CN114317542A - Screening probe for inducing epithelial-mesenchymal transition drug of tumor cells as well as preparation method and application of screening probe - Google Patents

Abstract

The invention belongs to the technical field of medicines, and discloses a screening probe for inducing a tumor cell epithelial-mesenchymal transition drug, which is characterized in that: the screening probe is modified at the 5' end of an aptamer SYL3C

And modifying 6-carboxyfluorescein at the 3' end of the aptamer SYL3C to form the probe. The probe of the invention identifies the down-regulated biomarker epithelial adhesion molecules in the epithelial-mesenchymal transition process according to the administered fine particlesAnd (4) screening the epithelial-mesenchymal transition positive drugs by the cell viability normalization fluorescence intensity signal reduction. In addition, the chemosensitizer for blocking the epithelial-mesenchymal transition is co-administered with the epithelial-mesenchymal transition inducer TGF-beta, and the chemosensitizer is screened according to the recovery condition of the cell viability normalized fluorescence intensity signal after administration.

Description

Screening probe for inducing epithelial-mesenchymal transition drug of tumor cells as well as preparation method and application of screening probe

Technical Field

The invention belongs to the field of medicines, and relates to a screening probe for inducing a tumor cell epithelial-mesenchymal transition drug, and a preparation method and application thereof.

Background

The inhibition of tumor metastasis and drug resistance are difficult problems in clinical tumor treatment. Epithelial-mesenchymal transition (EMT) is one of the important factors leading to tumor metastasis and drug resistance. EMT refers to the property of epithelial tumor cells to exhibit mesenchymal cells with the ability to migrate and invade after undergoing various physiological changes. Key biochemical and morphological changes in EMT include down-regulation of epithelial adhesion molecule (EpCAM) protein expression, loss of cell-cell contact, and the like. The activation of the EMT program is considered to be an important marker of tumor progression, and can induce the improvement of the metastatic capacity of epithelial tumor cells, and endow the epithelial tumor cells with the characteristics of tumor stem cells. Many studies have shown that chemotherapy drugs can trigger the metastasis and resistance characteristics of epithelial tumor cells through the EMT process, resulting in a decrease in drug efficacy. Therefore, screening of chemotherapeutic drugs inducing epithelial tumor cells to produce side effects of EMT, and chemotherapeutic sensitizers that antagonize the EMT process are very important for clinical tumor treatment.

Traditional chemotherapeutic drug screening focuses on its selective anti-tumor activity and often neglects the study of EMT effects. Meanwhile, the current research on the EMT process mainly depends on western blot experiments for biomarkers of the EMT process, and the whole process is complicated and time-consuming. Therefore, it is necessary to develop a simple and effective screening method for EMT-related drugs.

The aptamer is a single-stranded DNA or RNA sequence, and forms a secondary structure and a tertiary structure by depending on the complementarity of bases among strands, so as to realize the specific binding with a target. Compared with the traditional antibody, the aptamer is excellent in water solubility and chemical stability, easy to chemically modify and free of immunogenicity. The aptamer SYL3C can realize high-affinity and strong-specificity combination with the EMT biomarker EpCAM protein, but the effect of combination with the biomarker cannot be intuitively obtained only by adopting the aptamer.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides a screening probe for inducing a tumor cell epithelial-mesenchymal transition drug and a preparation method thereof.

The purpose of the invention is realized by the following technical scheme:

a screening probe for inducing the epithelial-mesenchymal transition of tumor cells features that the 5' end of aptamer SYL3C is modified

A probe (designated as TPE-SYL3C-FAM) formed by 6-Carboxyfluorescein (6-Carboxyfluorescein, 6-FAM) is modified at the 3' end of the aptamer SYL 3C.

Specifically, the screening probe is obtained by modifying amino at the 5 'end of aptamer SYL3C and modifying 6-carboxyfluorescein at the 3' end to obtain H₂N-SYL 3C-FAM; then H is added₂N-SYL 3C-a probe obtained by modifying Tetraphenylethylene (TPE) at the 5' end of FAM.

Those skilled in the art can base their general knowledge on H₂N-SYL3C-FAM is an amino group modified at the 5 'end and a 6-FAM modified at the 3' end of an aptamer SYL 3C.

Another objective of the present invention is to provide a method for preparing the screening probe for the drug for inducing epithelial-mesenchymal transition of tumor cells, comprising: using a mixed solvent of water and DMSO as a reaction solvent, H₂N-SYL3C-FAM and [1- (4-carboxyphenyl) -1,2, 2-triphenyl]And (3) reacting ethylene N-hydroxysuccinimide ester to obtain the probe TPE-SYL 3C-FAM.

Said H₂N-SYL3C-FAM and [1- (4-carboxyphenyl) -1,2, 2-triphenyl]The mass ratio of the ethylene N-hydroxysuccinimide ester is 2: 1-5: 1.

The reaction temperature is 4-37 ℃, and the reaction time is 12-24 h.

After the reaction is finished, the reaction solution is filled into a 3kD ultrafiltration centrifugal tube with the molecular weight cut-off, and the probe TPE-SYL3C-FAM is obtained by ultrafiltration and centrifugation.

Specifically, the preparation method of the screening probe for the drug for inducing epithelial-mesenchymal transition of the tumor cells comprises the following steps: the preparation concentration is 5-10.5 mg/mL^-1H of (A) to (B)₂N-SYL3C-FAM in aqueous solution, as per H₂N-SYL3C-FAM and [1- (4-carboxyphenyl) -1,2, 2-triphenyl]The mass ratio of the ethylene N-hydroxysuccinimide ester is 2: 1-5: 1, and the addition concentration is 20 mg/mL^-1Is [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] s]And (3) carrying out stirring reaction on a DMSO solution of ethylene N-hydroxysuccinimide ester, carrying out ultrafiltration and centrifugation on the reaction solution to obtain a probe TPE-SYL3C-FAM, and dispersing the obtained probe TPE-SYL3C-FAM by using a PBS buffer solution.

A method for preparing [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] ethylene N-hydroxysuccinimide ester, comprising the steps of:

dissolving triphenylethylene bromide, 4-ethoxycarbonylphenylboronic acid, potassium carbonate, tetrakis (triphenylphosphine) palladium and tetra-n-octylammonium bromide in a toluene-ethanol-water mixed solvent, and reacting under the protection of nitrogen to obtain 4- (1,2, 2-triphenylethylene) ethyl benzoate;

dissolving ethyl 4- (1,2, 2-triphenylethylene) benzoate and lithium hydroxide monohydrate in a mixed solvent of water and tetrahydrofuran, and reacting under the protection of nitrogen to obtain 1- (4-carboxybenzene) -1,2, 2-triphenylethylene;

and (3) dissolving pyridine, 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene and N, N' -disuccinimidyl carbonate in acetonitrile, and reacting to obtain [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] ethylene N-hydroxysuccinimide ester.

