NL2032008B1 - Fluorescent labeling kit for a tumor cell nucleus and labeling method thereof - Google Patents

Abstract

The present invention relates to a fluorescent labeling kit for a tumor cell nucleus and a labeling method thereof. Specifically, the present invention provides a method for 5 fluorescent labeling of a cell nucleus by a graphene-based tumor cell nuclear targeting fluorescent nanoprobe (GTTN), and a kit of the quantum dots for stable fluorescent labeling of a cell nucleus. By the method and kit, simple and practicable, eflicient and stable fluorescent labeling for the cell nucleus can be realized, and a stable tracing method of the cell nucleus is provided for long-term observational study of cells. The 10 method and kit can be used for efficient fluorescent labeling of the cell nucleus of cells, tissues, etc.

Description

FLUORESCENT LABELING KIT FOR A TUMOR CELL NUCLEUS AND

LABELING METHOD THEREOF

TECHNICAL FIELD

[01] The present invention relates to the technical fields of graphene-based tumor tumor cell nuclear targeting fluorescent nanoprobes and cell fluorescent labeling. In particular, the present invention provides a method for direct stable fluorescent labeling of a tumor nucleus by a new graphene-based tumor cell nuclear targeting fluorescent nanoprobe (GTTN), and a kit containing the GTTN for stable fluorescent labeling of a tumor nucleus.

BACKGROUND ART

[02] In traditional nuclear fluorescent labeling techniques, organic fluorescent dyes, such as 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), are mostly used for nuclear labeling. Both DAPI and PI bind strongly to the chromosomal DNA in the nucleus, so that they can be used to locate the nucleus and observe structural and morphological changes.

[03] At present, with the rapid development of materials science, fluorescent nanoprobes have been gradually applied in biomedical field due to unique optical characteristics. Compared with the organic fluorescent dyes, the fluorescent nanoprobes feature better photostability, high fluorescence intensity, wide excitation spectrum and narrow emission spectrum, and are not easy to be photobleached. These characteristics determine that the fluorescent nanoprobes apply to the fields of biological detection and imaging, and can be used as an excellent multivariate marker.

[04] At present, the fluorescent dyes commonly used for nuclear fluorescent labeling, such as DAPI and PI, all have a major shortcoming, i.e., poor photostability. Under continuous irradiation of excitation light, photobleaching will occur rapidly, making it difficult to maintain the fluorescent labeling state of the nucleus for a long time.

Therefore, it is necessary to develop a tracing method and product for long-term stable fluorescent labeling of the cell nucleus.

[05] Moreover, there is no research to directly realize selective localization of the nucleus by merely using the unique chemical advantages of nanomaterials. All the previous researches have crosslinked the nanomaterials with guiding molecules for nuclear localization, such as antibodies and nuclear localization signals, to realize localization labeling of the nucleus by quantum dots, resulting in complex steps, high cost and low efficiency in fluorescent labeling of the cell nucleus. Moreover, only the nanomaterials and the nucleus markers such as DAPI or PI are used in combination to label the nucleus at home and abroad, but there is no research on directly utilizing the unique optical properties of nanomaterials to realize long-term stable tracing and labeling of the cell nucleus without mediation of any special targeting molecule.

SUMMARY

[06] One objective of the present invention is to provide a fluorescent labeling kit for atumor cell nucleus to overcome the technical problems in the prior art.

[07] Another objective of the present invention is to provide a method for fluorescent labeling by the kit.

[08] To achieve the aforesaid purposes, the present invention adopts the following technical solution:

[09] In order to overcome the above-mentioned shortcomings of the conventional fluorescent dyes DAPI or PI and the inorganic dye quantum dots in nuclear staining, various researches are performed in the present invention, and an unique sulfonic group- based graphene quantum dot structure is synthesized. The preparation method is as shown in the patent (Sulfonic Group-based Graphene Quantum Dot Bioluminescence

Probe and Application Thereof (201510394852.8)). The structure efficiently and specifically binds specific histones and DNA in the nucleus, and labels a sample under a fluorescence microscope (or a confocal microscope), and the cell nucleus exhibits stable fluorescence under 405 nm excitation irradiation, so as to realize stable selective tracing labeling of the cell nucleus of a biological sample.

