含荧光团的卤素取代化合物(A-1)-Cl和(A-2)-Cl，可以直接商业购买，与3-氨乙基尿嘧啶衍生物在等当量的有机碱，例如4-二甲氨基吡啶，二乙胺，二异丙基乙胺或者吡啶的存在下进行碳-氮偶联反应。反应可在N,N-二甲基甲酰胺(DMF)，二氯甲烷(DCM)，吡啶等有机溶剂中室温或者加热便可制备获得用于检测肿瘤的荧光探针，反应的温度一般为室温至200℃反应3小时至240小时，较好地在室温至120℃反应3-72小时即可完成。所取得的目标产物可以通过硅胶柱分离、重结晶、或者使用半制备液相色谱分离获得。
方法B：用于检测肿瘤的荧光探针(含有荧光团A-3～A-8)的制备：
方法B：含有荧光团A-3至A-8的荧光探针
方法B中，带荧光团的羧酸原料(A-3)-OH、(A-4)-OH、(A-5)-OH、(A-6)-OH、(A-7)-OH、(A-8)-OH可以直接商业购买，或者遵循文献的方制备获得(Bioorg MedChem.2015Apr15；23(8):1758-62.)，带荧光团的羧酸原料与3-氨乙基尿嘧啶衍生物在耦合试剂存在下进行酰胺缩合。所使用的耦合试剂可以是肽合成反应中常用的多肽缩合试剂，例如N，N’-二环己基碳二亚胺(DCC)，1-羟基苯并三唑(HOBt),六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)或O-苯并三氮唑-N,N,N,N-四甲基脲四氟硼酸酯(TBTU)，O-苯并三氮唑-四甲基脲六氟磷酸盐(HBTU)等的存在下进行缩合反应进行酰胺缩合，所使用的溶剂可以是DMF，DCM，THF(四氢呋喃)，NMP(N-甲基吡咯烷酮)以及它们的任意混合溶剂，反应温度一般为零℃至100℃，缩合反应一般添加等当量的有机碱，例如4-二甲氨基吡啶，二乙胺，二异丙基乙胺等。反应时间一般可在3小时至72小时内完成。
在上述所有方法中，3-氨乙基尿嘧啶衍生物原料可以遵循文献的类似方法，由1-位保护的相应的尿嘧啶与环乙亚胺反应在3-位引入氨乙基，然后在酸性条件下水解脱去1-位保护基制备获得(J.Org.Chem.37(1972)2165-2168)。
反应式4和反应式7中的n为0，1，2，3或4，3-氨基尿嘧啶衍生物原料中的Z选自氢原子，乙酰基，C1-C8的直链烷基，F，Cl，Br，I，NH2，CN，NO2。该原料的制备方法可以遵循文献报道的方法(J.Org.Chem.37(1972)2165-2168)由1-位保护的相应的尿嘧啶与环乙亚胺反应在3-位引入氨乙基，然后在酸性条件下水解脱去1-位保护基。上述制备方法中，相应的含有(A-1)至(A-8)荧光团的原料均可以商业购买，或者遵循文献报道的反应条件顺利制备获得。
下面以实施例进一步说明本发明，但不限制本发明。
实施例1
用于检测肿瘤的荧光探针(I-1)的制备：
160mg 3-氨乙基尿嘧啶与200mg含荧光团的卤素取代化合物(A-1)-Cl溶于5毫升N,N-二甲基甲酰胺中，然后在室温加入202mg三乙胺，反应在氮气环境中在60℃加热12小时。反应完成后冷却至室温，加入20毫升二氯甲烷搅拌，然后将反应液用10毫升饱和氯化铵、10毫升饱和盐水依次洗涤。水相用二氯甲烷(2x10)反萃2次，得有机相溶剂用无水硫酸钠干燥，减压旋干，硅胶色谱法(二氯甲烷：甲醇＝15:1)纯化，得黄色固体目标产物(222mg)。
收率：70％
1H NMR(400MHz,DMSO)δ11.12(s,1H),9.45(s,1H),8.57(d,1H,J＝8.6Hz,),7.41(m,1H),6.55(d,1H,J＝8.6Hz,),5.50(m,1H),4.08(m,2H),3.71(m,2H).
