CN117586174B - 用于诊断结直肠癌的近红外荧光探针及其制法和应用 - Google Patents
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Abstract
本发明公开了一种用于诊断结直肠癌的近红外荧光探针及其制备方法和应用，所述近红外荧光探针可以特异性识别HSP90，制备方法包括将YQ‑HSI，2‑(7‑氮杂苯并三氮唑)‑N,N,N',N'‑四甲基脲六氟磷酸酯HATU和荧光染料MPA溶于溶剂二甲基亚砜中，然后加入N,N‑二异丙基乙胺在室温震荡反应，纯化，得到的近红外荧光探针可以应用于结直肠癌的诊断。
Description
技术领域
本发明涉及一种近红外荧光探针及其制法和应用，尤其涉及一种用于诊断结直肠癌的近红外荧光探针及其制备方法和应用。
背景技术
热休克蛋白（Heat shock protein,HSP）是从细菌到哺乳动物中广泛存在的一类热应激蛋白。当机体处于应激环境如高温、寒冷、疾病、感染等时，就会刺激此类蛋白快速合成，从而维持细胞的稳定，增强自身的保护。它是人体内重要的管家蛋白，其伴侣功能可在每个细胞区间观察到，并与蛋白质的翻译、迁移、定位、稳定和降解存在内在联系。按照分子量的大小，热休克蛋白主要分为五类，分别为HSP60、HSP70、HSP90、HSP110以及小分子热休克蛋白。
热休克蛋白90（Heat shock protein 90，HSP90）是一种功能强大的分子伴侣蛋白，也是研究最为广泛的热休克蛋白，主要负责各种客户分子的稳定性和完整性，包括各种转录因子、激酶以及类固醇激素蛋白。HSP90在正常细胞中的表达约为1-2%。不幸的是，在机体处于恶劣微环境下，例如缺氧和营养缺乏，热休克因子1(Heat shock transcriptionfactor 1,HSF1)被激活，诱导肿瘤细胞中HSP90的表达，约占总蛋白的4-6%。HSP90过表达时，可能导致客户蛋白的错误折叠和不正确的积累，进而影响相关的信号通路，最终导致生理功能异常，产生癌症、免疫性疾病、神经退行性疾病等。此外，大量的HSP90客户蛋白在肿瘤细胞的生长和增殖中发挥着重要作用，包括各种与癌症直接相关的激酶（Akt、Raf-1、Her-2、CDK-4)和转录因子(p53、Stat3)。因此HSP90也参与肿瘤细胞的增殖、侵袭、转移等过程的调控。这也使得它在肿瘤诊断中有着更为重要的地位。
目前，结直肠癌(Colorectal Cancer,CRC)是世界上最常见的恶性肿瘤，临床上最常用的结直肠癌的诊断方法有白光肠镜、病理组织学、影像学检查等。但是，白光肠镜其检查时间长且需活体取样，无法对整个结直肠进行全面检查；影像学检查是临床上结直肠癌的补充检查方法，但是缺乏分子特异性，分期诊断有限。
发明内容
发明目的：本发明旨在提供一种能够精确诊断结直肠癌的近红外荧光分子探针；本发明的另一目的在于提供一种用于诊断结直肠癌的近红外荧光探针的制备方法；本发明还旨在提供一种用于诊断结直肠癌的近红外荧光探针的应用。
技术方案：本发明所述的用于诊断结直肠癌的近红外荧光探针，结构式如下：
其中，n为大于等于0的整数。
进一步地，所述用于诊断结直肠癌的近红外荧光探针能特异性识别HSP90。
所述用于诊断结直肠癌的近红外荧光探针的制备方法，合成路线如下：
其中，n为大于等于0的整数。
进一步地，当近红外荧光探针的结构式中n=2时，其制备方法包括如下步骤：
将YQ-HSI，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯HATU和荧光染料MPA溶于溶剂中，然后加入N,N-二异丙基乙胺在室温震荡反应，纯化；合成路线如下：
进一步地，YQ-HSI，HATU和荧光染料MPA的质量比为1~2：1~2：2~3，震荡反应时间为1~3h，N,N-二异丙基乙胺的加入量为1~2 μL，纯化方法采用液相分离纯化，溶剂为二甲基亚砜。
所述用于诊断结直肠癌的近红外荧光探针可应用在光学成像中。
有益效果：与现有技术相比，本发明具有如下显著优点：所述近红外荧光探针在结直肠癌肿瘤细胞上的亲和力强，在相应的细胞水平的表达水平高，在皮下瘤模型的近红外活体成像的靶向性好。
附图说明
图1为MPA-YQHSI-2的结构式；
图2为MPA-YQHSI-2的质谱图；
图3为MPA-YQHSI-2的HPLC图；
图4为细胞表达量结果图；
图5为流式细胞术结果图；
图6为空白荷瘤鼠成像图；
图7为HCT116荷瘤鼠成像图；
图8为HT29荷瘤鼠成像图。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1 制备化合物MPA-YQHSI-2
本实施例提供MPA-YQHSI-n(n＝2)荧光探针的制备方法，MPA为近红外荧光染料（参发明专利CN101440282A）。
将2.0 mgYQ-HSI，2.0mg HATU和3.0mg 荧光染料MPA溶于到300μL二甲基亚砜(DMSO)中，然后加入1.0μL N ,N-二异丙基乙在室温震荡反应1h。反应结束后用制备液相进行分离纯化，制备液相条件如下所示：使用了Agilent 1220Infinity II系列HPLC系统配备Agilent ZORBAX SB-C18半制备柱(9.4×250mm，5um)，梯度淋洗60分钟，流速2mL/min，其中流动相A为超纯水(0 .01％TFA)，B为乙腈(0.01％TFA)。淋洗梯度设定为：0~5分钟时95％A和5％B，15分钟时80％A和20％B，45分钟时50％A和50％B，60分钟时5％A和95％B，最后收集得到产物经MPA-YQHSI-2，其结构式如图1所示，质谱图如图2所示，HPLC如图3所示。