In the step (1), the molar ratio of triphenylbromoethylene to 4-ethoxycarbonylphenylboronic acid is 1: 1.1-1: 2.4; the molar ratio of the triphenylbromoethylene to the potassium carbonate is 1: 12-1: 26; the molar ratio of the triphenylbromoethylene to the tetrakis (triphenylphosphine) palladium to the tetra-n-octylammonium bromide is 1 (0.04-0.1) to (0.08-0.2).

The toluene-ethanol-water mixed solvent is prepared from toluene, ethanol and water according to a volume ratio of (4-5) to 1:1.

The reaction temperature is 90-120 ℃, and the reaction time is 12-48 h.

After the reaction is finished, dichloromethane is used for extraction, the organic solvent is evaporated in a rotary mode, petroleum ether and dichloromethane are used as eluent, and the ethyl 4- (1,2, 2-triphenylvinyl) benzoate is obtained through flash column chromatography purification.

In the step (2), the molar ratio of the ethyl 4- (1,2, 2-triphenylvinyl) benzoate to the lithium hydroxide monohydrate is 1: 30-62; the volume ratio of the water to the tetrahydrofuran is 1:1.

The reaction temperature is 90-120 ℃, and the reaction time is 24-48 h.

After the reaction is finished, dichloromethane is used for extraction, the organic solvent is evaporated in a rotary mode, ethyl acetate and petroleum ether are used as eluent, and the mixture is purified through flash column chromatography to obtain 1- (4-carboxybenzene) -1,2, 2-triphenylethylene.

In the step (3), the molar ratio of the pyridine to the 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene to the N, N' -disuccinimidyl carbonate is 1 (0.2-1) to 0.2-0.8.

The reaction temperature is 60-90 ℃, and the reaction time is 2-12 h.

After the reaction is finished, ethyl acetate is extracted, the organic solvent is evaporated, and the [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] ethylene N-hydroxysuccinimide ester is obtained by flash column chromatography purification with ethyl acetate and petroleum ether as eluent in a ratio of 1:10(v: v).

The probe TPE-SYLC-FAM provided by the invention is used as a washing-free probe, and meets the requirement of high-throughput drug screening. The working principle of the probe TPE-SYL3C-FAM (figure 2) is as follows: fluorescence of TPE is dependent on aggregation-induced emission (AIE). The probe TPE-SYL3C-FAM designed based on the AIE principle has almost no fluorescence emission in a dispersed state, and can emit strong and stable fluorescence in an aggregated or solid state. The probe TPE-SYL3C-FAM takes EpCAM which is expressed and reduced in the epithelial-mesenchymal transition process as a target, a specific aptamer SYL3C is used for identifying the target, the local concentration of the probe near the target is induced to increase, and the AIE performance of Tetraphenylethylene (TPE) in the probe is used for inducing the probe to emit fluorescence. Meanwhile, in order to reduce the interference of environmental factors on the fluorescence intensity, an internal standard group FAM in the probe TPE-SYL3C-FAM is used as an internal standard signal, the fluorescence intensity is constant, the interference of the environmental factors (such as light quenching, instrument parameter fluctuation or excessive probe local concentration) can be avoided, the FAM is used for normalizing the TPE signal, the ratio of the TPE fluorescence intensity to the FAM fluorescence intensity is used as a fluorescence report value, and the accuracy and precision of the analysis method are improved.

The invention also aims to provide application of the screening probe TPE-SYL3C-FAM for inducing the tumor cell epithelial-mesenchymal transition drug in screening EMT-related drugs, and EMT-related drugs can be screened depending on the change of the fluorescence intensity of the probe.

The EMT related medicine is a medicine for inducing epithelial tumor cells to generate EMT side effects and a chemotherapy sensitizer for antagonizing the EMT process.

Specifically, the chemotherapeutic drug is paclitaxel, doxorubicin hydrochloride, gambogic acid, and cisplatin, but is not limited to the above drugs; the chemosensitizer is ferulic acid, bisdemethoxycurcumin, dihydroartemisinin, metformin and tetraethylthiuram disulfide, but is not limited to the chemosensitizer.

Screening chemotherapy drugs (namely epithelial-mesenchymal transition positive drugs) for inducing epithelial tumor cells to generate EMT side effects according to the decrease of cell viability normalized fluorescence intensity signals after administration: after incubation of tumor cells with different chemotherapeutic drugs, EpCAM protein on the cell surface was measured with TPE-SYL 3C-FAM. Due to the fact that target protein EpCAM is down-regulated in the EMT process, the aggregation degree of the probe is reduced, and the fluorescence intensity of TPE is down-regulated. Therefore, the EMT cells have a reduced fluorescence intensity compared to normal cells, and drugs that significantly reduce the fluorescence intensity of cells have the side effect of epithelial-mesenchymal transition.

The chemosensitizer antagonizes the EMT process (namely, the blocking of the epithelial-mesenchymal transition) and the TGF-beta as the epithelial-mesenchymal transition inducer are co-administered, the chemosensitizer is screened according to the recovery condition of the cell viability normalized fluorescence intensity signal after administration, and the fluorescence intensity of the chemosensitizer is higher than that of the TGF-beta group cells because the EMT passage is blocked by the chemosensitizer.

It is another object of the present invention to provide a method for screening an EMT-related drug using the screening probe,

when the EMT-related drug is a drug for inducing epithelial tumor cells to generate EMT side effects, the drug comprises the following components: inoculating passable tumor cells into black 96-well plate, placing at 37 deg.C and 5% CO₂Culturing for 24h in a cell culture box, removing the culture medium, adding a drug to be detected with the concentration of 5 mu M, incubating for 24h at 37 ℃, washing with PBS, adding 100 mu L of probe solution with the concentration of 10nM prepared by PBS buffer solution, and incubating; taking PBS buffer solution without the drug to be detected as a blank control group; the fluorescence intensity I is measured by a multifunctional microplate reader detector (lambda ex is 306nm, lambda em is 445nm)₄₄₅/I₅₂₄(ii) a Determining the cell activity by adopting an MTT method; in order to eliminate the interference of the cytotoxic action of the drug, the fluorescence intensities of the drug to be detected and the blank control group are normalized:

MTT normalized fluorescence intensity ═ fluorescence intensity/cell viability;

compared with a blank control group, if the MTT normalized fluorescence intensity of the drug to be detected is reduced, the drug to be detected induces the epithelial-derived cancer cells to generate EMT transformation.

