WO2022016770A1 - 一种利用核仁形态变化快速区分癌与正常组织的rna荧光探针 - Google Patents

一种利用核仁形态变化快速区分癌与正常组织的rna荧光探针 Download PDF

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WO2022016770A1
WO2022016770A1 PCT/CN2020/133634 CN2020133634W WO2022016770A1 WO 2022016770 A1 WO2022016770 A1 WO 2022016770A1 CN 2020133634 W CN2020133634 W CN 2020133634W WO 2022016770 A1 WO2022016770 A1 WO 2022016770A1
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rna
cancer
nucleoli
normal tissues
tissues
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于晓强
高鹏
何秀全
孟芳芳
贺峻祎
刘志强
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Shandong University
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Definitions

  • the invention relates to a fluorescent probe capable of simultaneously displaying RNA in living cells and normal tissues or cancer tissues and its application, in particular to an RNA fluorescent probe for rapidly distinguishing cancer and normal tissues by utilizing morphological changes of nucleoli and its preparation method Application of rapid pathological diagnostic reagents in tumor surgery.
  • Surgical resection of tumor lesions is currently an effective treatment method.
  • the surgical options for benign tumors and cancers are completely different, and must be strictly distinguished: local excision for benign tumors; extensive radical resection plus lymph node dissection for malignant tumors. If the cancer is misdiagnosed as benign, it may require a second operation on the patient; otherwise, it is a major medical malpractice. Therefore, the intraoperative pathological diagnosis to help the surgeon decide the surgical plan is indispensable, and it plays an important role in judging the nature of the tumor and determining the surgical mode.
  • Intraoperative diagnosis is the real-time, on-site sampling of patients after general anesthesia is performed on the operating table. The advantages are that the pathological samples are large and accurate, and the sampling process will not cause medical risks such as internal bleeding.
  • the report to determine whether the tumor is benign or malignant must be given within 30 minutes, so the intraoperative diagnosis is also called rapid diagnosis. It is precisely because of the strict time requirement that a diagnostic report must be issued within 30 minutes, for more than 100 years, there has been no other staining method except HE staining that can provide valuable diagnostic information in such a short time, causing disease. It is difficult for a physician to make a rapid pathological diagnosis based only on the HE staining results.
  • HE staining The principle of HE staining is to use two dyes, hematoxylin and eosin, to undergo acid-base reaction with the substances in the tissue to complete the staining. Due to the limitation of this staining principle, HE staining can only display the structure related to pH in the tissue. Compared with normal cells, the changes at the level of many biomolecules in tumor cells did not lead to significant changes in the acidity and alkalinity of pathological tissues, so HE staining could not show these changes at the molecular level.
  • RNA Ribonucleic acid plays an important role in biological evolution, especially in the translation of genetic information, and its main function is to realize the expression of genetic information on proteins.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • nucleolus the place where rRNA occurs, has been discovered for more than 100 years, its biological function has not been fully elucidated. It is generally accepted that the nucleolus is simply the site of transcription, processing, and assembly of rRNA in the nucleus. With the development of technology, the changes of nucleoli in cancerous tissue cells have attracted more and more attention from the medical community. Recent studies have found that not only do the size and shape of the nuclei change in cancer cells compared to normal cells, but also characteristic changes in the size, shape and number of nucleoli. Nucleolar abnormalities are a common feature of cancer cells, the study found.
  • giant nucleoli have become an important basis for the diagnosis of large cell lung cancer and Hodgkin's disease; large-sized nucleoli are also found in prostate intraepithelial neoplasia cells; the opposite is also true: ultra-small-sized nucleoli are cervical intraepithelial neoplasia lesions and small A diagnostic basis for degenerative development of lung cancer.
  • the following table summarizes the characteristics of changes in the nucleolus associated with cancer.
  • RNA fluorescent probes that can display nucleolar morphology in cells, especially in pathological tissues, and can use nucleolar morphological changes to rapidly distinguish cancer from normal tissues has great biological significance. academic and medical value.
  • the biological structure in the pathological tissue can be displayed under the light microscope, in which the nucleus is blue-black, the cytoplasm is pink, and the cartilage is blue, etc.
  • Pathologists make according to the staining map. diagnosis. The information is based on: cell atypia, including cell shape, size, and regularity; the size of the nucleus, especially the ratio of the nucleus to the cytoplasm; the size, shape, number of nucleoli, and the pH, etc.
  • the overall outline of the nucleolus cannot be clearly given, resulting in the interpretation of images related to the nucleolus by the doctor's experience.
  • RNA fluorescent probes that can image RNA and nucleoli in isolated living cells in vitro, their application in tissues is not ideal.
  • SYTO RNA-Select not only has an unpublished molecular structure, but its utility in tissues has not been widely demonstrated. If we can break through the bottleneck, we can design and develop RNA fluorescent probes that can rapidly, in situ, and highly selectively stain RNA in human pathological tissue sections, especially frozen sections, and further accurately image the nucleolus, and then prepare for oncology surgery.
  • Medium and rapid pathological diagnostic reagents are very likely to solve the common technical problem that the rapid diagnosis of benign and malignant tumors can only rely on the centuries-old HE staining method, and break the long-standing dilemma of orphan diagnosis.
  • the problem to be solved by the present invention is to provide an RNA fluorescent probe that utilizes nucleolar morphological changes to rapidly distinguish cancer from normal tissues and its application in the preparation of rapid pathological diagnostic reagents in tumor surgery.