ESI-MS:(m/z)319.1[M+1]+
实施例2
用于检测肿瘤的荧光探针(I-2)的制备：
按照实施例1的方法，将3-氨乙基尿嘧啶换成170mg3-氨乙基-5-甲基尿嘧啶，反应在60℃加热12小时，最后获得目标产物250mg。
收率：75％
1H NMR(400MHz,DMSO)δ11.12(s,1H),9.45(s,1H),8.57(d,1H,J＝8.6Hz,),7.78(br,1H),6.55(d,1H,J＝8.6Hz,),4.08(m,2H),3.71(m,2H),2.40(s,3H).
ESI-MS:(m/z)333.1[M+1]+
实施例3
用于检测肿瘤的荧光探针(I-3)的制备：
按照实施例1的方法，将3-氨乙基尿嘧啶换成175mg3-氨乙基-5-氟尿嘧啶，反应在60℃加热12小时，最后获得目标产物235mg。
收率：70％
1H NMR(400MHz,DMSO)δ11.12(s,1H),9.45(s,1H),8.57(d,1H,J＝8.6Hz,),7.88(d,1H,J＝5.0Hz),6.55(d,1H,J＝8.6Hz,),4.08(m,2H),3.71(m,2H).
ESI-MS:(m/z)337.1[M+1]+
实施例4
用于检测肿瘤的荧光探针(I-4)的制备：
3-氨乙基尿嘧啶(160mg)与450mg带荧光团的羧酸原料(A-4)-OH(n＝1)溶于8毫升N,N-二甲基甲酰胺溶液中，然后在室温加入202mg三乙胺，4-二甲氨基吡啶123mg，N，N'-二环己基碳二亚胺206mg，反应在氮气环境中在室温12小时。反应完成后，加入20毫升二氯甲烷搅拌，然后将反应液用10毫升饱和氯化铵、10毫升饱和盐水依次洗涤。水相用二氯甲烷(2x10)反萃2次，得有机相溶剂用无水硫酸钠干燥，减压旋干，硅胶色谱法(二氯甲烷：甲醇＝10:1)纯化，得红色固体目标产物(303mg)。
收率：55％
1H NMR(400MHz,MeOD)δ8.45(td,J＝13.5,1.5Hz,1H),7.48–7.17(m,9H),6.37(dd,J＝13.6,4.9Hz,2H),5.50(m,1H),3.85(t,J＝6.8Hz,2H),4.08(m,2H),3.71(m,2H),3.62(d,J＝1.8Hz,3H),3.56(d,J＝4.1Hz,1H),3.50(d,J＝3.0Hz,1H),2.63(t,J＝6.5Hz,2H),1.68(d,J＝1.1Hz,12H).
ESI-MS:(m/z)552.3[M]+
实施例5
用于检测肿瘤的荧光探针(I-5)的制备：
按照实施例4的方法，将其中的160mg 3-氨乙基尿嘧啶替换成170mg3-氨乙基-5-甲基尿嘧啶，其它同实施例4，最后获得目标产物334mg。
收率：60％
1H NMR(400MHz,MeOD)δ8.45(td,J＝13.5,1.5Hz,1H),7.78(br,1H),7.48–7.17(m,8H),6.37(dd,J＝13.6,4.9Hz,2H),4.08(m,2H),3.85(t,J＝6.8Hz,2H)3.71(m,2H),3.50(d,J＝3.0Hz,1H),3.62(d,J＝1.8Hz,3H),3.56(d,J＝4.1Hz,1H),2.63(t,J＝6.5Hz,2H),2.40(s,3H),1.68(d,J＝1.1Hz,12H).
