CN116359187A - Use of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione as fluorescent imaging agent - Google Patents
Use of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione as fluorescent imaging agent Download PDFInfo
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Abstract
The invention discloses a new application of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione, namely an application of the compound in the fields of fluorescence imaging reagent, namely that the compound is an androstane natural product from ciscens, has the advantages of sensitivity and rapidness in cell staining, small cytotoxicity, strong fluorescent signal, capability of in vivo imaging and the like, and has practical application value.
Description
Technical Field
The invention belongs to the technical field of biological analysis and detection, and particularly relates to application of a natural compound androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione in cell and animal living body fluorescence imaging.
Background
Along with the continuous deep research of biomedicine, a visual biological imaging technology plays an increasingly important role in the fields of life science and medicine, compared with other biological imaging technologies, fluorescence imaging has the characteristics of low cost and rapid imaging, has molecular-level sensitive single-molecule imaging, and can mark and trace the growth of tumors. However, the use of fluorescent dyes and quantum dots as fluorescent probes currently has some significant drawbacks, such as low light penetration depth, possible biological tissue destruction, and autofluorescence of biological samples, which limit their further application in the field of biological imaging. Currently, our knowledge is still limited in several ways: one is a biomarker suitable for imaging; secondly, selecting an imaging target and a contrast enhancement material; thirdly, chemical methods used to image probe biochemistry. In addition, many difficulties are also encountered in developing imaging agents, including: poor transport of probes targeting tissues or tumors; the biological toxicity is high; the stability of the probe is poor; low in vivo signal enhancement intensity, etc. Therefore, it is very important to develop a fluorescent imaging compound with strong fluorescent signal, high stability and low toxicity.
The Cinnamomum (Epigynum aurum) is one of Cinnamomum genus of oleaceae family, and is produced in southward of Yunnan province, and androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione is natural small molecular compound derived from Cinnamomum plant, and its molecular formula is C 19 H 18 O 3 Molecular weight 294.13, which belongs to the class of androstanes. Because of its small molecular weight and more double bonds in the structure, it is more easily passed through the cell barrier because of its good lipid solubility, but it has not been proposed to use it in the field of fluorescence imaging so far.
Disclosure of Invention
In order to solve the problems of the technology, the invention provides a new application of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione, namely an application of the androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione as a fluorescence imaging reagent in the field of cell or biological living body fluorescence imaging, wherein the chemical structural formula of the androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione is as follows:
the invention utilizes a fluorescence spectrophotometer to measure the optimum excitation wavelength of the compound to be 450nm and the optimum emission wavelength to be 550nm.
The application of the compound is verified by in vitro and in vivo experiments, in the in vitro experiments, a human epidermic cell line HaCat, a human acute lymphoblastic leukemia cell line BALL-1 and a solution of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione with a certain concentration are respectively adopted for mixed incubation, fluorescent signals of the compound under the wavelength of 450nm are detected, and the concentration is nontoxic to normal cells. The compound was intravenously injected into the tail of Balb/c nude mice for in vivo imaging experiments, and after 7 days, the brain, heart, liver, spleen, lung, kidney and testis of the Balb/c nude mice were not damaged by dissection. Based on the test results, the compound is shown to be applicable to the field of cell imaging and the field of biological living body imaging.
Compared with the prior art, the invention has the following advantages:
1. the invention develops a new biological application for the cismus monomer compound androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione, and opens up a new research field;
2. experiments show that the cissus mongolica monomer compound androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione can image normal cells and tumor cells at 60 mu mol/L, and has good inhibiting effect on leukemia cells without toxicity to the normal cells;
3. the cissus mongolica monomer compound androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione belongs to natural products, and has high safety; and the separation and purification process is simple, the cost is low, and the method is suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is an excitation spectrum (upper graph) and an emission spectrum (lower graph) of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione;
FIG. 2 shows the proliferation-inhibiting activity of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione on normal cells (human epidermal cells) HaCat (upper panel) and acute lymphoblastic leukemia cells BALL-1 (lower panel); p <0.05 (, P <0.01 (, P <0.001 (, x), and P <0.0001 (, x), indicating a significant difference compared to the negative control group (sample concentration of 0);
FIG. 3 is a fluorescence plot of human epidermal cells HaCat, acute lymphoblastic leukemia cells BALL-1 incubated with androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione for 24h and 48 h;
FIG. 4 is a graph of in vivo imaging results of Balb/c nude rat tail intravenous androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione compounds;
FIG. 5 shows HE staining of organs after 50. Mu.l of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione compound at a concentration of 2mg/mL was intravenously injected into Balb/c rat tail.