  • the RNA fluorescent probe for rapidly distinguishing cancer and normal tissues by utilizing nucleolar morphological changes is characterized in that: the chemical name of the RNA fluorescent probe is: (E)-1-(3-aminopropyl) -4-(2-(9-ethyl-carbazol-3-enyl)vinyl)pyridine dibromide, referred to as CAPY-AP, whose chemical structural formula is shown in formula (I); the RNA fluorescent probe It can simultaneously display the distribution of RNA and nucleoli in living cells and normal tissues or cancer tissues.
  • the judging index for quickly distinguishing cancer and normal tissues by using nucleolar morphological changes is that there is only a single inconspicuous nucleolus in normal tissues, while cancer tissues There are multiple nucleoli and large nucleoli in the
  • RNA fluorescent probe for rapidly distinguishing cancer and normal tissue by utilizing nucleolar morphological changes is as follows:
  • RNA fluorescent probe for quickly distinguishing cancer and normal tissues by utilizing nucleolar morphological changes of the present invention is used to label or display the distribution of RNA and nucleoli in living cells.
  • the living cells are preferably SiHa cancer cells.
  • RNA fluorescent probe for quickly distinguishing cancer and normal tissue by utilizing nucleolar morphological changes of the present invention is used to label or display the distribution of RNA and nucleolus in normal tissue or cancer tissue.
  • the normal tissue is preferably a frozen pathological section of normal breast tissue
  • the cancer tissue is preferably a frozen pathological section of breast cancer tissue.
  • the mammary gland refers to human mammary gland normal tissue or pathological tissue.
  • RNA fluorescent probe for rapidly distinguishing cancer from normal tissue by utilizing nucleolar morphological changes of the present invention is used in the preparation of a rapid pathological diagnosis reagent in tumor surgery.
  • the fluorescent probe CAPY-AP of the present invention was combined with the commercial nuclear fluorescent probe Hoechst 33342, and the staining process provided by the present invention was used to carry out experiments in frozen sections of breast tumor pathological tissues, and a highly clear fluorescent image was obtained.
  • the fluorescent probe CAPY-AP of the present invention can stain cytoplasmic RNA, and the shape of the nucleolus is clearly visible. It shows that the fluorescent probe of the present invention has good permeability and can penetrate the inside of the tissue and enter the nucleus to be targeted to the nucleolus, which overcomes the functions that other existing RNA probes cannot achieve. In addition, by comparing the frozen section HE images and fluorescent images of normal breast and breast cancer tissues, it is found that the fluorescent staining of the present invention shows obvious advantages in terms of resolution and richness of details.
  • the RNA fluorescent probe for rapidly distinguishing cancer and normal tissues by utilizing the morphological changes of nucleoli can simultaneously display the distribution of RNA and nucleoli in living cells and tissues, laying a foundation for its application in the preparation of rapid pathological diagnostic reagents in tumor surgery foundation.
  • the fluorescent probe CAPY-AP of the present invention can image RNA and nucleoli with high fidelity in living cells and tissues.
  • the probe CAPY-AP can clearly image nucleoli, and give the size, number and morphology of nucleoli in the tissue.
  • the carbazole pyridine salt compound CAPY-AP of the present invention has ultra-high RNA affinity and ultra-high permeability, and can easily image RNA and nucleoli in tissue sections .
  • the probe of the present invention has the characteristics of good membrane permeability, strong color rendering and strong photostability, indicating that it has a wide range of applications as RNA and nucleolar fluorescent probes, especially for imaging nucleoli in tissues , is expected to be developed as a simple and intuitive biological detection reagent for RNA and nucleolus-related physiological and pathological studies.
  • Figure 1 Confocal fluorescence images of probe CAPY-AP staining active SiHa cells.
  • Fluorescence images showed obvious green light distribution in the cytoplasm and nucleolus, which clearly indicated that the probe CAPY-AP of the present invention could specifically image cytoplasmic RNA and nucleolus in living cells.
  • Figure 2 Confocal fluorescence images of SiHa cells stained with CAPY-AP after RNase treatment of fixed SiHa cells.
  • Figure 3 Comparison of HE staining and fluorescent staining of frozen sections of cancer and adjacent normal tissue from breast patients (pathology number: 57977.18).
  • CAPY-AP and Hoechst 33342 stained the frozen sections of breast cancer and adjacent normal tissues, under a laser scanning confocal microscope, the tissues were irradiated with 488 nm and 405 nm laser light respectively, and the superimposed images of the two channels were collected. Obtain fluorescent pictures. Frozen sections of the same tissue were stained with HE to obtain HE-stained pictures.
  • Figure 4 Comparison of HE staining and fluorescence staining of frozen sections of cancer and adjacent normal tissue from breast patients (pathology number: 60551.18).
  • Figure 5 Comparison of HE staining and fluorescent staining of frozen sections of cancer and adjacent normal tissue from breast patients (pathology number: 61864.18).
  • the involved tissue frozen sections were provided by the cooperative unit, Department of Pathology, Qilu Hospital, Shandong University, and the materials, cells, reagents, etc. used were obtained from commercial sources unless otherwise specified.
  • Example 2 SiHa cell culture.