ESI-MS:(m/z)556.3[M]+
实施例6
用于检测肿瘤的荧光探针(I-6)的制备：
按照实施例4的方法，将其中的160mg 3-氨乙基尿嘧啶换成175mg3-氨乙基-5-氟尿嘧啶，其它同实施例4，最后获得目标产物331mg。
收率：58％
1H NMR(400MHz,MeOD)δ8.45(td,J＝13.5,1.5Hz,1H),7.88(d,1H,J＝5.0Hz),7.48–7.17(m,8H),6.37(dd,J＝13.6,4.9Hz,2H),4.08(m,2H),3.85(t,J＝6.8Hz,2H),3.71(m,2H),3.50(d,J＝3.0Hz,1H),3.62(d,J＝1.8Hz,3H),3.56(d,J＝4.1Hz,1H),,2.63(t,J＝6.5Hz,2H),1.68(d,J＝1.1Hz,12H).
ESI-MS:(m/z)570.3[M]+
实施例7
用于检测肿瘤的荧光探针(I-7)的制备：
按照实施例4的方法，将其中的450mg带荧光团的羧酸原料(A-4)-OH(n＝1)换成(A-4)-OH(n＝4)(494mg)，与3-氨乙基尿嘧啶165mg反应，其它同实施例4，最后获得目标产物430mg。
收率：72％
ESI-MS:(m/z)594.3[M]+
Elemental analysis:
Calculated for C36H44ClN5O3,C,68.61；H,7.04；N,11.11；found C,68.63；H,7.01；N,11.10.
实施例8
用于检测肿瘤的荧光探针(I-8)的制备：
按照实施例4的方法，将其中的带荧光团的羧酸原料(A-4)-OH(n＝1)换成(A-4)-OH(n＝4)(494mg)，其中的160mg3-氨乙基尿嘧啶换成170mg3-氨乙基-5-甲基尿嘧啶，其它同实施例4，最后获得目标产物401mg。
收率：66％
ESI-MS:(m/z)608.4[M]+
Elemental analysis:
Calculated for C37H46ClN5O3,C,68.98；H,7.20；N,10.87；found C,68.98；H,7.22；N,10.85.
实施例9
用于检测肿瘤的荧光探针(I-9)的制备：
按照实施例4的方法，将其中的带荧光团的羧酸原料(A-4)-OH(n＝1)换成(A-4)-OH(n＝4)(494mg)，其中的160mg3-氨乙基尿嘧啶换成175mg3-氨乙基-5-氟尿嘧啶，其它同实施例4，最后获得目标产物355mg。
收率：58％
ESI-MS:(m/z)612.3[M]+
Elemental analysis:
Calculated for C36H43ClFN5O3,C,66.70；H,6.69；N,10.80；found C,66.71；H,6.66；N,10.83.
实施例10
用于检测肿瘤的荧光探针(I-10)的制备：
按照实施例4的方法，将其中带荧光团的羧酸原料(A-4)-OH(n＝1)换成478mg(A-5)-OH(n＝1)其它同实施例4，最后获得目标产物375mg。
收率：65％
ESI-MS:(m/z)578.3[M]+
Elemental analysis:
Calculated for C35H40ClN5O3,C,68.45；H,6.56；N,11.40；found C,68.46；H,6.55；N,11.43.
实施例11
用于检测肿瘤的荧光探针(I-11)的制备：
按照实施例4的方法，将其中带荧光团的羧酸原料(A-4)-OH(n＝1)换成478mg(A-5)-OH(n＝1)，并将3-氨乙基尿嘧啶换成170mg3-氨乙基-5-甲基尿嘧啶，其它同实施例4，最后获得目标产物343mg。
收率：58％
ESI-MS:(m/z)592.3[M]+
Elemental analysis:
Calculated for C36H42ClN5O3,C,68.83；H,6.74；N,11.15；found C,68.84；H,6.75；N,11.14.
