RNA FLUORESCENT PROBE FOR RAPIDLY DISTINGUISHING CANCER TISSUE FROM NORMAL TISSUE BASED ON NUCLEOLAR MORPHOLOGICAL CHANGES

Abstract

An RNA fluorescent probe for rapidly distinguishing cancer tissue from normal tissue based on nucleolar morphological changes, the probe being (E)-1-(3-aminopropyl)-4-(2-(9-ethyl-9H-carbazol-3-yl)vinyl)pyridine-1-ium dibromide, abbreviated as CAPY-AP. The probe can target RNA in culture cells and normal tissue as well as cancer tissue and then display nucleolar morphology. The judging criteria of distinguishing the cancer tissue from the normal tissue based on the nucleolar morphological changes is only single and unconspicuous nucleolus in most cells of normal tissue, while the enlarged nucleoli and/or multiple nucleoli exist in many cells of cancer tissue. Compared with other existing RNA probes, the probe has super-high RNA affinity and super-high permeability, and can rapidly image the RNA and nucleoli in tissue sections. Additionally, the probe has characteristics of good membrane permeability, strong fluorescence and good photostability, and is expected to be applied in preparation of intraoperative pathological diagnostic reagents for tumors.

Claims

1. An RNA fluorescent probe for rapidly distinguishing cancer tissue from normal tissue based on nucleolar morphological changes, wherein a chemical name of the RNA fluorescent probe is (E)-1-(3-aminopropyl)-4-(2-(3-(9-ethyl-carbazol))vinyl)pyridine dibromide salt, abbreviated as CAPY-AP; a chemical structure of the RNA fluorescent probe is shown in formula (I): ##STR00004##

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. Application of the RNA fluorescent probe for rapidly distinguishing cancer tissue from normal tissue based on nucleolar morphological changes according to claim 1 in rapid intraoperative pathological diagnosis for tumors.

7. A method for preparing the RNA fluorescent probe for rapidly distinguishing cancer tissue from normal tissue based on nucleolar morphological changes according to claim 1, comprising the steps of firstly mixing 4-methylpyridine with 3-bromopropylamine hydrobromide to react to obtain compound 1; performing a Vilsmeier reaction to synthesize compound 2; mixing compound 1 and compound 2 to prepare a final product by a reflux reaction with piperidine as a catalyst; and finally obtaining a purified red solid product CAPY-AP by column chromatography, wherein the structures of compound 1 and compound 2 are as follows: ##STR00005##

8. A method for rapid staining cultured cells, wherein the method comprises staining cells with the RNA fluorescent probe of claim 1, labeling or displaying distributions of RNA and nucleoli in the cells.

9. The method according to claim 8, wherein the cells are cancer cells or normal cells.

10. A method for rapidly distinguishing cancer tissue from normal tissue based on nucleolar morphological changes, wherein the method comprises staining all cells in a tissue section with the RNA fluorescent probe of claim 1, labeling or displaying distributions of RNA and nucleoli in all cells.

11. The method of claim 10, wherein the tissue section is a normal tissue section or a pathological section.

12. The method of claim 10, wherein the tissue section is a tissue section of mammary gland.

13. The method of claim 10, wherein judging criteria of distinguishing the cancer tissue from the normal tissue based on the nucleolar morphological changes are that there is only single and unconspicuous nucleolus in most cells of normal tissue, while the enlarged nucleoli and/or multiple nucleoli exist in many cells of cancer tissue.

14. A rapid pathological diagnostic reagent for rapidly distinguishing cancer tissue from normal tissue based on nucleolar morphological changes, comprising the RNA fluorescent probe according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows confocal fluorescent images of live SiHa cells stained with CAPY-AP.

[0022] Obtaining method: the SiHa cells are incubated with CAPY-AP for 30 min, and then the cells are irradiated with a 488 nm laser under a confocal laser scanning microscope to obtain micrographs. FIG. 1a is the image of green channel at 500-600 nm from CAPY-AP excited by the 488 nm laser. FIG. 1B is a differential interference contrast (DIC) micrograph scanned by a bright field laser. FIG. 1c is the merge image of FIG. 1a and FIG. 1b.

[0023] Fluorescence images show obvious green light distribution in cytoplasmic and nucleolar areas, which clearly indicates that CAPY-AP can specifically image cytoplasmic RNA and the nucleoli in culture cells.

[0024] FIG. 2 shows confocal fluorescent images of fixed SiHa cells stained with CAPY-AP after RNase treatment.