When the EMT-related drug is a chemosensitizer that antagonizes the EMT process, it includes: inoculating passable tumor cells into black 96-well plate, placing at 37 deg.C and 5% CO₂Culturing for 24h, discarding the culture medium, starving the cells for 48h with a fetal calf serum-free culture medium, washing with PBS, and continuing to contain 5 ng/mL^-1TGF-beta 1 and 1-10 mu M of cells treated with a culture medium without fetal bovine serum containing the chemosensitizer to be tested for 48 hours to only contain 5 ng/mL^-1Culturing cells for 48h by using a TGF-beta 1 fetal calf serum-free culture medium as a control group; discarding the culture medium, washing with PBS, adding 100 μ L of 10nM probe solution prepared from PBS buffer solution, and incubating; the fluorescence intensity I is measured by a multifunctional microplate reader detector (lambda ex is 306nm, lambda em is 445nm)₄₄₅/I₅₂₄(ii) a Determining the cell activity by adopting an MTT method; in order to eliminate the interference of the cytotoxic action of the drug, the fluorescence intensity of the chemosensitizer to be detected and the control group is normalized:

MTT normalized fluorescence intensity ═ fluorescence intensity/cell viability;

compared with the control group, if the MTT normalized fluorescence intensity of the chemosensitizer to be detected is increased, the chemosensitizer to be detected antagonizes the EMT process.

The tumor cells are A549 cells and HepG2 cells. The culture medium of the A549 cells is a DMEM culture medium containing 10% fetal calf serum, and the culture medium of the HepG2 cells is an RPMI-1640 culture medium containing 10% fetal calf serum.

The incubation condition of the A549 tumor cells is 10nM TPE-SYL3C-FAM incubation for 15min at room temperature; the incubation condition of HepG2 tumor cells is 10nM TPE-SYL3C-FAM incubation for 15min at 4 DEG C

The drug to be detected is prepared by PBS buffer solution.

The method for measuring the cell viability by the MTT method comprises the following steps: after the fluorescence intensity was measured, the 96-well plate was removed, and 100. mu.L of 500. mu.g/mL was added to each well in the dark^-1MTT solution at 37 deg.C with 5% CO₂The cell culture box is cultured for continuous dark incubation for 4h, liquid in each hole is discarded, 100 mu L of DMSO is added into each hole, the absorbance of each hole is measured at 492nm by adopting an enzyme labeling instrument, and the cell activity is calculated:

cell survival (%) — average absorbance of administration group/average absorbance of control group × 100.

Drawings

FIG. 1: screening probe TPE-SYL3C-FAM synthetic scheme.

FIG. 2: a functional diagram of the screening probe TPE-SYL 3C-FAM.

FIG. 3: mass spectrum (A) of ethyl 4- (1,2, 2-triphenylvinyl) benzoate and¹H-NMR spectrum (B).

FIG. 4: mass spectrum (A) of 1- (4-carboxylbenzene) -1,2, 2-triphenylethylene and¹H-NMR spectrum (B).

FIG. 5: [1- (4-carboxyphenyl) -1,2, 2-triphenyl radical]Mass spectrum of ethylene N-hydroxysuccinimide ester (A) and¹H-NMR spectrum (B).

FIG. 6: TPE-SYL3C-FAM ultraviolet-visible spectrum (A) and fluorescence spectrum (B).

FIG. 7: TPE-SYL3C-FAM grafting standard curve.

FIG. 8: TPE-SYL3C-FAM performance characterization: water solubility (A), different proportions of tetrahydrofuran-water fluorescence spectrogram (B), different proportions of tetrahydrofuran-water maximum fluorescence intensity (C), and 99% of TPE-SYL3C-FAM particle size (D) in tetrahydrofuran water solution.

FIG. 9: TPE-SYL3C-FAM quantitatively identified a standard curve for EpCAM protein.

FIG. 10: fluorescence stability (A), high salt concentration stability (B), oxidation/reduction environmental stability (C) and acid-base environmental stability (D) of TPE-SYL 3C-FAM.

FIG. 11: TPE-SYL3C-FAM were incubated in A549 cells at 4 ℃ for 15, 30 and 60min (A-C), respectively; TPE-SYL3C-FAM was incubated in A549 cells at room temperature for 15, 30 and 60min (D-F), respectively; TPE-SYL3C-FAM were incubated in A549 cells at 37 ℃ for 15, 30 and 60min (G-I), respectively; TPE-SYL3C-FAM were incubated in HepG2 cells at 4 ℃ for 15, 30 and 60min (J-K), respectively; TPE-SYL3C-FAM was incubated in HepG2 cells at room temperature for 15, 30 and 60min (M-O), respectively; TPE-SYL3C-FAM was incubated in HepG2 cells at 37 ℃ for 15, 30 and 60min (P-R), respectively.

FIG. 12: cytotoxicity assay of probes: effect of different concentrations of probe on cell viability of HepG2 cells and a549 cells (a); selecting the effect of the probe on HepG2 cytotoxicity under experimental conditions (B); effect of the probe on a549 cytotoxicity under the selected experimental conditions (C).

FIG. 13: the ability of the probe to target recognition of a549 cells.

FIG. 14: the probe targets the ability to recognize HepG2 cells.

FIG. 15: the probe targets the ability to recognize HEK-293T cells.

FIG. 16: the probe recognizes the capacity of EMT to transform HepG2 cells (A) and A549 cells (B).

FIG. 17: TPE-SYL3C-FAM is used for screening A549 cells (A) and HepG2 cells (B) for chemotherapeutic drugs for inducing epithelial tumor cells to generate EMT process.