实施例12
用于检测肿瘤的荧光探针(I-12)的制备：
按照实施例4的方法，将其中带荧光团的羧酸原料(A-4)-OH(n＝1)换成478mg(A-5)-OH(n＝1)，并将3-氨乙基尿嘧啶换成175mg3-氨乙基-5-氟尿嘧啶，其它同实施例4，最后获得目标产物423mg。
收率：71％
ESI-MS:(m/z)596.3[M]+
Elemental analysis:
Calculated for C35H39ClFN5O3,C,66.50；H,6.22；N,11.08；found C,66.51；H,6.22；N,11.09.
实施例13
用于检测肿瘤的荧光探针(I-13)的制备：
按照实施例4的方法，将其中带荧光团的羧酸原料(A-4)-OH(n＝1)换成520mg(A-5)-OH(n＝4)，其它同实施例4，最后获得目标产物410mg。
收率：66％
ESI-MS:(m/z)620.4[M]+
Elemental analysis:
Calculated for C38H46ClN5O3,C,69.55；H,7.07；N,10.67；found C,69.57；H,7.06；N,10.68.
实施例14
用于检测肿瘤的荧光探针(I-14)的制备：
按照实施例4的方法，将其中带荧光团的羧酸原料(A-4)-OH(n＝1)换成520mg(A-5)-OH(n＝4)，将160mg3-氨乙基尿嘧啶换成170mg3-氨乙基-5-甲基尿嘧啶，其它同实施例4，最后获得目标产物443mg。
收率：70％
ESI-MS:(m/z)634.4[M]+
Elemental analysis:
Calculated for C39H48ClN5O3,C,69.88；H,7.22；N,10.45；found C,69.89；H,7.22；N,10.46.
实施例15
用于检测肿瘤的荧光探针(I-15)的制备：
按照实施例4的方法，将其中带荧光团的羧酸原料(A-4)-OH(n＝1)换成520mg(A-5)-OH(n＝4)，将160mg3-氨乙基尿嘧啶换成175mg3-氨乙基-5-氟尿嘧啶，其它同实施例4，最后获得目标产物453mg。
收率：71％
ESI-MS:(m/z)638.4[M]+Elemental analysis:
Calculated for C38H45ClFN5O3,C,67.69；H,6.73；N,10.39；found C,67.67；H,6.72；N,10.36.
实施例1-实施例15对部分用于检测肿瘤的荧光探针的制备进行了详细的介绍，按实施例1的方法可以对式(I)化合物中Z选自乙酰基，C2、C3、C4、C5、C6、C7、C8的直链烷基，Cl，Br，I，NH2，CN，NO2；R选自(A-2)进行制备；按实施例4的方法可以对式(I)化合物中Z选自乙酰基，C2、C3、C4、C5、C6、C7、C8的直链烷基，Cl，Br，I，NH2，CN，NO2；R选自(A-3)、(A-6)、(A-7)或(A-8)进行反应，得到相应的化合物；
R还可以选自表1列举的具有不同激发波长的荧光团：
表1
荧光团名称 激发波长(nm) 发射波长(nm) 中文名称
Hydroxycoumarin 325 386 羟基香豆素
Aminocoumarin 350 445 氨基香豆素
Methoxycoumarin 360 410 甲氧基香豆素
Cascade Blue (375)；401 423 级联蓝
Pacific Blue 403 455 太平洋蓝
Pacific Orange 403 551 太平洋橙
Lucifer yellow 425 528 荧光黄
NBD 466 539 硝基苯并恶二唑
R-Phycoerythrin(PE) 480；565 578 藻红蛋白