[0025] Obtaining method: the fixed SiHa cells treated with RNase and then stained with CAPY-AP. Under the confocal laser scanning microscope, the cells are irradiated with the 488 nm laser to obtain micrographs. FIG. 2a is the image of a green channel at 500-600 nm from CAPY-AP excited by the 488 nm laser. FIG. 2b is a differential interference contrast (DIC) micrograph scanned by a bright field laser. FIG. 2c is the merge image of FIG. 2a and FIG. 2b.

[0026] The results show that the fluorescence of CAPY-AP in the cells treated with RNase is relatively weak, and the fluorescence in the nucleoli and cytoplasm is almost invisible.

[0027] FIG. 3 shows a comparison of H&E staining and fluorescence staining of frozen sections of human mammary cancer tissue and its adjacent normal tissue (Pathology No. 57977.18).

[0028] Obtaining method: after the frozen sections of human mammary cancer tissue and its adjacent normal tissue are stained with CAPY-AP and Hoechst 33342, the tissue is irradiated with 488 nm and 405 nm lasers respectively under the confocal laser scanning microscope, and superposition images of two channels (blue channel: 420-450 nm, green channel: 500-600 nm) are obtained. The frozen sections of the same tissue are stained with H&E to obtain H&E staining images. Amongst, the enlarged images come from two box regions of fluorescent staining images of mammary cancer tissue.

[0029] FIG. 4 shows a comparison of H&E staining and fluorescence staining of frozen sections of human mammary cancer tissue and its adjacent normal tissue (Pathology No. 60551.18).

[0030] Obtaining method: after the frozen sections of human mammary cancer tissue and its adjacent normal tissue are stained with CAPY-AP and Hoechst 33342, the tissue is irradiated with 488 nm and 405 nm lasers respectively under the confocal laser scanning microscope, and superposition images of two channels (blue channel: 420-450 nm, green channel: 500-600 nm) are obtained. The frozen sections of the same tissue are stained with H&E to obtain H&E staining images. Amongst, the enlarged images come from two box regions of fluorescent staining images of mammary cancer tissue.

[0031] FIG. 5 shows a comparison of H&E staining and fluorescence staining of frozen sections of human mammary cancer tissue and its adjacent normal tissue (Pathology No. 61864.18).

[0032] Obtaining method: after the frozen sections of human mammary cancer tissue and its adjacent normal tissue are stained with CAPY-AP and Hoechst 33342, the tissue is irradiated with 488 nm and 405 nm lasers respectively under the confocal laser scanning microscope, and superposition images of two channels (blue channel: 420-450 nm, green channel: 500-600 nm) are obtained. The frozen sections of the same tissue are stained with H&E to obtain H&E staining images. Amongst, the enlarged images come from two box regions of fluorescent staining images of mammary cancer tissue.

[0033] According to the results in FIG. 3, FIG. 4 and FIG. 5, it is found that CAPY-AP can specifically image the cytoplasmic RNA and nucleoli in frozen sections of human mammary pathological tissue. In addition, the probe can clearly image the sizes, shapes, location and numbers of nucleoli in two enlarged images. At the same time, it was also found that there were enlarged nucleoli and/or multiple nucleoli in mammary cancer tissue, while the nucleoli in the normal tissue are smaller and less conspicuous. However, the nucleoli in the cancer tissue and the normal tissue cannot be displayed clearly in the H&E staining images.

DETAILED DESCRIPTION

[0034] The following describes the present invention in detail with reference to the specific accompanying drawings and embodiments. Described in the following embodiments are only exemplary implementations of the present invention. It should be clear that the following instructions are just in order to explain the present invention, not make any restrictions in the form of the present invention. Any simple modification, equivalent change or modification to the implementation according to the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0035] In the following embodiments, tissue frozen sections involved are provided by the cooperating organization, the department of pathology, Qilu Hospital of Shandong University, and materials, cells, reagents, etc. used are obtained commercially unless otherwise specified.