  • the SiHa cells were cultured in adherent culture medium containing 10% fetal calf serum, in a 37 °C, 5% CO 2 humidified incubator in culture medium was changed every 2-3 days passaged once. After the cells have grown to the logarithmic phase, splicing culture: 1 Soak the coverslip in absolute ethanol for 30 minutes, dry in an alcohol lamp and put it in a disposable 35mm petri dish for use; 2 Put the overgrown cells in the 100mL cell flask The cells were washed three times with PBS, digested with 1 mL of 0.25% trypsin for 3-5 minutes, carefully poured out the trypsin, added with fresh culture medium, pipetted evenly and counted the cells.
  • the concentration was 1 ⁇ 10 5 per ml, and then inoculated into the above-mentioned petri dish containing coverslips, and cultured in a 5% CO 2 incubator to allow the cells to grow on the sheets. After the SiHa cells had grown and covered the coverslip, they were used for the experiment.
  • Example 3 Observation of staining of active SiHa cells with probe CAPY-AP.
  • a 5mM solution of probe CAPY-AP in DMSO was prepared as a stock solution.
  • the SiHa cell slides covered the coverslip, remove the medium in the petri dish, rinse the coverslip containing the SiHa cells 3 times with clean PBS, stain the cells with 5 ⁇ M CAPY-AP, and put them in a CO 2 incubator. Incubate for 30min.
  • the stained slides were taken out, the excess probes were washed away, and the cells growing side down were covered on a glass slide, and the staining position, fluorescence distribution and brightness of the cells were observed under a laser scanning confocal fluorescence microscope (the cells were irradiated with a 488 nm laser). changes, etc.
  • Example 4 Observation of staining of SiHa cells treated with ribonuclease (RNase) by probe CAPY-AP (RNase digestion experiment).
  • Preparation of fixed cells First, soak the coverslip (climbing slide) with SiHa cells in Example 2 in 4% paraformaldehyde solution for 30 minutes, and then permeabilize it with 0.5% Triton X-100 at room temperature for 2 minutes to obtain the cells to be stained. Fixed cells.
  • Example 5 Fluorescent staining was performed on the frozen sections of cancer and adjacent normal tissues of breast patients (pathology number: 57977.18), and the fluorescent pictures were compared with the corresponding HE staining images, and the respective diagnostic criteria were given.
  • Frozen sections of cancer and adjacent normal tissues of intraoperative breast patients were obtained from the Department of Pathology, Qilu Hospital, Shandong University. It was immersed in a PBS buffer solution (containing 10% EtOH) containing 10 ⁇ M probe CAPY-AP and 10 ⁇ M Hoechst 33342 for 10 min, and then observed under a laser scanning confocal microscope (irradiated tissue with 488 nm and 405 nm laser light, respectively) Obtain the fluorescent staining pictures and compare them with the corresponding HE staining pictures provided by the pathology department.
  • Figure 3 Fluorescence images show distinct green light distribution in the cytoplasmic and nucleolar regions, but no fluorescence in the extracellular matrix. It is clearly indicated that the probe of the present invention can specifically image cytoplasmic RNA and nucleolus in human breast cancer tissue. In addition, the number, size and morphology of nucleoli can be clearly displayed from the enlarged fluorescence images. By comparing the frozen section HE images and fluorescence images of normal breast and breast cancer tissues, it was found that the fluorescence staining results showed obvious advantages. The structure of the nucleolus in the tissue can be clearly presented, while the nucleolus is not clear in the HE images of the cryosection. In addition, we also found that there is only a single inconspicuous nucleolus in normal breast tissue, but there are multiple nucleoli and large nucleoli in breast cancer tissue.
  • Example 6 Fluorescent staining was performed on the frozen sections of cancer and adjacent normal tissues of breast patients (pathology number: 60551.18), and the fluorescent pictures were compared with the corresponding HE staining images, and the respective diagnostic criteria were given.
  • Frozen sections of cancer and adjacent normal tissues of intraoperative breast patients were obtained from the Department of Pathology, Qilu Hospital, Shandong University. It was immersed in a PBS buffer solution (containing 10% EtOH) containing 10 ⁇ M probe CAPY-AP and 10 ⁇ M Hoechst 33342 for 10 min, and then observed under a laser scanning confocal microscope (irradiated tissue with 488 nm and 405 nm laser light, respectively) Obtain the fluorescent staining pictures and compare them with the corresponding HE staining pictures provided by the pathology department.
  • Example 7 Fluorescent staining was performed on the frozen sections of cancer and adjacent normal tissues of breast patients (pathology number: 61864.18), and the fluorescent pictures were compared with the corresponding HE staining images, and the respective diagnostic criteria were given.
  • Frozen sections of cancer and adjacent normal tissues of intraoperative breast patients were obtained from the Department of Pathology, Qilu Hospital, Shandong University. It was immersed in a PBS buffer solution (containing 10% EtOH) containing 10 ⁇ M probe CAPY-AP and 10 ⁇ M Hoechst 33342 for 10 min, and then observed under a laser scanning confocal microscope (irradiated tissue with 488 nm and 405 nm laser light, respectively) Obtain the fluorescent staining pictures and compare them with the corresponding HE staining pictures provided by the pathology department.