FIG. 18: TPE-SYL3C-FAM is used for screening A549 cells (A) and HepG2 cells (B) for chemotherapeutic sensitizers for blocking EMT processes.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the drawings and the specific embodiments, but should not be construed as limiting the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

The preparation method of the screening probe TPE-SYL3C-FAM for inducing the epithelial-mesenchymal transition drug of the tumor cells is shown in figure 1, and the steps are as follows:

step (1), dissolving triphenylbromoethylene (700mg), 4-ethoxycarbonylphenylboronic acid (450mg), potassium carbonate (3.6g), tetrakis (triphenylphosphine) palladium (120mg) and tetra-n-octylammonium bromide (100mg) in a toluene-ethanol-water (4:1:1, v: v: v) mixed solvent (60mL), heating and refluxing at 90 ℃ for 12h under nitrogen protection, extracting with 40mL of dichloromethane for 3 times, combining dichloromethane phases, performing rotary evaporation, and purifying by flash column chromatography (eluent is petroleum ether: dichloromethane: 1:4, v: v), to obtain ethyl 4- (1,2, 2-triphenylvinyl) benzoate (yield 86.1%); FIG. 3 shows the structural characterization of ethyl 4- (1,2, 2-triphenylvinyl) benzoate, [ M + Na]⁺Precise theoretical molecular weight m/z 427.1674, found m/z 427.1675, molecular formula: c₂₉H₂₄O₂；

Step (2), dissolving ethyl 4- (1,2, 2-triphenylvinyl) benzoate (400mg), lithium hydroxide monohydrate (1.26g) and water (20mL) in tetrahydrofuran (20mL), heating and refluxing at 90 ℃ for 36h under the protection of nitrogen, cooling the reaction solution to room temperature, adjusting the pH of the reaction solution to 4.0 with 1M HCl, extracting 3 times with 20mL dichloromethane, combining dichloromethane phases, performing rotary evaporation, and purifying by flash column chromatography (eluent is ethyl acetate: petroleum ether ═ 1:8, v: v) to obtain 1- (4-carboxybenzene) -1,2, 2-triphenylethylene (yield 43.8%); FIG. 4 shows the structural characterization results of 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene, [ M + H ]]⁺Precise theoretical molecular weight m/z 377.1542, found m/z 377.1534, molecular formula: c₂₇H₂₁O₂；

Step (3) pyridine (250. mu.L), 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene (250mg) and N, N' -disuccinimidylAminocarbonate (200mg) was dissolved in acetonitrile (50mL), heated at 85 ℃ under reflux for 3h, extracted 3 times with 25mL ethyl acetate, the ethyl acetate phases combined, rotary evaporated and purified by flash column chromatography (eluent ethyl acetate: petroleum ether: 1:10, v: v) to give [1- (4-carboxyphenyl) -1,2, 2-triphenyl-phenyl ] -1,2, 2-triphenyl-carbonate]Ethylene N-hydroxysuccinimide ester (yield 71.4%); FIG. 5 is [1- (4-carboxyphenyl) -1,2, 2-triphenyl)]Structural characterization of ethylene N-hydroxysuccinimide ester, [ M + Na ]]⁺Precise theoretical molecular weight m/z 496.1525, found m/z 496.1520, molecular formula: c₃₁H₂₄NO₄；

Step (4) of adding H₂N-SYL3C-FAM lyophilized powder (14OD, note: 1OD ssDNA ═ 33. mu.g ssDNA) was dissolved in 45. mu.L of water to give 10.27 mg. multidot.mL^-1H of (A) to (B)₂N-SYL3C-FAM solution of H₂N-SYL3C-FAM solution and 5. mu.L of 20 mg/mL^-1Is [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] s]Mixing ethylene N-hydroxysuccinimide ester in DMSO solution, stirring at 4 deg.C overnight, loading the reaction solution into ultrafiltration centrifuge tube (0.5mL, cut-off molecular weight 3kD), ultrafiltering at 15000rpm for 20min, and dispersing the precipitate with 100 μ L of PBS buffer solution with pH of 7.4 to obtain 4.34 mg/mL^-1The yield of TPE-SYL3C-FAM was about 94% from the TPE-SYL3C-FAM probe solution.

FIG. 6 shows the structural characterization results of TPE-SYL 3C-FAM. In the UV-visible spectrum (FIG. 6A), the UV absorption peak of the nucleotide and TPE molecules in SYL3C-FAM can be observed, and the UV absorption peak of the internal standard group FAM in SYL3C-FAM can be observed in the small graph; in the fluorescence spectrum (fig. 6B), the fluorescence emission peaks of FAM and TPE were observed, but the maximum emission wavelength of TPE was blue-shifted.

Taking [1- (4-carboxyl phenyl) -1,2, 2-triphenyl]And adding a proper amount of ethylene N-hydroxysuccinimide ester into DMSO to dissolve the ethylene N-hydroxysuccinimide ester to obtain a probe stock solution. The probe stock solution was diluted with water to obtain working solutions of 0.5, 1, 5, 10, 25, 50. mu.M concentration series. In [1- (4-carboxyphenyl) -1,2, 2-triphenyl]Measuring the absorbance of ethylene N-hydroxysuccinimide ester at a maximum absorption wavelength of 319nm, performing regression calculation on the absorbance A and the concentration C, and fitting [1- (4-carboxyphenyl) -1,2, 2-triphenyl ester]Regression equation of concentration-absorbance of ethylene N-hydroxysuccinimide esterTo obtain the regression equation (fig. 7): a ═ 0.0103C-0.0172 (R)²0.9986). And substituting the absorbance A of the TPE-SYL3C-FAM under the wavelength of 319nm into a regression equation to obtain the grafting rate of the TPE-SYL3C-FAM, which is 94.16%.

To characterize the water solubility of TPE-SYL3C-FAM, the fluorescence intensity of TPE in water and Tetrahydrofuran (THF) was measured separately. Taking appropriate amount of TPE, preparing into solution with appropriate concentration with pure water or pure tetrahydrofuran, and measuring fluorescence intensity under different emission wavelengths by fluorescence emission spectrometry, the result is shown in FIG. 8A (THF-TPE represents tetrahydrofuran as solvent, H₂O-TPE means that the solvent is water). The fluorescence intensity of the TPE in pure water is strong, which indicates that the TPE is in an aggregation state in the water, and a intramolecular movement Restriction (RIM) passage is opened; the fluorescence intensity of TPE in THF is weak, indicating that TPE is dispersed in THF.

To characterize the AIE performance of TPE-SYL3C-FAM, the 10. mu.L concentration from step (4) was taken as 4.34 mg. multidot.mL^-1TPE-SYL3C-FAM Probe solution, 400. mu.L of tetrahydrofuran/water (THF/H) in different volume ratios₂O) mixed solvents, and optical characteristics of the probes in different solvents were measured by fluorescence emission spectroscopy using fluorescence intensities at different emission wavelengths, and the results are shown in fig. 8B. Water is a benign solvent for TPE-SYL3C-FAM, and THF is a poor solvent for TPE-SYL 3C-FAM. THF/H in various ratios₂O may cause TPE-SYL3C-FAM to aggregate to varying degrees: the larger the water content is, the better the dispersibility of TPE-SYL3C-FAM is, and the weaker the fluorescence intensity is; the larger the THF content, the poorer the dispersibility of TPE-SYL3C-FAM and the stronger the fluorescence intensity. To sum up, NH is described₂And (2) the connection between the-SYL 3C-FAM and the TPE is successful, and the TPE-SYL3C-FAM is changed from fat solubility to water solubility, so that the solubility inversion is realized, and the target detection is facilitated.