[0036] Example 1: Synthesis of CAPY-AP

##STR00003##

[0037] The specific synthesis is as follows:

[0038] synthesis of compound 1: 4-methylpyridine (0.93 g, 10 mmol) and 3-bromopropylamine hydrobromide (2.41 g, 11 mmol) were dissolved in 20 mL ethanol and heated at 85° C. for a reflux reaction for 12 hours. At the end of the reaction, white solid appeared. After filtration and washing with petroleum ether, pure white compound 1 was obtained. The yield was 70%. .sup.1H NMR (300 MHz, DMSO-d.sub.6), δ (ppm): 8.938; (d, J=6 Hz, 2H), 8.047; (d, J=6.3 Hz, 2H), 7.797; (s, 3H), 4.628-4.582; (t, J=6.9 Hz, 2H), 2.860-2.794; (m, 2H), 2.622; (s, 3H), 2.220-2.124; (m, 2H).

[0039] Synthesis of compound 2: POC1.sub.3 (9.2 mL, 100 mmol) was slowly added into dry DMF (7.7 mL, 100 mmol) under an ice bath condition. After stirring for 30 min, viscosity appeared. Then N-ethylcarbazole (2.94 g, 10 mmol) dissolved in CHCl.sub.3 was added into the system. After stirring for 1 hour continuously, the mixture was heated at 80° C. for a reaction for 12 hours. After cooling to room temperature, the mixture was poured into ice water and the pH of the system was adjusted to be neutral with NaOH. Then extraction with CH.sub.2Cl.sub.2 was conducted, and an organic phase was washed with water twice and dried with anhydrous magnesium sulfate for 3 hours. Finally, ethyl acetate/petroleum ether (1:8, V/V) was used as an eluent, and separation purifying by column chromatography was conducted to obtain a light-yellow pure substance. The yield was 50%. .sup.1H NMR (300 MHz, DMSO-d6), δ (ppm): 10.07 (s, 1H), 8.79; (s, 1H), 8.31; (d, J=7.8 Hz, 1H), 8.02-7.989; (m, 1H), 7.80; (d, J=6.6 Hz, 1H), 7.72; (d, J=8.1 Hz, 1H), 7.55; (t, J=7.65 Hz, 1H), 7.31; (d, J=7.5 Hz, 1H), 4.53; (t, J=7 Hz, 2H), 1.34; (t, J=7.05 Hz, 3H).

[0040] Synthesis of compound CAPY-AP: compound 1 (0.52 g, 1.67 mmol) and compound 2 (0.45 g, 2 mmol) were dissolved in 15 mL ethanol to obtain a yellow transparent solution, and 3-4 drops of piperidine were added to make the solution turn red quickly. Reaction was heated to reflux overnight, and then red solid precipitates were obtained. After cooling, filtration, and washing with ethanol, dichloromethane/methanol (20:1) was used as an eluent to obtain a purified red solid product by column chromatography, namely CAPY-AP with a yield of 40%. .sup.1H NMR (300 MHz, DMSO-d.sub.6): δ8.94 (d, J=6.9 Hz, 2H), 8.61; (s, 1H), 8.30-8.19; (m, 4H), 7.93-7.90; (m, 4H), 7.86; (d, J=11.1 Hz, 1H), 7.77; (d, J=8.7 Hz, 1H), 7.75-7.29; (m, 2H), 7.29; (t, J=7.5 Hz, 1H), 4.59; (t, J=6.9 Hz, 2H), 4.52; (m, 2H), 2.88; (m, 2H), 2.22; (m, 2H) 1.34; (t, J=7.1 Hz, 3H). .sup.13C NMR (75 MHz, DMSO.sup.-d.sub.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.sub.24H.sub.27Br.sub.2N.sub.3), found: 356.2184 [M—2Br—H].sup.2+.

[0041] Example 2: SiHa cells culture.

[0042] SiHa cells were cultured in adherent conditions in a culture media containing 10% fetal bovine serum and incubated in a 37° C. and 5% CO.sub.2 saturated humidity incubator. The culture media was replaced every 2-3 days for passage once. When the cells grew to a logarithmic stage, cover glass-graft culture was conducted: (1) a cover glass was soaked in anhydrous ethanol for 30 min, and then was dried with an alcohol lamp and put into a disposable 35 mm culture dish for standby application; and (2) the overgrown cells in a 100 mL cell flask were washed with PBS three times and digested with 1 mL of 0.25% trypsin for 3-5 min, the trypsin was carefully poured out, fresh culture media was added, blowing was conducted evenly, the cells were counted, the cell density was controlled with the adding amount of the culture media to make the final concentration of the cells be 1×10.sup.5 per ml, then the cells were seeded into the culture dish containing the cover glass and cultured in a 5% CO.sub.2 incubator for growing on the cover glass. After the SiHa cells grew on the cover glass and grew over the cover glass, they were used in the experiment.