Detailed Description
The androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione used in the embodiment of the invention is prepared and obtained according to the existing phytochemical separation and purification technology. The invention is further illustrated in the following data in connection with experimental examples and the accompanying drawings. The examples of these experiments are only for illustrating the present invention, and are not intended to limit the scope of application of the present invention. After reading the description of the present invention, those skilled in the art will recognize that various changes, modifications and variations are equivalent, which are within the scope of the invention as defined in the appended claims, and that the reagents used in the examples, unless otherwise indicated, are those of conventional commercial products or those formulated according to conventional methods, and that the methods in the examples, unless otherwise indicated, are all conventional experimental methods.
In the examples RPMI1640 medium, DMEM medium, fetal Bovine Serum (FBS), phosphate Buffer (PBS), penicillin and streptomycin were all purchased from Gibco corporation; DMSO was purchased from beijing solibao biotechnology limited; cell count (CCK-8) detection kit was purchased from Shanghai Baisai Biotechnology Co.
Example 1: fluorescence spectrometry of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione
A certain amount of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione is precisely weighed and dissolved in DMSO to prepare a sample solution with the concentration of 1mg/mL, a clean four-sided light-passing 10mm quartz cuvette is filled with about 2/3 volume of the sample solution, and then the excitation spectrum and the emission spectrum of the solution are tested by using a HITACHI F-4600 fluorescence spectrophotometer. Excitation spectrum detection conditions: the emission wavelength Em is 900nm, the excitation wavelength Ex is 200-880nm, the Ex slit is 10nm, and the Em slit is 20nm; emission spectrum detection conditions: the excitation wavelength Ex is 430nm, the emission wavelength Em is 450-900nm, the Ex slit is 10nm, the Em slit is 20nm, and the excitation spectrum and the emission spectrum of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione are shown in figure 1;
as can be seen from FIG. 1, the optimum excitation wavelength of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione is about 450nm, and the optimum emission wavelength is about 550nm.
Example 2: toxicity experiment 1 and experimental material of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione on normal cells and tumor cells
Preparing a solution: preparing 30mmol/L mother liquor of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione with cell-grade DMSO under aseptic condition, sealing, storing in a refrigerator at-20deg.C for use in dark, and diluting into sample solutions with different concentrations according to final concentration requirement after dosing in experiment;
cell line: human acute lymphoblastic leukemia cells BALL-1 and human epidermal cells HaCat were purchased from Kunming cell Bank of the national academy of sciences of China, and these cell lines were cultured in RPMI1640 and DMEM medium containing 10% fetal bovine serum+1% penicillin and streptomycin, respectively, at 37℃and 5% CO 2 Culturing in an incubator for standby;
2. experimental method
Taking human epidermal cells HaCat in exponential growth phase, and preparing into 2×10 concentration with complete culture medium 4 The cell suspension of each hole is inoculated into a 96-well plate, 200 mu L of each hole is divided into a drug group and a negative control group, 5 compound holes are arranged in each group (or each drug concentration), after 24h of culture, 200 mu L of culture medium containing different concentrations of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione (the final concentration is 10 mu mol/L, 20 mu mol/L, 40 mu mol/L, 80 mu mol/L and 120 mu mol/L) is respectively used for the drug groupReplacement of the original medium, replacement of the original medium with 200 μl of fresh complete medium was performed in the negative control group; after the culture is continued for 22 hours, 20 mu L of CCK-8 reagent is added into each hole, the mixture is put back into an incubator for further incubation for 4 hours, then an enzyme-labeled instrument is used for measuring the absorbance value (OD value) of each hole at the wavelength of 490nm, the influence of different concentration drugs on the cell survival rate is calculated according to a formula, the survival rate= (OD value of a drug group/OD value of a negative control group) ×100%, and the result is shown in the upper graph of fig. 2;
acute lymphoblastic leukemia cells in exponential growth phase BALL-1 were grown at 5×10 4 Density of wells/wells was seeded in 96-well plates and the assay was divided into drug and negative control groups with a total volume of 200 μl per well. The final concentration of the liquid medicine is 10 mu M, 20 mu M, 40 mu M, 80 mu M and 120 mu M, the same volume of culture medium is added into a negative control group, 5 compound holes are arranged in each group (or each drug concentration), 20 mu L of CCK-8 solution is added into each hole after the culture is carried out for 44 hours, the enzyme label instrument detects the absorbance of each hole at 490nm wavelength after the culture is continued for 4 hours, and the survival rate, the survival rate= (OD value of the drug group/OD value of the negative control group) ×100% is calculated, and the result is shown in the lower graph of fig. 2;
as shown in FIG. 2, the compounds had no damaging effect on normal cells at drug concentrations of 120. Mu. Mol/L. In addition, androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione significantly reduced the viability of tumor cells in a dose-dependent manner within 48 hours; in summary, it is worth emphasizing that androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione is non-toxic to normal cells and has a growth inhibitory effect on leukemic cells.