Red 613 480；565 613 溶剂红
PerCP 490 675 多甲藻黄素叶绿素蛋白
Fluorescein 495 519 荧光素
BODIPY-FL 503 512 氟化硼二吡咯
Cy2 489 506 花青素Cy2
Cy3 (512)；550 570；(615) 花青素Cy3
Cy3B 558 572；(620) 花青素Cy3B
Cy3.5 581 594；(640) 花青素Cy3.5
Cy5 (625)；650 670 花青素Cy5
Cy5.5 675 694 花青素Cy5.5
Cy7 743 767 花青素Cy7
TRITC 547 572 四甲基异硫氰酸罗丹明
X-Rhodamine 570 576 罗丹明
Lissamine Rhodamine B 570 590 丽丝胺罗丹明B
Texas Red 589 615 德克萨斯红
Allophycocyanin(APC) 650 660 别藻蓝蛋白
试验例1：用于检测肿瘤的荧光探针(简称荧光探针)荧光光谱的测定实验
将荧光探针用DMSO溶解，PBS缓冲液稀释成50μM，用Thermo ScientificVarioskan LUX荧光酶标仪测定荧光探针的荧光光谱，以确定本发明荧光探针的荧光激发和发射波长。
本发明所提供的荧光探针中，
A-1系列的荧光探针的激发波长范围为：470nm至475nm，发射波长范围为：540nm和555nm；
A-2系列的荧光探针的激发波长范围为：400nm至415nm，发射波长范围为：500nm和550nm；
A-3系列的荧光探针化合物的激发波长范围为：350nm至390nm，发射波长范围为：500nm和520nm；
A-4系列的荧光探针的激发波长范围为：510nm至550nm，发射波长范围为：560nm和580nm；
A-5系列的荧光探针的激发波长范围为：620nm至645nm，发射波长范围为：660nm和680nm；
A-6系列的荧光探针的激发波长范围为：470nm至510nm，发射波长范围为：520nm和560nm；
A-7系列的荧光探针的激发波长范围为：360nm至380nm，发射波长范围为：410nm和450nm；
A-8系列的荧光探针的激发波长范围为：510nm至525nm，发射波长范围为：530nm和560nm；
本发明代表性荧光探针的激发波长与发射波长：表-2：
试验例2：肿瘤检测试剂的制备
1，称取1mg荧光探针，根据其分子量使用分析纯二甲基亚砜(DMSO)将荧光探针配制成1mM溶液，然后用PBS稀释成1-100μM的检测试剂。
2，当所使用的荧光探针在稀释过程中出现不溶解时，可通过提高DMSO的体积百分比使探针充分溶解配制透明溶液。DMSO的最高体积百分比控制在10％以下。
3，将配制成的荧光探针试剂溶液，分成两个浓度组备用。一般使用5μM/10μM，10μM/20μM,50μM/100μM二倍浓度组合。测试肿瘤细胞时将选定的浓度组合中两个不同浓度的探针试剂，针对肿瘤细胞作同时测试，以提高检测的精密度和可信度。
4，所配制的荧光探针试剂，一般在使用前配制，或者配置后置于-20℃冷冻避光保存。
试验例3：本发明的用于检测肿瘤的荧光探针在检测肿瘤中的应用
细胞培养(正常细胞和肿瘤细胞)：
从美国模式菌种收集中心(ATCC)购得人正常乳腺细胞MCF10A和人乳腺癌细胞MCF7。分别按照相应的细胞培养条件进行培养并使用。具体培养条件如下：MCF10A：RPMI-1640培养基，10％胎牛血清，95％空气，5％二氧化碳，37℃；MCF7：MEM培养基，10％胎牛血清，10ug/mL胰岛素95％空气，5％二氧化碳，37℃。
肿瘤检测：
1，将荧光探针先用DMSO配制成1mM溶液，然后用PBS稀释成50μM和100μM两个浓度待用。