[10] The fluorescent labeling kit for a tumor cell nucleus is a GTTN solution with a particle size range of 3-5 nm and a concentration of 10-30 mg/L.

[11] Further, a solvent of the solution is deionized water, normal saline or a medium buffer solution for cell culture.

[12] A method for fluorescent labeling of cell nucleus adopts the kit in the solution and includes the following specific steps:

[13] 1) immobilizing a biological sample with 4% paraformaldehyde;

[14] 2) co-incubating GTTN with the biological sample at a room temperature for 5 min, with a surface of the biological sample covered by the GTTN, and washing off excess GTTN from the surface of the biological sample (the surface is covered) by a

PBS buffer system; and

[15] 3) observing the cell nucleus of the biological sample under 405 nm excitation light irradiation.

[16] The biological sample is stored at a room temperature, 4°C or -70°C. The fluorescent labeling object includes cells, tissues or live animals.

[17] The present invention further provides the following kit, including: (1) a labeling solution, which is a quantum dot solution with a particle size range of 2-3 nm.

The GTTN is packaged in a form of mother solution (100 mg/L) which may be prepared into a 10 mg/L working solution, and the GTTN therein can be prepared in the medium buffer solution for cell culture (such as DMEM, RPMI 1640 medium), and may be further prepared in a normal saline buffer solution, or other buffer solutions suitable for nucleus labeling recognized in the field; a working temperature is 37°C, or a room temperature. The kit further includes (2) a stationary solution, (3) a mounting medium, etc. conventionally used for cell labeling, immunohistochemistry, etc. Moreover, the kit may further include (4) a package insert. In addition to specifying the reagents contained in the kit, the package insert further specifies how to perform the fluorescent labeling through the above-mentioned method.

[18] Beneficial effects: Through experimental verification, the inventor finds that compared with the existing traditional organic nuclear fluorescent labeling techniques,

the present invention features the following advantages and effects:

[19] 1. The imaging is clear and stable. The fluorescently-labeled nucleus of the present invention features strong photoquenching resistance, and the kit can be used for long-term stable labeling of the cell nucleus.

[29] 2. The labeling method is simple and quick.

[21] 3. The scope of application is wide. Upon various experimental verifications of the labeling method and the kit of the present invention, the nuclear targeting performance of the GTTN is applicable to a variety of cells and animal tissue sections.

Therefore, the method and kit can be further applied in flow cytometry and histological staining.

[22] 4. The method is simple, easy to operate and practicable. Compared with the existing traditional fluorescent labeling techniques, in the present invention, it is unnecessary to crosslink the GTTN with guiding molecules for nuclear localization, such as antibodies and nuclear localization signals, but the selective localization of nucleus is directly realized by using the unique chemical advantages of GTTN, saving the intermediate step.

BRIEF DESCRIPTION OF THE DRAWINGS

[23] FIG. 1 shows a fluorescence stability contrast experiment between GTTN and

DAPI A: Changes of fluorescence intensities of two fluorescent dyes, after continuous laser irradiation at different time points under a laser scanning confocal microscope, under the precondition that the cell nuclei are dyed by the GTTN and the DAPI respectively after 4T1 cells (mouse breast cancer cells) are immobilized by a 4% paraformaldehyde solution. Scale: 10 um. B, C: Statistical graphs of normalized fluorescence intensities of GTTN and DAPL

[24] FIG. 2 shows efficient targeting of GTTN to the nuclei of different cells. After cells are immobilized by a 4% paraformaldehyde solution, the nuclei are dyed by the

GTTN. A: 4TI cells; B: C17.2 cells (mouse neural stem cells); C: GES-1 cells (human gastric mucosa cells). Scale: 10 um.

[25] FIG. 3 shows efficient targeting of GTTN to the nuclei of tumor tissues (mouse subcutaneous tumor model). After tissue sections are immobilized by a 4% paraformaldehyde solution, the nuclei are dyed by the GTTN. A: Liver cancer tissue; B:

Breast cancer tissue; C: Cervical cancer tissue; D: Lung cancer tissue. Scale: 10 um.

[26] FIG. 4 shows efficient targeting of GTTN to the nuclei of normal tissues. After tissue sections are immobilized by a 4% paraformaldehyde solution, the nuclei are dyed by the GTTN. A: Heart; B: Liver; C: Spleen; D: Lung; E: Kidney; F: Brain. Scale: 10 um.