实施例2 HSP90探针在结直肠癌细胞系上的表达情况
将HCT116、HT29、SW480细胞平铺于六孔板中，待细胞长满，弃培养基，用1× PBS漂洗细胞2次，去尽残留培养基。加入4× SDS样品缓冲液，刮落细胞，转移到Ep管，煮沸样品5min，离心12000 g，5 min，取上清。取2-3μl的蛋白溶液即为上样量。加入足够量的电泳液后开始上样。电泳条件：压缩胶80 V，30 min；分离胶120 V，1.5 h。电泳至溴酚兰刚跑出即可终止电泳，进行转膜。依据胶的大小剪取膜和滤纸，将转移槽置于冰浴中，放入三明治结构，加转膜缓冲液。转膜电泳条件：220 mA，2 h。转膜结束后，切断电源，取出膜，放入5%的脱脂奶粉中封闭1.5 h。一抗过夜孵育。孵育结束，TBST洗膜三遍每次7 min，再在室温条件下孵育二抗1 h，洗膜三遍每次7 min，曝光显影，参见图4。结果显示HSP90蛋白在HCT116中表达最高，其次是SW480，HT29为低表达。
实施例3 HSP90探针对HCT116细胞的亲和力
将培养好的人结直肠癌细胞HCT116平铺于24孔板中，待细胞长满，弃培养基，用1× PBS漂洗细胞2次，去尽残留培养基。一共三组，每组三个孔，三组分别加入PBS、50 μM探针、50μM MPA。培养箱孵育4 h。将细胞消化下来，用1× PBS洗2次，洗去多余的探针。通过流式细胞术检测其平均荧光强度，荧光强度越强则证明对细胞的亲和力越高，参见图5。体外亲和力结果显示实施例1制备的HSP90近红外荧光探针可以特异性识别细胞中的HSP90。
实施例4 HSP90探针在空白荷瘤鼠体内的光学成像
取已有的HSP90探针原溶液（10 mg/mL）1 μg，溶于100 μL生理盐水中，通过尾静脉注射的方法，分别给三只空白裸鼠（体重约20 g）注射实施例1制备的HSP90探针100 μL，并于给药后2 h、4 h、6 h、12 h以及24 h进行光学信号采集。观察探针在小鼠体内的分布以及肿瘤区域的富集。HSP90探针在三只荷瘤鼠中的成像效果基本一致，从2 h的成像图中可以看出探针在肝脏中有聚集，后续经过代谢肝脏中的荧光强度渐无。显像结果如图6所示，说明探针主要是经肝代谢。
实施例5 HSP90探针在结直肠腺癌HCT116荷瘤鼠体内的光学成像
取已有的HSP90探针原溶液（10 mg/mL）1 μg，溶于100 μL生理盐水中，通过尾静脉注射的方法，分别给三只结直肠癌HCT116皮下瘤裸鼠（体重约20 g）注射实施例1制备的HSP90探针100 μL，并于给药后2 h、4 h、6 h、12 h以及24 h进行光学信号采集。观察探针在小鼠体内的分布以及肿瘤区域的富集。HSP90探针在三只荷瘤鼠中的成像效果基本一致，从2 h的成像图中可以看出探针在肿瘤部位和肝脏中有所聚集，后续肿瘤中探针的聚集逐渐增多，肝脏中逐渐降低。显像结果如图7所示，说明探针对HCT116皮下荷瘤鼠的靶向性强。
实施例6 HSP90探针在结直肠腺癌HT29荷瘤鼠体内的光学成像
取已有的HSP90探针原溶液（10 mg/mL）1 μg，溶于100 μL生理盐水中，通过尾静脉注射的方法，分别给三只结直肠癌HT29皮下瘤裸鼠（体重约20 g）注射实施例1制备的HSP90探针100 μL，并于给药后2 h、4 h、6 h、12 h以及24 h进行光学信号采集。观察探针在小鼠体内的分布以及肿瘤区域的富集。HSP90探针在三只荷瘤鼠中的成像效果基本一致，从2 h的成像图中可以看出探针在肿瘤部位和肝脏中有所聚集，后续肿瘤中探针的聚集逐渐增多，肝脏中逐渐降低。显像结果如图8所示，说明探针对HT29皮下荷瘤鼠的靶向性强。
Claims (6)
1.一种用于诊断结直肠癌的近红外荧光探针，其特征在于，其为特异性识别HSP90的近红外荧光探针，该探针的结构式如下：
。
2.一种权利要求1所述的用于诊断结直肠癌的近红外荧光探针的制备方法，其特征在于，包括如下步骤：
将YQ-HSI，2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯HATU和荧光染料MPA溶于溶剂中，然后加入N,N-二异丙基乙胺在室温震荡反应，纯化；合成路线如下：
。
3.根据权利要求2所述的用于诊断结直肠癌的近红外荧光探针的制备方法，其特征在于，YQ-HSI，HATU和荧光染料MPA的质量比为1~2：1~2：2~3。
4.根据权利要求2所述的用于诊断结直肠癌的近红外荧光探针的制备方法，其特征在于，N,N-二异丙基乙胺的加入量为1~2 μL。
5.根据权利要求2所述的用于诊断结直肠癌的近红外荧光探针的制备方法，其特征在于，震荡反应时间为1~3h。
6.根据权利要求2所述的用于诊断结直肠癌的近红外荧光探针的制备方法，其特征在于，纯化方法采用液相分离纯化。
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