2，在细胞培养箱中，在不同的96孔板中分别铺设1X104正常乳腺细胞MCF10A和乳腺癌细胞MCF7。待细胞过夜贴壁后，分别加入50μM和100μM两个浓度的荧光探针溶液培养30分钟。每个浓度设复孔5个，同时设空白对照组不添加细胞只添加荧光探针溶液。冰冷PBS洗3遍，转移到酶标板中测量荧光强度。
平均荧光强度＝(每个药物浓度下测得的每个复孔荧光强度取平均值)-(空白对照组各个复孔测得的荧光强度平均值)。取小数点后2位。
荧光探针在正常细胞和对应的癌细胞内的荧光强度在两种探针浓度下的测试结果如下：表-3：
表3中结果显示，本发明荧光探针在正常乳腺细胞内的荧光强度与相应的乳腺癌细胞相比，癌细胞对荧光探针的吸收平均超过正常细胞的4倍以上。显示本发明荧光探针能够敏感识别正常细胞和癌细胞，从而能够应用于对癌细胞的检测。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明,对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
本发明还提供了所述荧光探针在制备用于分析或检测肿瘤的制剂，以及在分析检测肿瘤中的用途。由于肿瘤细胞在增殖过程中，会需要摄取大量的核苷类物质从而高表达ENT转运蛋白，针对这种肿瘤增殖特异性生理现象，本发明荧光探针可以通过对肿瘤细胞、肿瘤组织切片的染色用于诊断肿瘤细胞的存在以及对治疗效果的评估。另外，某些核苷类或者蛋白激酶类化疗药物会迅速上调肿瘤细胞对ENT的表达，因此应用本发明的荧光探针可以针对这类药物进行细胞筛选研究。针对上述用途，通过使用1-100μM的本发明荧光探针，与细胞培养10-120min，便能针对细胞是否高表达ENT进行评估。检测探针荧光强度的方法可以是荧光分光光度计，荧光酶标仪，荧光显微镜以及共聚焦显微镜等，本发明采用荧光酶标仪实施荧光强度检测，所使用的激发波长根据具体的探针而定，具体探针的激发和发射波长如实验例1所示。
上述用于检测肿瘤的荧光探针对分布于细胞膜上的核苷转运蛋白尤其是平衡型核苷转运体ENT具有特异性识别能力，并随着ENT蛋白在细胞表达水平的不同而表现出不同荧光强度。因此本发明的探针分子能够对肿瘤细胞表达ENT的程度进行检验和检测。很多种类的癌细胞与正常细胞相比高表达ENT蛋白，对此类癌细胞可通过使用本发明的荧光探针实施体外或体内的监测评估。肿瘤细胞在对化疗药物产生耐药的过程中往往会自动下调细胞本身表达ENT蛋白，因此上述探针可用于检验和预测肿瘤治疗过程中是否形成耐药反应。另外，某些核苷类，或者蛋白激酶抑制剂类的抗肿瘤药物，通过对肿瘤细胞的刺激能够使其产生ENT蛋白的上调或者下调反应，据此，本发明的荧光探针亦可用于通过检测肿瘤细胞因未知药物产生的ENT变化来筛选不同的抗肿瘤药物。

Claims (5)

1.一种用于检测肿瘤的荧光探针，其结构如式(I)所示：
其中，
Z选自氢原子，乙酰基，C1-C8的直链烷基，F，Cl，Br，I，NH2，CN，NO2；
R选自(A-1)、(A-2)、(A-3)、(A-4)、(A-5)、(A-6)、(A-7)或(A-8)；
n＝0-4。
2.根据权利要求1所述荧光探针，其特征是所述：Z选自氢原子，甲基，F；R选自(A-1)、(A-2)、(A-3)、(A-4)或(A-5)；n＝1-4。
3.根据权利要求2所述荧光探针，其特征是所述：Z选自氢原子，甲基，F；R选自(A-1)、(A-4)或(A-5)；n＝1-4。
4.根据权利要求3所述荧光探针，其特征是所述：Z选自氢原子，甲基，F；R选自(A-1)。
5.权利要求1-4之一的荧光探针在制备肿瘤检测试剂中的用途。
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