The maximum fluorescence intensity of the probe TPE-SYL3C-FAM in different volume ratios of tetrahydrofuran/water solution is shown in FIG. 8C. The fluorescence intensity of TPE-SYL3C-FAM is almost unchanged when the THF proportion is lower than 40%, which shows that the probe is well dispersed in the solvent at the moment, and the energy is freely and rotatably dissipated by covalent single bonds; when the THF ratio exceeds 40%, the fluorescence intensity of the probe gradually increases, and the fluorescence intensity of the probe is maximized at a THF ratio of 99%. TPE-SYL3C-FAM has larger Stokes shift (139 nm, 306nm and 445nm) in aqueous solution, and can reduce the self-absorption of the probe in the water environment.

To further verify the AIE performance of TPE-SYL3C-FAM, the particle size of the probes in different solvents was characterized by a Malvern particle size and Dynamic Light Scattering (DLS) function of a potentiometric analyzer. The particle size of the probe was determined in water and 99% THF at room temperature, respectively. TPE-SYL3C-FAM formed particles with a particle size of 316.5nm in 99% THF (FIG. 8D), while the DLS signal of TPE-SYL3C-FAM could not be detected in water, indicating that the probe emits fluorescence due to aggregation, further illustrating the AIE effect of the probe.

Dissolving 50 mu g of epithelial cell adhesion molecules (EpCAM) by using 1mL of PBS buffer solution to obtain an EpCAM stock solution; diluting EpCAM stock solution step by PBS buffer solution to obtain series concentration (0-300 ng. mL)^-1) EpCAM working solution of (1). And (3) diluting a proper amount of the TPE-SYL3C-FAM probe prepared in the step (4) with PBS to prepare a 20nM probe solution, mixing 100 mu L of the probe solution with 400 mu L of series EpCAM working solutions with concentration to obtain a buffer solution with the probe concentration of 4nM, incubating for 1h at 4 ℃, setting emission wavelengths to be 445nM and 524nM respectively by a fluorescence emission spectrometry, and measuring the fluorescence intensity. As shown in FIG. 9A, the fluorescence intensity of the TPE module in the probe was positively correlated to the EpCAM concentration at an emission wavelength of 445nm (R²0.9776 EpCAM concentration vs. I_445nmPlotted). Emission wavelengths are respectively set to 445nm and 524nm, the linear correlation coefficient of the fluorescence intensity of TPE and the EpCAM concentration can be known to be better by adopting an internal standard normalization method, and R²0.9917 (fig. 9B, EpCAM concentration vs. I_445nm/I_524nmPlotted). Therefore, the method facilitates the quantitative detection of EpCAM protein in a dose-dependent manner by adopting an internal standard method, and can be further used for evaluating the expression of EpCAM in cells and screening EMT related drugs.

The probe TPE-SYL3C-FAM was dissolved in buffer media of different pH or different composition to evaluate its fluorescence and structural stability. And (3) taking the TPE-SYL3C-FAM solution (10 mu L) in the step (4), adding solutions with different ionic strengths (0, 0.5, 1 and 2M sodium chloride solutions), an oxidizing solvent (30% hydrogen peroxide), a reducing solvent (0.1M ascorbic acid) and PBS solutions (pH3-10) (each 400 mu L) with different pH values, incubating for 30min at room temperature, and measuring the fluorescence intensity by a fluorescence emission spectroscopy method. As a result, as shown in FIG. 10, the fluorescence intensity of TPE-SYL3C-FAM dispersed in PBS buffer solution with pH 7.4 is almost unchanged after continuous excitation for 30min at the emission wavelength of 445nm (FIG. 10A), which shows that the fluorescent probe designed based on AIE has stronger light stability than the traditional probe; the fluorescence intensity of the probe TPE-SYL3C-FAM is not changed obviously after the probe TPE-SYL3C-FAM is incubated for 30min under different physiological conditions, such as a series of ionic strength aqueous solutions (0-2M NaCl, figure 10B), an oxidizing solvent (30% hydrogen peroxide), a reducing solvent (0.1M ascorbic acid) (figure 10C) and different pH environments (pH3-10, figure 10D), which shows that the probe TPE-SYL3C-FAM can overcome various physiological barriers and keep the structure and fluorescence stability.

Cell culture conditions: human non-small cell lung cancer cell line (A549), human liver cancer cell line (HepG2), and human kidney epithelial cell line (HEK-293T) were purchased from the China center for type culture Collection. A549 cells and HEK-293T cells adopt DMEM culture medium containing 10% Fetal Bovine Serum (FBS), HepG2 cells adopt RPMI-1640 culture medium containing 10% Fetal bovine serum, and the culture conditions are as follows: placing at 37 deg.C with 5% CO₂Culturing in a cell culture box, and carrying out passage after digestion when the cell grows to be 80% over the wall of a cell culture bottle.

To improve the sensitivity of the probe to detect EpCAM in the cells, the conditions of the probe experiment were optimized for HepG2 and A549 cells, including probe concentration (0, 1, 10, 50, 100, 200nM), incubation time (15, 30, 60min) and incubation temperature (4 deg.C, room temperature, 37 deg.C). The specific operation is as follows: HepG2 and A549 cells were inoculated into black 96-well plates (5000 cells/well), respectively, and placed at 37 ℃ with 5% CO₂Culturing in a cell culture box, and removing the culture medium after 24 hours; starving the cells for 48h in a fetal calf serum-free medium, and washing 1 time with PBS; and (3) diluting the TPE-SYL3C-FAM probe solution prepared in the step (4) with a PBS buffer solution to prepare probe solutions with different concentrations, respectively adding tumor cells for incubation, and observing the cell surface target recognition of the fluorescent probe by using a fluorescent microscope with a GFP channel. The results are shown in FIG. 11: (1) the concentration of the probe: in A549 and HepG2 cells, the fluorescence intensity at 4 ℃ and 37 ℃ is increased along with the increase of the concentration of TPE-SYL3C-FAMThe fluorescence intensity was gradually increased, but both fluorescence intensities reached a maximum at 10nM and 50nM, respectively, at room temperature. (2) Incubation temperature: in the HepG2 cell line, the fluorescence intensity is higher than other temperatures at 4 ℃; in the A549 cell line, the fluorescence intensity was maximal at room temperature. (3) Incubation time: the fluorescence intensity of the A549 cell line is not obviously changed within 60min, and the fluorescence intensity is slightly reduced for the HepG2 cell line. Therefore, the incubation condition of the A549 cell line is 10nM TPE-SYL3C-FAM incubation for 15min at room temperature; the incubation condition of the HepG2 cell line was 10nM TPE-SYL3C-FAM incubated at 4 ℃ for 15 min. The fluorescent responses of the A549 and the HepG2 cells at 4 ℃ and 37 ℃ are concentration-dependent, namely the higher the concentration of the probe is, the larger the fluorescent output value is; whereas the fluorescence intensities of A549 and HepG2 cells reached maximum values at 10nM and 50nM, respectively, at room temperature, which may be due to the interaction of probe concentration and cellular uptake.