[27] FIG. 5 shows a discussion on a nuclear targeting mechanism of GTTN. A: After the GTTN is mixed with histones (specific proteins in the nucleus) of different concentrations and incubated for different times, adsorption rates of the histones and the

GTTN are detected. B: Changes of a fluorescence intensity of the GTTN after an interaction with different amounts of DNA (0.2, 0.4, 0.6, 0.8, 0.9, 1 mL).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[28] The method and kit of the present invention will be described in detail below by embodiments, but the scope of protection required by the application is not limited to the embodiments.

[29] Example 1:

[30] Fluorescence stability contrast experiment between GTTN and DAPI:

[31] In order to research the photostability of GTTN, DAPI, a traditional nuclear fluorescent dye, was used as a reference. The method specifically included the following steps:

[32] 1. 4TI cells (mouse breast cancer cells) were cultured in a DMEM medium containing 10% fetal bovine serum at 37°C in an environment of 5% CO:.

[33] 2. The cells were washed by a PBS solution twice, 2-5 min each time.

[34] 3. The cells were immobilized by a 4% paraformaldehyde-PBS stationary solution for 15 min.

[35] 4. The cells were washed by the PBS solution twice, 2-5 min each time.

[36] 5. The treated cells were divided into two groups; GTTN (10 mg/L) and DAPI (2 mg/L) were dropped respectively, and the cells were incubated at a room temperature for 5 min.

[37] 6. The cells were washed by the PBS solution twice. 38] 7. A mounting medium was dropped.

[39] 8. The cells were placed under a confocal microscope and excited with 405 nm excitation light, and nuclear imaging was observed. The cells were continuously irradiated by laser, and images were collected every 5 min to observe photobleaching of the dye.

[40] Results: The details are shown in FIG. 1. From FIG. 1, it can be seen that after the GTTN was continuously irradiated by laser for 40 min, the fluorescence was not attenuated but significantly enhanced; at the same time, obvious photobleaching occurred to the DAPI under the same conditions, and the fluorescence of the DAPI almost disappeared after 40 min. The GTTN exhibited advantages as a nuclear labeling dye, and no photoquenching occurred.

[41] Example 2:

[42] Efficient targeting of GTTN to the nuclei of different cells:

[43] In order to research the properties of GTTN to the nuclei of different cells, several different types of cells were used for experiment. The method specifically included the following steps:

[44] 1. 4TI cells, C17.2 cells (mouse neural stem cells) and GES-1 cells (human gastric mucosa cells) were cultured in a DMEM or RPMI 1640 medium containing 10% fetal bovine serum at 37°C in an environment of 5% CO».

[45] 2. The cells were washed by a PBS solution twice, 2-5 min each time.

[46] 3. The cells were immobilized by a 4% paraformaldehyde-PBS stationary solution for 15 min.

[47] 4. The cells were washed by the PBS solution twice, 2-5 min each time.

[48] 5. GTTN (10 mg/L) was dropped in the treated cells, and the cells were incubated at a room temperature for 5 min.

[49] 6. The cells were washed by the PBS solution twice.

[50] 7. A mounting medium was dropped. [S1] 8. The cells were placed under a confocal microscope and excited with 405 nm excitation light, and nuclear imaging was observed.

[52] Results: The details are shown in FIG. 2. From FIG. 2, it can be seen that the

GTTN-labeled nuclei of the present invention exhibited strong fluorescence, and similar results were obtained for the 4T1 cells, C17.2 cells and GES-1 cells, demonstrating that the labeling method of the present invention applied to labeling of the nuclei of various immobilized cells (such as normal cells and tumor cells). The method can be used for specific stable fluorescent labeling of the nuclei.

[53] Example 3:

[54] Efficient targeting of GTTN to the nuclei of different tumor and normal tissues:

[55] In order to research the properties of GTTN to the nuclei of different tumor and normal tissues, several different types of tumor and normal tissues were used for experiment. The method specifically included the following steps:

[56] 1. Different types of tumor and normal tissue sections were used, such as liver cancer tissue, breast cancer tissue, cervical cancer tissue, lung cancer tissue, heart, liver, spleen, lung, kidney and brain. [S7] 2. The tissue sections were immobilized by a 4% paraformaldehyde-PBS stationary solution for 15 min.