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Abstract

本发明公开了一种利用核仁形态变化快速区分癌与正常组织的RNA荧光探针，该探针为(E)-1-(3-氨基丙基)-4-(2-(9-乙基-咔唑-3-烯基)乙烯基)吡啶二溴盐，简称CAPY-AP；所述RNA荧光探针能同时显示活细胞和正常组织或癌组织中的RNA与核仁分布，所述利用核仁形态变化快速区分癌与正常组织的判断指标是正常组织中只有单个不明显的核仁，而癌组织中存在多核仁以及大核仁的现象；与现有其他RNA探针相比，本发明所述的探针具有较高的RNA亲和力以及超高的渗透性，能够轻松地对组织切片内的RNA和核仁成像。另外，本发明所述探针具有膜通透性好、显色强、光稳定性较强的特点，有望在制备肿瘤术中快速病理诊断试剂中得到深入应用。

Description

一种利用核仁形态变化快速区分癌与正常组织的RNA荧光探针 技术领域
本发明涉及一种能同时显示活细胞和正常组织或癌组织中RNA的荧光探针及其应用，尤其涉及一种利用核仁形态变化快速区分癌与正常组织的RNA荧光探针及其在制备肿瘤术中快速病理诊断试剂中的应用。
背景技术
重大疾病的诊断试剂、方法和技术，历来是国际高科技角逐的主要领域。我国是人口大国，居民中肿瘤发病率常年居高不下，平均每天有万人以上被确诊罹患肿瘤疾病，其中很多人被确诊为恶性肿瘤(癌症)。据2019年1月份最新统计数据显示，2015年我国平均每天有超过1万人被确诊为癌症，即每分钟有7.5人被确诊为癌症。近十多年来，恶性肿瘤发病率每年保持3.9％增幅，而死亡率每年保持2.5％增幅。
手术切除肿瘤病变组织是目前普遍采用的有效治疗手段。但良性肿瘤和癌症的手术方案完全不同，必须严格区分：良性肿瘤行局部切除；恶性肿瘤行大范围根治性切除加淋巴结清扫。如果癌症误判为良性，则可能需要对患者二次手术；反之则是重大医疗事故。因此帮助外科医生决定手术方案的术中病理诊断不可或缺，它在判断肿瘤性质、确定手术术式(mode)中发挥着重要的作用。术中诊断是患者在手术台上全身麻醉开刀后，进行实时、现场取材，其优势是病理样本取材大、部位准确、取材过程不会造成内出血等医疗风险。但由于患者正在手术台等待病理诊断结果，确定肿瘤良恶性的报告必须在30分钟之内给出，因此术中诊断也被称为快速诊断。正是由于必须在30分钟之内出具诊断报告这一严格的时间要求，一百多年来，除HE染色外，尚无其他染色方法可在如此短时间内提供有价值的诊断信息，造成病理科医生仅依据HE染色结果就得做出快速病理诊断的困难局面。
众所周知，疾病诊断是一个高难度的工作，利用孤证做诊断是医学上的大忌，但由于缺乏可用的技术，在肿瘤术中快速病理诊断中却只能如此，这不仅增加了病理科医生的诊断难度，而且给病理医生带来巨大的心理压力，是病理医生工作中的痛点。因此亟需创立一个与HE并行的术中快速病理诊断新技术，解决术中快速判断肿瘤良恶性只能依靠已使用百年的HE染色方法这一个共性技术难题，打破目前孤证诊断困局。但百年来再没有一项新技术能够突破30分钟做出病理诊断的瓶颈。
HE染色的原理是利用苏木素和伊红两种染料与组织内的物质发生酸碱反应而完成染色，由于这一染色原理的限制，HE染色只能显示组织内与酸碱度有关的结构。相比于正常细胞，肿瘤细胞发生的诸多生物分子水平的变化并未导致病理组织酸碱性的显著变化，因此HE染 色不能显示这些分子层次上的变化。
核糖核酸(RNA)在生物进化中扮演着重要的角色，尤其在遗传信息的翻译中起着决定性的作用，主要功能是实现遗传信息在蛋白质上的表达。在细胞中，根据结构与功能的不同，RNA主要分三类，即信使RNA(mRNA)，转运RNA(tRNA)，核糖体RNA(rRNA)，近年的研究显示，RNA在肿瘤的生物学行为中发挥重要作用。
作为rRNA发生地的核仁虽然已经发现了有100多年，但是它的生物功能并没有得到完全阐明。人们普遍认为，核仁只是细胞核中rRNA转录、加工、装配的场所。随着技术的发展，癌变组织细胞中核仁的变化越来越引起医学界的高度重视。最近的研究发现，和正常细胞相比，癌症细胞中不仅是细胞核的尺寸和形状发生变化，其核仁的大小、形状和数量也发生了特征性的变化。研究发现，核仁异常是癌细胞的普遍特征。例如，巨大核仁已成为大细胞肺癌与Hodgkin病的一个重要诊断依据；前列腺上皮内瘤细胞中也有大尺寸核仁现象；相反的也有：超小尺寸核仁则是宫颈上皮内瘤病变和小细胞退行发育肺癌的一个诊断依据。下表中汇总了与癌症关联的细胞核仁的变化特征。
表1与癌症相关联的细胞核仁变化
Figure PCTCN2020133634-appb-000001
因此，基于RNA与核仁在肿瘤学领域的重要性，开发能够显示细胞中尤其是病理组织中核仁形态、并能够利用核仁形态变化快速区分癌与正常组织的RNA荧光探针具有巨大的生物学与医学价值。