The biocompatibility of TPE-SYL3C-FAM with HepG2 or A549 cell lines was evaluated by MTT method. Inoculating the cells into a 96-well plate (5000 cells/well), adding 150 μ L of probe solution with corresponding concentration into each well after 80% fusion (taking TPE-SYL3C-FAM probe solution prepared in step (4), diluting with PBS buffer solution to prepare probe solution with concentration of 0, 1, 10, 50, 100, 200nM), standing at 37 deg.C, and adding 5% CO₂The cell culture box is continuously incubated for 24 hours; the 96-well plate was removed, and 500. mu.g/mL of 100. mu.L was added to each well in the dark^-1MTT solution at 37 deg.C with 5% CO₂The cell culture box is continuously incubated for 4 hours in a dark place, liquid in each hole is discarded, 100 mu L of DMSO is added into each hole, the absorbance of each hole is measured by a microplate reader at 492nm, and the survival rate of the cells is calculated:

cell survival rate (%). ratio of average absorbance in administration group/average absorbance in control group. times.100

Fig. 12A shows: after 24h incubation, the cell activity of the two cells is more than 70% under all the measurement concentrations of the probe, which shows that TPE-SYL3C-FAM has good biocompatibility and is suitable for biosensing.

Considering that the optimal incubation condition of the A549 cell line is 10nM TPE-SYL3C-FAM incubation for 15min at room temperature; the optimal incubation condition for the HepG2 cell line was 10nM TPE-SYL3C-FAM incubation at 4 ℃ for 15 min. The toxicity of the probe under the optimal incubation conditions for the drug was evaluated. The results are shown in FIGS. 12B, 12C, indicating that: under the working condition of the probe (the concentration of the probe is 10nM, the action time is 15min), compared with the control group, the activity of HepG2 and A549 cells is not statistically different, which indicates that the probe is not cytotoxic under the working condition.

HepG2, A549 and HEK-293T cells were seeded in 6-well plates (1.0X 10, respectively)⁵Cells/well), culturing overnight at 37 ℃, adding probe solutions with a series of concentrations (taking the TPE-SYL3C-FAM probe solution prepared in the step (4) to dilute with PBS buffer solution to prepare probe solutions with concentrations of 0, 1, 10, 50, 100 and 200nM respectively), incubating for 1h at 4 ℃, and washing with PBS buffer solution. Images were acquired using a nikon fluorescence microscope equipped with 4', 6-diamidino-2-phenylindole (DAPI) and texas red filters. All images were evaluated for target recognition ability of the probe with the same 100 μm scale using fluorescence microscopy. The results are shown in FIGS. 13-15. As can be seen from FIG. 13, the fluorescence intensity of the A549 cell results gradually increased with increasing probe concentration, indicating that TPE-SYL3C-FAM targets EpCAM-positive A549 cells. As can be seen in FIG. 14, the fluorescence intensity of the results of HepG2 cells gradually increased with increasing probe concentration, indicating that TPE-SYL3C-FAM targets EpCAM positive cells HepG2 cells. As can be seen from FIG. 15, no significant fluorescence was observed at the probe concentration of 0-200nM in HEK293T cells, indicating that TPE-SYL3C-FAM could not target the HEK-293T cells, which are EpCAM negative cells, demonstrating the specificity of the probe.

To evaluate whether the probe can detect EMT transformed cells, TGF-beta 1 is used for inducing EMT of HepG2 and A549 cells, and then the probe is added for incubation to observe the fluorescence intensity. The specific operation is as follows: HepG2 or A549 cells were seeded in black 96-well plates (5000 cells/well) and placed at 37 ℃ with 5% CO₂Culturing in the cell culture box, removing culture medium after 24 hr, starving the cells with fetal calf serum-free culture medium for 48 hr, washing with PBS for 1 time, and continuing to contain 5 ng/mL^-1Cells were treated for 48h with TGF- β 1 in fetal bovine serum free medium to induce EMT of the cells; the medium was discarded, washed 1 time with PBS, and 150. mu.L of 10nM TPE-SYL3C-FAM in PBS was added at the optimum temperature (optimum temperature for A549)Incubating at room temperature and optimum temperature of HepG2 of 4 deg.C for 15min, and detecting fluorescence intensity with multifunctional microplate reader (λ ex ═ 306nm, λ em ═ 445 nm); as a control, TGF-. beta.1 was not added. The result is shown in figure 16, because the level of EpCAM protein on the cell surface is down-regulated after the EMT of HepG2 and A549 cells is induced by TGF-beta 1, the fluorescent intensity of the probe is reduced as shown by the result of detecting the level of EpCAM protein on the cell surface by TPE-SYL3C-FAM, and the effectiveness of the probe in identifying the EMT cells is verified.

HepG2 or A549 cells were seeded in black 96-well plates (5000 cells/well) and placed at 37 ℃ with 5% CO₂Culturing for 24h in a cell culture box, removing the culture medium for screening chemotherapeutic drugs which can induce EMT of epithelial tumor cells, adding serial concentrations (concentration of 0.01, 0.1, 1 and 5 mu M) of drugs to be tested prepared by PBS buffer solution: paclitaxel (Paclitaxel), Doxorubicin hydrochloride (Doxorubicin), Gambogic acid (Gambogic acid) and Cisplatin (cissplatin), taking PBS buffer solution without drug as blank control, incubating at 37 ℃ for 24h, adding 100 μ L of TPE-SYL3C-FAM PBS solution with concentration of 10nM, incubating at optimum temperature (the optimum temperature of A549 is room temperature, the optimum temperature of HepG2 is 4 ℃) for 15min, and detecting fluorescence intensity (λ) with multifunctional microplate reader_ex＝306nm，λ_em445 nm). Since chemotherapeutic drugs are cytotoxic to tumor cells and can reduce cell numbers, the change in fluorescence intensity of the probe may result from two aspects: (1) a decrease in cell number results in a decrease in EpCAM protein concentration; (2) EpCAM protein was down-regulated during EMT.