[0043] Example 3: Staining live SiHa cells with CAPY-AP.

[0044] First, DMSO solution of a 5 mM CAPY-AP was prepared as a stock solution. After SiHa cells grew over a cover glass, culture medium in culture dish was removed, the cover glass with the SiHa cells was rinsed with clean PBS 3 times, and the cells were stained with 5 μM CAPY-AP, and incubated in a CO.sub.2 incubator for 30 min. After staining, the cover glass with cells was taken out, excess probes were washed off, the cover glass with the cell growth side facing down covers a glass slide, and the staining locations, fluorescence distribution and brightness changes of the cells were observed under a confocal laser scanning microscope (the cells were irradiated with a 488 nm laser).

[0045] Results are shown in FIG. 1. FIG. 1a is the image of the green channel at 500-600 nm from CAPY-AP excited by the 488 nm laser. FIG. 1B is a differential interference contrast (DIC) micrograph scanned by a bright field laser. FIG. 1c is the merge image of FIG. 1a and FIG. 1B. Fluorescence images show obvious green light distribution in cytoplasmic and nucleolar areas, which clearly indicates that CAPY-AP can specifically image cytoplasmic RNA and nucleoli in culture cells.

[0046] Example 4: Staining observation of RNase-treated SiHa cells (RNase digestion assay) with CAPY-AP.

[0047] Preparation of fixed cells: first, the cover glass covered with the SiHa cells prepared in Example 2 was immersed in a 4% paraformaldehyde solution for 30 min, and then 0.5%

[0048] Triton X-100 was used for permeation at room temperature for 2 min to obtain the fixed cells.

[0049] A group of the above fixed cells was taken out and digested in an incubator for 2 hours after RNase with a 25 μg/mL concentration was added, then washed with PBS 3 times and stained with a 5 μM CAPY-AP in a CO.sub.2 incubator for 30 min. The cover glass with stained cells was taken out, excess dyes were washed off, the cover glass with a cell growth side facing down covers a glass slide, and the staining locations, fluorescence distribution and brightness changes of the cells were observed under a confocal laser scanning microscope (the cells were irradiated with a 488 nm laser) and recorded.

[0050] Results are shown in FIG. 2. FIG. 2a is the fluorescent image of a green channel at 500-600 nm from CAPY-AP excited by the 488 nm laser. FIG. 2b is a differential interference contrast (DIC) micrograph scanned by a bright field laser. FIG. 2c is the merge image of FIG. 2a and FIG. 2b. It is found that the fluorescence of the cells treated with the RNase is weak, and the fluorescence in the nucleoli and cytoplasm is almost invisible. Since the RNase can hydrolyze RNA in the cells, it can be certified and verified that the probe of the present invention can selectively target the RNA in the cells.

[0051] Example 5: Fluorescence staining was conducted on frozen sections of human mammary cancer tissue and its adjacent normal tissue (Pathology No. 57977.18), and fluorescence images were compared with corresponding H&E staining images, and respective diagnostic criteria were given at the same time.

[0052] The frozen sections of the mammary cancer tissue and its adjacent normal tissue from intraoperative pathological samples of breast cancer patients (Pathology No. 57977.18) were obtained from the department of pathology, Qilu Hospital of Shandong University. In order to stain, the frozen sections were immersed into a 10% EtOH PBS buffer solution with a 10 μM CAPY-AP and a 10 μM Hoechst 33342 for 10 min, and then observed under a confocal laser scanning microscope (the tissue was irradiated with 488 nm and 405 nm lasers respectively) to obtain fluorescence images. The fluorescence images were compared with the corresponding H&E images.

[0053] Results are shown in FIG. 3 and Table 2.

[0054] In FIG. 3, the fluorescence images show obvious green light distribution in cytoplasmic and nucleolar areas, while no fluorescence exists in extracellular matrices. It clearly indicates that the probe of the present invention can specifically image cytoplasmic RNA and nucleoli in human mammary cancer tissue and normal mammary tissue. In addition, the sizes, shapes, location and numbers of the nucleoli can be clearly shown in magnified fluorescence images. By comparing the H&E images and the fluorescence images of the frozen sections of the normal mammary tissue and mammary cancer tissue, it is obvious that fluorescence staining has great advantages. The morphologies of the nucleoli in the tissue can be clearly shown, whereas the nucleoli were not clearly seen in the H&E images. In addition, we also have found a phenomenon that only single inconspicuous nucleoli exist in the normal mammary tissue, while enlarged nucleoli and/or multiple nucleoli exist in the mammary cancer tissue.