[58] 3. The tissue sections were washed by a PBS solution twice, 2-5 min each time.

[59] 4. SYTO17 (5 uM, a nuclear labeling dye, 605 nm excitation wavelength) was dropped in the treated tissue sections, and the tissue sections were incubated at a room temperature for 5 min, and then GTTN (10 mg/L) was dropped, and the tissue sections were incubated at a room temperature for 5 min.

[60] 5. The tissue sections were washed by the PBS solution twice.

[61] 6. A mounting medium was dropped.

[62] 7. The tissue sections were placed under a confocal microscope and excited with 405 nm excitation light, and nuclear imaging was observed.

[63] Results: The details are shown in FIGS. 3 and 4. From FIGS. 3 and 4, it can be seen that the GTTN-labeled nuclei of the present invention exhibited strong fluorescence, and similar results were obtained for the liver cancer tissue, breast cancer tissue, cervical cancer tissue, lung cancer tissue, heart, liver, spleen, lung, kidney and brain, demonstrating that the labeling method of the present invention applied to labeling of the nuclei of various immobilized tissue sections. The method can be used for specific stable fluorescent labeling of the nuclei.

[64] Example 4:

[65] Discussion on nuclear targeting mechanism of GTTN:

[66] In order to research the cause of direct targeting of GTTN to the nuclei, the following two experiments were designed:

[67] Binding experiment of GTTN and histones in the nuclei:

[68] 1. Histones were dissolved in a water, the solution was placed in a high-speed centrifuge and centrifuged at 20,000 rpm for 20 min to remove impurities, and supernatant was collected for experiment.

[69] 2. The histones were mixed with GTTN, wherein a final concentration of the histones was 0.5 or 2 mg/mL, and a final concentration of the GTTN was 60 mg/L.

[70] 3. The solution was incubated in PBS (37°C, pH7.4) for different times (10 min, 30 min, 2 h).

[71] 4. Each sample was placed in the high-speed centrifuge and centrifuged at 20,000 rpm for 20 min, and a polymer of the histones and the GTTN was collected.

[72] 5. The polymer was redissolved in the PBS, and a concentration of the histones was detected by a Bradford test.

[73] Binding experiment of GTTN and DNA:

[74] 1. Ater DNA was dissolved in a water, GTTN interacted with different amounts of DNA (0.2, 0.4, 0.6, 0.8, 0.9, 1 mL), wherein a final concentration of the DNA was 1 mmol/L, and a final concentration of the GTTN was 60 mg/L.

[75] 2. Each solution was incubated in PBS (37°C, pH7.4) for 30 min.

[76] 3. A fluorescence change of the mixture was detected by a fluorescence spectrophotometer.

[77] Results: The details are shown in FIG. 5. From FIG. 5, it can be seen that the

GTTN and the DNA were obviously bound, and there was obvious dose effect and time effect, indicating that the GTTN specifically bound to specific DNA and histones in the nuclei, so the GTTN was well suitable for labeling the nuclei.

Claims (5)

Conclusions 1. Fluorescence labeling kit for a tumor cell nucleus, wherein the kit is a GTTN solution with a particle size range of 3-5 nm and a concentration of 10-30 mg/L. The tumor cell nucleus fluorescent labeling kit of claim 1, wherein a solvent of the solution is deionized water, normal saline or a cell culture medium buffer solution. The tumor cell nucleus fluorescent labeling kit according to claim 2, wherein the cell culture medium buffer solution is a DMEM high glucose medium or an RPMI 1640 medium. A method for causing a cell nucleus to fluoresce, using the kit according to claim 1 or 2, and comprising the following specific steps: a. immobilizing a biological sample with 4% paraformaldehyde; b. co-incubating GTTN with the biological sample at room temperature for 5 minutes, with a surface of the biological sample covered by GTTN, and washing off excess GTTN from the surface of the biological sample by a PBS buffer system; and c. observing the cell nucleus of the biological sample under irradiation with 405 nm excitation light. The method of claim 4, wherein the biological sample is cells, animal tissues or living animals.