In order to eliminate the interference of the cytotoxic effect of the antitumor drug, the fluorescence intensity of each administration group is normalized:

MTT normalized fluorescence intensity ═ fluorescence intensity/cell viability

As shown in fig. 17, the fluorescence intensity of the probe decreased with the increase of the dose by normalizing the fluorescence result of TPE with MTT result using 4 chemotherapeutics causing EMT of epithelial tumor cells, such as paclitaxel, doxorubicin hydrochloride, gambogic acid and cisplatin, as model drugs.

HepG2 or A549 cells were seeded in black 96-well plates (5000 cells/well) and placed at 37 ℃ with 5% CO₂In a cell culture incubatorCulturing for 24h, removing culture medium to screen chemosensitizer capable of reversing EMT process, starving cells for 48h with fetal calf serum-free culture medium, washing with PBS buffer solution for 1 time, and adding TGF-beta 1 (final concentration 5 ng. mL)^-1) And the drug to be tested at the series of concentrations ( final concentration 1, 5, 10 μ M) formulated in PBS: ferulic Acid (Ferulic Acid), Bisdemethoxycurcumin (Bisdeoxyucucuculin), Dihydroartemisinin (dihydroartemisinine), Metformin (Methformin) or tetraethylthiuram disulfide (Disulfiam) in fetal calf serum-free medium treated for 48h to contain TGF-. beta.1 alone (final concentration 5 ng. mL)^-1) The cells cultured by the fetal calf serum-free culture medium for 48 hours are TGF-beta groups, and the cells cultured by the fetal calf serum-free culture medium without TGF-beta 1 and the drug to be detected for 48 hours are blank control groups; discarding the culture medium, washing with PBS for 1 time, adding 100 μ L of 10nM PBS solution of TPE-SYL3C-FAM, incubating at the optimum temperature (the optimum temperature of A549 is room temperature, the optimum temperature of HepG2 is 4 deg.C) for 1h, and detecting the fluorescence intensity (λ) with multifunctional microplate reader_ex＝306nm，λ_em445 nm). The results are shown in figure 18, 5 EMT chemosensitizers such as ferulic acid, bisdemethoxycurcumin, dihydroartemisinin, metformin or tetraethylthiuram disulfide are taken as model drugs, TGF-beta 1 is respectively combined with the 5 drugs, the fluorescence intensity of the probe is gradually recovered along with the increase of the dosage, and the probe is proved to be capable of effectively identifying the chemosensitizer of EMT.

Example 2

Step (1), triphenylbromoethylene (700mg), 4-ethoxycarbonylphenylboronic acid (700mg), potassium carbonate (5.4g), tetrakis (triphenylphosphine) palladium (120mg) and tetra-n-octylammonium bromide (100mg) were dissolved in a toluene-ethanol-water (5:1:1, v: v: v) mixed solvent (60mL), heated under reflux at 100 ℃ under nitrogen for 24 hours, extracted with dichloromethane for 3 times, and then purified by flash column chromatography (eluent petroleum ether: dichloromethane ═ 1:4, v: v) to give ethyl 4- (1,2, 2-triphenylvinyl) benzoate.

Dissolving 4- (1,2, 2-triphenylvinyl) ethyl benzoate (400mg), lithium hydroxide monohydrate (1.89g) and water (30mL) in tetrahydrofuran (30mL), and heating and refluxing for 24h at 100 ℃ under the protection of nitrogen; the reaction was cooled to room temperature, the pH of the reaction was adjusted to 4.0 with 1M HCl, extracted 3 times with dichloromethane, and purified by flash column chromatography (eluent ethyl acetate: petroleum ether ═ 1:8, v: v) to give 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene;

step (3), pyridine (80 μ L), 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene (340mg) and N, N' -disuccinimidyl carbonate (200mg) were dissolved in acetonitrile (80mL), heated under reflux at 90 ℃ for 2h, extracted with ethyl acetate 3 times, and purified by flash column chromatography (eluent ethyl acetate: petroleum ether ═ 1:10, v: v) to give [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] ethylene N-hydroxysuccinimide ester;

step (4) of adding H₂N-SYL3C-FAM lyophilized powder (14OD) was dissolved in water (70. mu.L), and [1- (4-carboxyphenyl) -1,2, 2-triphenyl) was added]DMSO solution of ethylene N-hydroxysuccinimide ester (20 mg. mL)^-110 μ L) was stirred at room temperature overnight, the reaction solution was put into an ultrafiltration centrifugal tube (0.5mL, cut-off molecular weight 3kD), and subjected to ultrafiltration centrifugation at 15000rpm to obtain TPE-SYL3C-FAM as a probe, which was dispersed in PBS buffer.

Example 3

Step (1), dissolving triphenylbromoethylene (700mg), 4-ethoxycarbonylphenylboronic acid (900mg), potassium carbonate (5g), tetrakis (triphenylphosphine) palladium (240mg) and tetra-n-octylammonium bromide (200mg) in a mixed solvent (60mL) of toluene-ethanol-water (4:1:1, v: v: v), heating and refluxing at 120 ℃ for 48h under nitrogen protection, extracting with dichloromethane for 3 times, and purifying by flash column chromatography (eluent is petroleum ether: dichloromethane ═ 1:4, v: v) to obtain ethyl 4- (1,2, 2-triphenylvinyl) benzoate;

step (2), dissolving ethyl 4- (1,2, 2-triphenylvinyl) benzoate (400mg), lithium hydroxide monohydrate (2.52g) and water (40mL) in tetrahydrofuran (40mL), heating and refluxing at 120 ℃ for 48 hours under the protection of nitrogen, cooling the reaction solution to room temperature, adjusting the pH of the reaction solution to 4.0 with 1M HCl, extracting with dichloromethane for 3 times, and purifying by flash column chromatography (eluent is ethyl acetate: petroleum ether ═ 1:8, v: v) to obtain 1- (4-carboxybenzene) -1,2, 2-triphenylethylene;

step (3), pyridine (250 μ L), 1- (4-carboxyphenyl) -1,2, 2-triphenylethylene (380mg) and N, N' -disuccinimidyl carbonate (250mg) were dissolved in acetonitrile (100mL), heated under reflux at 90 ℃ for 12h, extracted with ethyl acetate 3 times and purified by flash column chromatography (eluent ethyl acetate: petroleum ether ═ 1:10, v: v) to give [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] ethylene N-hydroxysuccinimide ester;

step (4) of adding H₂Dissolving N-SYL3C-FAM (14OD) lyophilized powder in water (90 μ L), adding [1- (4-carboxyphenyl) -1,2, 2-triphenyl benzene)]DMSO solution of ethylene N-hydroxysuccinimide ester (20 mg. mL)^-110 μ L), stirring overnight at 4 deg.C, loading the reaction solution into an ultrafiltration centrifuge tube (0.5mL, cut-off molecular weight 3kD), ultrafiltering and centrifuging at 15000rpm to obtain TPE-SYL3C-FAM, and dispersing the probe with PBS buffer solution.