利用HE(苏木素/伊红)染色，可以将病理组织中的生物构造在光学显微镜下显示出来，其中细胞核着蓝黑色，细胞浆着粉红色，软骨着蓝色等，病理专家依据染色图谱做出诊断。其依据的信息有：细胞的异型性，包括细胞的形态、大小、以及是否规则；细胞核的大小、特别是细胞核与细胞浆的核浆比；核仁的大小、形态、数量，以及核仁的酸碱度等。但是由于染色的选择性差，无法清晰给出核仁的整体轮廓，导致与核仁有关的图像判读全凭医生的经验。遗憾的是，尽管目前有很多的RNA荧光探针能够在体外孤立培养的活细胞中成像RNA以及核仁，但是他们在组织中的应用并不理想。SYTO RNA-Select作为唯一可用于活细胞RNA成像的商用染料，不仅其分子结构未公布，其在组织中的实用性也尚未得到广泛证实。如能突破瓶颈，设计、开发出能够在人体病理组织切片，特别是冰冻切片中快速、原位、高 选择性地染色RNA、并进一步精准地成像核仁的RNA荧光探针，进而制备肿瘤术中快速病理诊断试剂，则极有可能解决术中快速判断肿瘤良恶性只能依靠已使用百年的HE染色方法这一个共性技术难题，打破长期存在的孤证诊断困局。
发明内容
针对现有技术的不足，本发明要解决的问题是提供一种利用核仁形态变化快速区分癌与正常组织的RNA荧光探针及其在制备肿瘤术中快速病理诊断试剂中的应用。
本发明所述的利用核仁形态变化快速区分癌与正常组织的RNA荧光探针，其特征在于：所述RNA荧光探针的化学名称为：(E)-1-(3-氨基丙基)-4-(2-(9-乙基-咔唑-3-烯基)乙烯基)吡啶二溴盐，简称CAPY-AP，其化学结构式如式(I)所示；所述RNA荧光探针能同时显示活细胞和正常组织或癌组织中的RNA与核仁分布，所述利用核仁形态变化快速区分癌与正常组织的判断指标是正常组织中只有单个不明显的核仁，而癌组织中存在多核仁以及大核仁的现象；
Figure PCTCN2020133634-appb-000002
上述利用核仁形态变化快速区分癌与正常组织的RNA荧光探针的制备方法是：
首先将4-甲基吡啶与3-溴丙胺氢溴酸盐混合反应得到化合物1。然后通过vilsmeier反应合成化合物2。将化合物1与2混合，哌啶做催化剂，回流反应制备终产物。最后通过柱色谱法获得纯化的红色固体产物CAPY-AP。反应式如下：
Figure PCTCN2020133634-appb-000003
本发明所述利用核仁形态变化快速区分癌与正常组织的RNA荧光探针在标记或显示活细胞内RNA与核仁分布的应用。
其中：所述活细胞优选为SiHa癌细胞。
本发明所述利用核仁形态变化快速区分癌与正常组织的RNA荧光探针在标记或显示正 常组织或癌组织中RNA与核仁分布的应用。
其中：所述正常组织优选是正常乳腺组织的冰冻病理切片，所述癌组织优选是乳腺癌组织的冰冻病理切片。进一步的，所述乳腺是指人体乳腺正常组织或病理组织。
本发明所述利用核仁形态变化快速区分癌与正常组织的RNA荧光探针在制备肿瘤术中快速病理诊断试剂中的应用。
实验结果证实，本发明所述的荧光探针CAPY-AP在标记SiHa细胞后，在激光扫描共聚焦显微镜下，用488nm的激光照射细胞得到显微图片显示，在细胞质和核仁区有明显的绿光分布，明确提示所述探针能够在活细胞中专一性的成像胞质RNA和核仁，并且在与经RNase消化实验后的细胞进行对比试验后也得到进一步的证实。另外，将本发明所述的荧光探针CAPY-AP与商用细胞核荧光探针Hoechst 33342组合，并用本发明提供的染色工艺在乳腺肿瘤病理组织的冰冻切片中进行实验，获得了高度清晰的荧光图像，并且将其与HE染色图像进行了对照研究。从实验得到的荧光图片中可以看出，本发明的荧光探针CAPY-AP能够染色胞质RNA，并且核仁的形态清晰可见。说明本发明所述的荧光探针具有好的渗透性能够穿过组织内部进入细胞核进而靶向到核仁中，克服了现有其他RNA探针无法实现的功能。另外，通过对比正常乳腺和乳腺癌组织的冰冻切片HE图像和荧光图像发现，在分辨率、细节的丰富程度上本发明的荧光染色显示出明显的优势。不仅从新角度提供HE染色所表达的全部信息，而且能够提供更多的细节信息，可以清楚地呈现出组织中核仁的结构，而这些信息在冰冻切片的HE图像中几乎是缺失的。通过对比申请人还有重要发现：正常乳腺组织中只有单个不明显的核仁，而乳腺癌组织中存在多核仁以及大核仁的现象。这一发现为利用核仁形态变化快速区分癌与正常组织提供了判断依据。总之，通过总结冰冻切片HE染色与荧光染色在乳腺组织中的诊断判据，可以看出本发明的荧光探针CAPY-AP及荧光染色结果具有显著的优势，仅利用核仁形态变化这一诊断参数就能轻易地区分乳腺癌组织与正常组织，实现了简单、量化、精准诊断。