Experimental example 3: fluorescence imaging experiments of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione in cells
Human epidermal cells HaCat in exponential growth phase were grown at 2X 10 5 Inoculating cell number per dish into small copolymerized dish, culturing for 24 hr with total volume of 2mL, sucking out stock culture solution, adding androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione solution with concentration of 60 μmol/L, adding control group into equal volume of culture medium, and adding 5% CO at 37deg.C 2 After culturing in incubator for 24h and 48h respectively, the culture was terminated, and the incubation was washed with cold PBS for 2 times, 1mL of 4% paraformaldehyde fixing solution was added to each dish, and after fixing at room temperature for 15min, the incubation was washed with cold PBS for two times, and then with 1mL of 0.5. Mu.g/mLDAPI solution of (C) was stained at room temperature in the dark for 15min, washed 2 times with cold PBS, and then 0.5mL of PBS was added to infiltrate the cells to prevent them from drying. Then photographing under a laser confocal fluorescence microscope, and exciting with 405nm and 450nm wavelength to acquire a cell fluorescence image.
The test result shows that blue fluorescence emitted by cell nucleus dye DAPI is observed at an excitation wavelength of 405nm, green fluorescence emitted by cells after sample treatment and a combined fluorescence image (mered) under two excitation light channels are observed at an excitation wavelength of 450nm, and the test shows that the fluorescence intensity is gradually increased along with the increase of the incubation time of the samples.
Acute lymphoblastic leukemia cells in exponential growth phase BALL-1 were grown at 5×10 5 Inoculating cell number per well into 6-well plate, adding 60 μmol/L androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione solution, adding equal volume of culture medium into control group, and adding 5% CO at 37deg.C in 2mL total volume per well 2 After culturing for 24h and 48h in an incubator respectively, stopping culturing; after the cell suspension is lightly blown uniformly, the cell suspension is respectively transferred into 2mL centrifuge tubes, the centrifugation is carried out for 5min at 1000rmp to collect cells, then the cells are washed for 2 times by using cold PBS, 1mL of 4% paraformaldehyde fixing solution is respectively added into each centrifuge tube, the cells are fixed for 15min at room temperature, the fixing solution is removed by centrifugation for 5min at 1000rmp, and the cell sediment is washed twice by using cold PBS. Then 1mL of DAPI solution with the concentration of 0.5 mug/mL is used for dyeing for 15min at room temperature in a dark way, then the solution is washed for 2 times by using cold PBS, 0.1mL of PBS is added for suspending and used for manufacturing a cell slide, a laser confocal fluorescence microscope is used for photographing, and excitation is carried out by adopting the wavelengths of 405nm and 450nm, so that a cell fluorescence image can be acquired.
The results are shown in FIG. 3, the experimental results show that blue fluorescence emitted by cell nuclear dye DAPI is observed at an excitation wavelength of 405nm, green fluorescence emitted by cells after sample treatment and a combined fluorescence image (Merged) under two excitation light channels are observed at an excitation wavelength of 450nm, and the experimental results show that the fluorescence intensity is also gradually increased along with the increase of the incubation time of the samples.
Experimental example 4: fluorescence imaging experiments of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione in Balb/c nude mice
12 6 week old Balb/c male nude mice (20.+ -. 2 g) were divided into 2 groups: after each group is adapted to 6 groups, 50 mu L of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione solution with the concentration of 2mg/mL is injected into the tail vein of the group, the solvent (cell-level DMSO) with the same volume is injected into the group, different time points of 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours and 8 hours are respectively selected after the injection, the experimental nude mice are subjected to gas anesthesia by isoflurane, then the nude mice are placed on an operation platform of a living body imager of a small animal, the excitation wavelength 465nm is set, the emission wavelength DsRed (580 nm) is set, and living body fluorescence distribution images can be acquired;
as a result, as can be seen from fig. 4, the administration group nude mice (right nude mice in the picture) were able to emit fluorescence in vivo at an excitation wavelength of 465nm and an emission wavelength of DsRed (580 nm) as compared with the control group (left nude mice in the picture), and the fluorescence intensity was increased and then decreased with the lapse of time.
Experimental example 5: male steroid-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione injection Balb/c nude mice 7 days later organ HE staining experiment
12 6 week old Balb/c male nude mice (20.+ -. 2 g) were divided into 2 groups: a dosing group and a control group, 6 animals in each group are adapted, after one week, 50 mu L of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione solution with the concentration of 2mg/mL is injected into the tail vein of the dosing group, the control group is injected with the solvent (cell-grade DMSO) with the same volume, water and food are normally provided after the dosing, weight change is recorded every day, brain, heart, liver, spleen, lung, kidney and testis are dissected after 7 days, pathological HE staining is carried out, and whether the damage to animal organs is caused by the drug is observed;
the results are shown in FIG. 5, and compared to the blank group, the dosing group (Treated) did not show inflammatory or necrotic cell aggregation and therefore did not cause damage to the brain, heart, liver, spleen, lung, kidney and testes of Balb/c nude mice.
Claims (1)
1. Use of androstane-4, 6,8 (9), 13 (14) -tetraene-3, 11, 16-trione as a fluorescent imaging agent.
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