Claims (10)

1. A screening probe for inducing epithelial-mesenchymal transition drugs of tumor cells is characterized in that: the screening probe is modified at the 5' end of an aptamer SYL3C

6-carboxyfluorescein is modified at the 3' end of the aptamer SYL3C to form a probe TPE-SYL 3C-FAM.

2. The screening probe for drugs inducing epithelial-to-mesenchymal transition in tumor cells according to claim 1, wherein: the screening probe is obtained by modifying amino at the 5 'end of an aptamer SYL3C and modifying 6-carboxyfluorescein at the 3' end to obtain H₂N-SYL3C-FAM, then H₂The 5' amino group of N-SYL3C-FAM is modified with tetraphenylethylene to obtain the probe TPE-SYL 3C-FAM.

3. A method for preparing a screening probe for the drug inducing epithelial-to-mesenchymal transition in tumor cells according to claim 1, wherein the probe comprises: the method comprises the following steps: using a mixed solvent of water and DMSO as a reaction solvent, H₂N-SYL3C-FAM and [1- (4-carboxyphenyl) -1,2, 2-triphenyl]And reacting ethylene N-hydroxysuccinimide ester to obtain the probe.

4. The method for preparing the screening probe of the drug for inducing epithelial-to-mesenchymal transition in tumor cells according to claim 3, wherein the probe is prepared by a method comprisingIn the following steps: said H₂N-SYL3C-FAM and [1- (4-carboxyphenyl) -1,2, 2-triphenyl]The mass ratio of the ethylene N-hydroxysuccinimide ester is 2: 1-5: 1.

5. The method for preparing a screening probe for a drug that induces epithelial-to-mesenchymal transition in a tumor cell according to claim 3, wherein: the reaction temperature is 4-37 ℃, and the reaction time is 12-24 h.

6. The method for preparing a screening probe for a drug that induces epithelial-to-mesenchymal transition in a tumor cell according to claim 3, wherein: after the reaction is finished, the reaction solution is filled into an ultrafiltration centrifugal tube with the cut-off molecular weight of 3kD, and the probe is obtained by ultrafiltration and centrifugation.

7. A method for preparing a screening probe for the drug inducing epithelial-to-mesenchymal transition in tumor cells according to claim 1, wherein the probe comprises: the method comprises the following steps: the preparation concentration is 5-10.5 mg/mL^-1H of (A) to (B)₂N-SYL3C-FAM in aqueous solution, as per H₂N-SYL3C-FAM and [1- (4-carboxyphenyl) -1,2, 2-triphenyl]The mass ratio of the ethylene N-hydroxysuccinimide ester is 2: 1-5: 1, and the addition concentration is 20 mg/mL^-1Is [1- (4-carboxyphenyl) -1,2, 2-triphenyl ] s]And (3) stirring and reacting the ethylene N-hydroxysuccinimide ester in a DMSO solution, and performing ultrafiltration and centrifugation on the reaction solution to obtain the probe.

8. The use of the screening probe for drugs inducing epithelial-mesenchymal transition in tumor cells according to claim 1 for screening for EMT-related drugs.

9. Use according to claim 8, characterized in that: the EMT related medicine is a medicine for inducing epithelial tumor cells to generate EMT side effects and a chemotherapy sensitizer for antagonizing the EMT process.

10. A method for screening an EMT-related drug using the screening probe for an epithelial-to-mesenchymal transition-inducing drug for tumor cells according to claim 1, wherein:

when the EMT-related drug is a drug for inducing epithelial tumor cells to generate EMT side effects, the drug comprises the following components: inoculating passable tumor cells into black 96-well plate, placing at 37 deg.C and 5% CO₂Culturing for 24h in a cell culture box, discarding the culture medium, adding a drug to be tested with a concentration of 5 μ M, incubating for 24h at 37 ℃, washing with PBS, adding a probe solution of claim 1 prepared from PBS buffer with a concentration of 10nM, and incubating; taking PBS buffer solution without the drug to be detected as a blank control group; the fluorescence intensity I is measured by a multifunctional microplate reader detector (lambda ex is 306nm, lambda em is 445nm)₄₄₅/I₅₂₄(ii) a Determining the cell activity by adopting an MTT method; and (3) carrying out normalization treatment on the fluorescence intensity of the drug to be detected and the blank control group:

MTT normalized fluorescence intensity ═ fluorescence intensity/cell viability;

compared with a blank control group, if the MTT normalized fluorescence intensity of the drug to be detected is reduced, the drug to be detected induces the epithelial-derived cancer cells to generate EMT conversion;

when the EMT-related drug is a chemosensitizer that antagonizes the EMT process, it includes: inoculating passable tumor cells into black 96-well plate, placing at 37 deg.C and 5% CO₂Culturing for 24h, discarding the culture medium, starving the cells for 48h with a fetal calf serum-free culture medium, washing with PBS, and continuing to contain 5 ng/mL^-1TGF-beta 1 and 1-10 mu M of cells treated with a culture medium without fetal bovine serum containing the chemosensitizer to be tested for 48 hours to only contain 5 ng/mL^-1Culturing cells for 48h by using a TGF-beta 1 fetal calf serum-free culture medium as a control group; discarding the medium, washing with PBS, adding 10nM probe solution prepared from PBS buffer solution, and incubating; the fluorescence intensity I is measured by a multifunctional microplate reader detector (lambda ex is 306nm, lambda em is 445nm)₄₄₅/I₅₂₄(ii) a Determining the cell activity by adopting an MTT method; and (3) carrying out normalization treatment on the fluorescence intensity of the chemosensitizer to be detected and the control group:

MTT normalized fluorescence intensity ═ fluorescence intensity/cell viability;

compared with the control group, if the MTT normalized fluorescence intensity of the chemosensitizer to be detected is increased, the chemosensitizer to be detected antagonizes the EMT process.

Images