本发明提供的利用核仁形态变化快速区分癌与正常组织的RNA荧光探针能同时显示活细胞以及组织中RNA与核仁的分布，为其在制备肿瘤术中快速病理诊断试剂中的应用奠定了基础。大量的实验证明，本发明所述的荧光探针CAPY-AP能够在活细胞和组织中高保真的成像RNA以及核仁。特别是在人体乳腺病理组织中，探针CAPY-AP能够清晰成像核仁，并且给出组织中核仁的大小、数量以及形态。与现有其他RNA探针相比，本发明所述的咔唑吡啶盐类化合物CAPY-AP具有超高的RNA亲和力以及超高的渗透性，能够轻松地对组织切片内的RNA和核仁成像。另外，本发明所述探针具有膜通透性好、显色强、光稳定性 较强的特点，预示其作为RNA和核仁荧光探针具有广泛的应用，尤其是成像组织中的核仁，有望开发为RNA和核仁相关的生理和病理学研究所用的简捷、直观的生物检测试剂。
附图说明
图1：探针CAPY-AP对活性SiHa细胞染色的共聚焦荧光照片。
获得方法：用CAPY-AP探针孵育SiHa细胞30min后，在激光扫描共聚焦显微镜下，用488nm的激光照射细胞得到显微图片。其中a图是探针CAPY-AP在488nm激发下，采集500-600nm绿色通道的照片，b图是明场激光扫描的微分干涉显微照片，c图为a、b两图的合并图(共定位图)。
荧光图像显示在细胞质和核仁区有明显的绿光分布，明确提示本发明的探针CAPY-AP能够在活细胞中专一性的成像胞质RNA和核仁。
图2：对固定的SiHa细胞经核糖核酸酶(RNase)处理后，用CAPY-AP对SiHa细胞进行染色的共聚焦荧光照片。
获得方法：用CAPY-AP对RNase处理后的固定SiHa细胞染色。在激光扫描共聚焦显微镜下，用488nm的激光照射细胞得到显微图片。其中a图是探针CAPY-AP在488nm激发下，采集500-600nm绿色通道的照片，b图是明场激光扫描的微分干涉显微照片，c图为a、b两图的合并图(共定位图)。
结果发现经RNase处理后的细胞在显微镜下的荧光较弱，几乎看不到核仁和细胞质中的荧光。
图3：乳腺病患(病理号：57977.18)癌症与癌旁正常组织冰冻切片的HE染色与荧光染色对比图。
获得方法：探针CAPY-AP与Hoechst 33342对乳腺癌与癌旁正常组织的冰冻切片染色后，在激光扫描共聚焦显微镜下，分别用488nm和405nm的激光照射组织，收取两个通道的叠加图得到荧光图片。同一组织冰冻切片用HE染色获得HE染色图片。
图4：乳腺病患(病理号：60551.18)癌症与癌旁正常组织冰冻切片的HE染色与荧光染色对比图。
获得方法：探针CAPY-AP与Hoechst 33342对乳腺癌与癌旁正常组织的冰冻切片染色后，在激光扫描共聚焦显微镜下，分别用488nm和405nm的激光照射组织，收取两个通道的叠加图得到荧光图片。同一组织冰冻切片用HE染色获得HE染色图片。
图5：乳腺病患(病理号：61864.18)癌症与癌旁正常组织冰冻切片的HE染色与荧光染色对比图。
获得方法：探针CAPY-AP与Hoechst 33342对乳腺癌与癌旁正常组织的冰冻切片染色后，在激光扫描共聚焦显微镜下，分别用488nm和405nm的激光照射组织，收取两个通道的叠加图得到荧光图片。同一组织冰冻切片用HE染色获得HE染色图片。
图3至图5的结果均发现荧光图像显示探针CAPY-AP能够在人体乳腺病理组织的冰冻切片中专一性的成像胞质RNA和核仁。另外，通过对荧光图中方框区域放大可以看到，探针可以清晰地成像核仁的形态、大小以及数量。同时发现乳腺癌组织中存在多核仁以及大核仁的现象，而正常组织中的核仁较小，不如癌组织中的明显。而在癌症与正常组织的HE染色图像中核仁并不明显。
具体实施方式
下面结合具体附图和实施例对本发明内容进行详细说明。如下所述例子仅是本发明的较佳实施方式而已，应该说明的是，下述说明仅仅是为了解释本发明，并非对本发明作任何形式上的限制，凡是依据本发明的技术实质对实施方式所做的任何简单修改，等同变化与修饰，均属于本发明技术方案的范围内。
下述实施例中，涉及的组织冰冻切片由合作单位山东大学齐鲁医院病理科提供，所使用的材料、细胞、试剂等，如无特殊说明，均从商业途径得到。
实施例1：探针CAPY-AP的合成。
Figure PCTCN2020133634-appb-000004
具体合成如下：
化合物1的合成：将4-甲基吡啶(0.93g,10mmol)、3-溴丙胺氢溴酸盐(2.41g,11mmol)溶解于20mL乙醇中，85℃加热回流反应12小时。反应结束后，出现白色固体。过滤，石油醚洗涤，得纯净的白色化合物1。产率70％。 1H NMR(300MHz,DMSO-d 6),δ(ppm):8.938(d,J＝6Hz,2H),8.047(d,J＝6.3Hz,2H),7.797(s,3H),4.628-4.582(t,J＝6.9Hz,2H),2.860-2.794(m,2H),2.622(s,3H),2.220-2.124(m,2H).