TABLE-US-00002 TABLE 2 Pathological Diagnostic Criteria for Mammary Tumor via H&E Staining and Fluorescence Staining of Frozen Sections Diagnostic Normal Mammary Methods Criteria Mammary Gland Cancer H&E Tissue structures Normal Abnormal The interpretation and cell morphologies morphologies morphologies of images is based on Sizes and morphologies Relatively uniform, The sizes and morphologies pathologist's experience, of cells and nuclei nuclear chromatin are different, the nuclei and only highly qualified is uniform, and are enlarged, chromatin is pathologists can issue nuclear membranes not uniform and in a frozen section reports are thin, smooth and thick block shape, and regular. nuclear membranes are thickened irregular Nuclear to Normal Increased cytoplasmic ratio Fluorescence Providing some Most cells contain Many Cells contain Judgment is visually information single inconspicuous enlarged nucleoli intuitive and accurate displayed by H&E nucleoli and multiple nucleoli staining, with emphasis on nucleolar changes for judgment

[0055] According to the respective diagnostic criteria of H&E and fluorescence in Table 2, it is visually intuitive to distinguish mammary cancer tissue from the normal tissue by nucleolar morphological changes in the fluorescent images.

[0056] Example 6: Fluorescence staining was conducted on frozen sections of human mammary cancer tissue and its adjacent normal tissue (Pathology No. 60551.18), and the fluorescence images were compared with corresponding H&E staining images.

[0057] The frozen sections of the mammary cancer tissue and its adjacent normal tissue from intraoperative pathological samples of breast cancer patients (Pathology No. 60551.18) were obtained from the department of pathology, Qilu Hospital of Shandong University. In order to stain, the frozen sections were immersed into a 10% EtOH PBS buffer solution with a 10 μM CAPY-AP and a 10 μM Hoechst 33342 for 10 min, and then observed under a confocal laser scanning microscope (the tissue was irradiated with 488 nm and 405 nm lasers respectively) to obtain fluorescence images. The fluorescence images were compared with the corresponding H&E images.

[0058] Results are shown in FIG. 4. The fluorescence images display that CAPY-AP can specifically image the cytoplasmic RNA and nucleoli in the frozen sections of the human mammary pathological tissue. In addition, enlarged images show the phenomenon that the enlarged nucleoli and/or multiple nucleoli exist in the mammary cancer tissue. The nucleoli in the normal tissue are smaller and less conspicuous than those in the cancer tissue. It is proved that the probe has a significant advantage in imaging the nucleoli of the pathological tissue compared with the H&E staining. The pathological diagnostic criteria for mammary tumor with fluorescent staining given in Example 5 have been proven again.

[0059] Example 7: Fluorescence staining was conducted on frozen sections of human mammary cancer tissue and its adjacent normal tissue (Pathology No. 61864.18), and the fluorescence images are compared with corresponding H&E staining images.

[0060] The frozen sections of the mammary cancer tissue and its adjacent normal tissue from intraoperative pathological samples of breast cancer patients (Pathology No. 61864.18) were obtained from the department of pathology, Qilu Hospital of Shandong University. In order to stain, the frozen sections were immersed into a 10% EtOH PBS buffer solution with a 10 μM CAPY-AP and a 10 μM Hoechst 33342 for 10 min, and then observed under a confocal laser scanning microscope (the tissue was irradiated with 488 nm and 405 nm lasers respectively) to obtain fluorescence images. The fluorescence images were compared with the corresponding H&E images.

[0061] Results are shown in FIG. 5. The fluorescence images display that CAPY-AP can specifically image the cytoplasmic RNA and nucleoli in the frozen sections of the human mammary pathological tissue. In addition, enlarged images show the phenomenon that the enlarged nucleoli and/or multiple nucleoli exist in the mammary cancer tissue. The nucleoli in the normal tissue are smaller and less conspicuous than those in the cancer tissue. It is proved that the probe has a significant advantage in imaging the nucleoli of the pathological tissue compared with the H&E staining. In addition, the nucleoli can hardly be seen in the H&E staining images, again proving that CAPY-AP has a significant advantage in imaging the nucleoli of the pathological tissue compared with the H&E staining. At the same time, the pathological diagnostic criteria for mammary tumor with fluorescent staining given in Example 5 have also been proven again.