化合物2的合成：冰浴条件下，将POCl 3(9.2mL,100mmol)缓慢加入到干燥的DMF(7.7mL,100mmol)中，搅拌30分钟后出现粘稠状。然后将溶解在CH 3Cl的N-乙基咔唑(2.94g,10 mmol)加入体系中，继续搅拌1小时后将混合物于80℃加热，反应12小时。冷却至室温后将混合物倒入冰水中，用NaOH调节体系pH至中性。然后用CH 2Cl 2萃取，将有机相水洗两次，并用无水硫酸镁干燥3小时。最后用乙酸乙酯/石油醚(1:8，V/V)作为洗脱液通过柱色谱分离提纯，获得淡黄色纯净物。产率为50％。 1H NMR(300MHz,DMSO-d6),δ(ppm):10.07(s,1H),8.79(s,1H),8.31(d,J＝7.8Hz,1H),8.02-7.989(m,1H),7.80(d,J＝6.6Hz,1H),7.72(d,J＝8.1Hz,1H),7.55(t,J＝7.65Hz,1H),7.31(d,J＝7.5Hz,1H),4.53(t,J＝7Hz,2H),1.34(t,J＝7.05Hz,3H).
化合物CAPY-AP的合成：将化合物1(0.52g,1.67mmol)和化合物2(0.45g,2mmol)溶于15mL乙醇中，得黄色透明溶液，加3-4滴哌啶，溶液迅速变红色。加热回流反应过夜，有红色固体析出。冷却，过滤，乙醇洗涤。最后用二氯甲烷/甲醇(20:1)作为洗脱液通过柱色谱法获得纯化的红色固体产物即为CAPY-AP，产率40％。 1H NMR(300MHz,DMSO-d 6):δ8.94(d,J＝6.9Hz,2H),8.61(s,1H),8.30-8.19(m,4H),7.93-7.90(m,4H),7.86(d,J＝11.1Hz,1H),7.77(d,J＝8.7Hz,1H),7.75-7.29(m,2H),7.29(t,J＝7.5Hz,1H),4.59(t,J＝6.9Hz,2H),4.52(m,2H),2.88(m,2H),2.22(m,2H)1.34(t,J＝7.1Hz,3H). 13C NMR(300MHz,DMSO-d 6),δ(ppm):154.26,144.43,143.36,141.5,140.64,129.21,126.94,126.86,126.71,123.71,123.24,122.62,121.75,120.97,120.43,120.43,120.16,114.36,110.34,110.19,56.87,37.74,36.14,28.96,14.21.HRMS:m/z(C 24H 27Br 2N 3),found:356.2184[M-2Br-H] 2+。
实施例2：SiHa细胞培养。
将SiHa细胞贴壁培养于内含10％胎牛血清培养液中，在37℃，5％CO 2的饱和湿度孵箱中培养，每2-3天换液传代1次。待细胞生长到对数期，接片培养：①将盖玻片于无水乙醇中浸泡30min，酒精灯烘干后放入一次性35mm培养皿中，备用；②将100mL细胞瓶中长满的细胞用PBS洗三遍，用1mL0.25％胰酶消化3-5分钟，小心地倒出胰酶，加入新鲜培养液吹打均匀并细胞计数，以培养液的添加量控制细胞密度，使细胞终浓度为每毫升1×10 5，然后接种至内上述含盖玻片的培养皿中，放入5％CO 2培养箱中培养，使细胞爬片生长。待SiHa细胞爬片生长并长满盖玻片后，用于实验。
实施例3：探针CAPY-AP对活性SiHa细胞的染色观察。
首先配制5mM探针CAPY-AP的DMSO溶液作为母液。待SiHa细胞爬片长满盖玻片后，移除其培养皿中的培养基，用干净的PBS冲洗含有SiHa细胞的盖玻片3次，用5μM CAPY-AP 染色细胞，在CO 2培养箱中孵育30min。染色后的爬片取出，洗去多余的探针，细胞生长面朝下盖在载玻片上，在激光扫描共聚焦荧光显微镜下(用488nm的激光照射细胞)观察细胞着色位置，荧光分布及亮度变化等。
结果见图1。其中a图是探针CAPY-AP在488nm激发下，采集500-600nm绿色通道的照片，b图是明场激光扫描的微分干涉显微照片，c图为a、b两图的合并图(共定位图)。荧光图像显示在细胞质和核仁区有明显的绿光分布，明确提示本发明的探针CAPY-AP能够在活细胞中专一性的成像胞质RNA和核仁。
实施例4：探针CAPY-AP对经核糖核酸酶(RNase)处理后的SiHa细胞(RNase消化实验)的染色观察。
固定细胞制备：首先将实施例2制备的长满SiHa细胞的盖玻片(爬片)浸泡于4％多聚甲醛溶液30min，然后用0.5％Triton X-100室温通透2min，得待染色的固定细胞。
取上述一组固定细胞，加入25mg/mL RNase在培养箱中消化2h，然后用PBS清洗3遍，并同时用5μM CAPY-AP染色细胞，在CO 2培养箱中孵育30min。将染色后的细胞爬片取出，洗去未结合的多余染液，细胞生长面朝下盖在载玻片上，在激光扫描共聚焦显微镜下观察(用488nm的激光照射细胞)，记录细胞中着色部位，荧光分布及亮度变化等。
结果见图2，其中a图是探针CAPY-AP在488nm激发下，采集500-600nm绿色通道的照片，b图是明场激光扫描的微分干涉显微照片，c图为a、b两图的合并图(共定位图)。发现经RNase处理后的细胞在显微镜下的荧光较弱，几乎看不到核仁和细胞质中的荧光。由于RNase能水解掉细胞中的RNA，因此，从此角度可以证实并验证本发明的探针能够选择性成像细胞中的RNA。
实施例5：对乳腺病患(病理号：57977.18)的癌症与癌旁正常组织的冰冻切片进行荧光染色，并将荧光图片与相应的HE染色图像进行对比，同时给出各自的诊断判据。
从山东大学齐鲁医院病理科获取术中乳腺病患(病理号：57977.18)的癌症与癌旁正常组织的冰冻切片。将其浸入到含有10μM探针CAPY-AP与10μM Hoechst 33342的PBS缓冲溶液(含10％EtOH)中染色10min，然后在激光扫描共聚焦显微镜下进行观察(分别用488nm和405nm的激光照射组织)获取荧光染色图片，并与病理科提供的相应的HE染色图片进行对比。
结果见图3和表2。
图3：荧光图像显示在细胞质和核仁区有明显的绿光分布，而在细胞外基质中没有荧光。 明确提示本发明的探针能够在人体乳腺癌组织中专一性的成像胞质RNA和核仁。另外，从放大的荧光图片中可以发现核仁的数量、大小以及形态都能够清晰显示出来。通过对比正常乳腺和乳腺癌组织的冰冻切片HE图像和荧光图像发现，荧光染色结果显示出明显的优势。可以清楚地呈现出组织中核仁的结构，而在冰冻切片的HE图像中核仁并不清楚。另外我们还发现：正常乳腺组织中只有单个不明显的核仁，而乳腺癌组织中存在多核仁以及大核仁的现象。
表2.冰冻切片HE染色与荧光染色乳腺组织的诊断判据。
Figure PCTCN2020133634-appb-000005
由表2中HE与荧光各自的诊断判据中发现，在荧光图像中仅利用核仁这一诊断参数就能轻易地区分乳腺癌组织与正常组织，相比HE诊断要容易的多。
实施例6：对乳腺病患(病理号：60551.18)的癌症与癌旁正常组织的冰冻切片进行荧光染色，并将荧光图片与相应的HE染色图像进行对比，同时给出各自的诊断判据。
从山东大学齐鲁医院病理科获取术中乳腺病患(病理号：60551.18)的癌症与癌旁正常组织的冰冻切片。将其浸入到含有10μM探针CAPY-AP与10μM Hoechst 33342的PBS缓冲溶液(含10％EtOH)中染色10min，然后在激光扫描共聚焦显微镜下进行观察(分别用488nm和405nm的激光照射组织)获取荧光染色图片，并与病理科提供的相应的HE染色图片进行对比。
结果见图4。荧光图像显示探针CAPY-AP能够在人体乳腺病理组织的冰冻切片中专一性的成像胞质RNA和核仁。另外，从放大的图片中可以看到乳腺癌组织中存在多核仁以及大核仁的现象。正常组织中的核仁较小，不如癌组织中的明显。证明该探针在成像病理组织的核仁方面与HE染色相比具有显著的优势。
实施例7：对乳腺病患(病理号：61864.18)的癌症与癌旁正常组织的冰冻切片进行荧光染色，并将荧光图片与相应的HE染色图像进行对比，同时给出各自的诊断判据。
从山东大学齐鲁医院病理科获取术中乳腺病患(病理号：61864.18)的癌症与癌旁正常组织的冰冻切片。将其浸入到含有10μM探针CAPY-AP与10μM Hoechst 33342的PBS缓冲溶液(含10％EtOH)中染色10min，然后在激光扫描共聚焦显微镜下进行观察(分别用488nm和405nm的激光照射组织)获取荧光染色图片，并与病理科提供的相应的HE染色图片进行对比。
结果见图5。荧光图像显示探针CAPY-AP能够在人体乳腺病理组织的冰冻切片中专一性的成像胞质RNA和核仁。另外，从放大的图片中可以看到乳腺癌组织中存在多核仁以及大核仁的现象。正常组织中的核仁较小，不如癌组织中的明显。证明该探针在成像病理组织的核仁方面与HE染色相比具有显著的优势。另外在HE染色图片中几乎看不到核仁，再次证明探针CAPY-AP在成像病理组织的核仁方面与HE染色相比具有显著的优势。

Claims (6)

  1. 一种利用核仁形态变化快速区分癌与正常组织的RNA荧光探针，其特征在于：所述RNA荧光探针的化学名称为：(E)-1-(3-氨基丙基)-4-(2-(9-乙基-咔唑-3-烯基)乙烯基)吡啶二溴盐，简称CAPY-AP，其化学结构式如式(I)所示；所述RNA荧光探针能同时显示活细胞和正常组织或癌组织中的RNA与核仁分布，所述利用核仁形态变化快速区分癌与正常组织的判断指标是正常组织中只有单个不明显的核仁，而癌组织中存在多核仁以及大核仁的现象；
    Figure PCTCN2020133634-appb-100001
  2. 权利要求1所述的利用核仁形态变化快速区分癌与正常组织的RNA荧光探针在标记或显示活细胞内RNA与核仁分布的应用。
  3. 如权利要求2所述的应用，其特征在于：所述活细胞为SiHa癌细胞。
  4. 权利要求1所述的利用核仁形态变化快速区分癌与正常组织的RNA荧光探针在标记或显示正常组织或癌组织中RNA与核仁分布的应用。
  5. 如权利要求4所述的应用，其特征在于：所述正常组织是正常乳腺组织的冰冻病理切片，所述癌组织是乳腺癌组织的冰冻病理切片。
  6. 权利要求1所述的利用核仁形态变化快速区分癌与正常组织的RNA荧光探针在制备肿瘤术中快速病理诊断试剂中的应用。
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