CN113030048A - FPR1 channel function-based carcinogenicity in-vitro detection method for chemicals - Google Patents
FPR1 channel function-based carcinogenicity in-vitro detection method for chemicals Download PDFInfo
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
Abstract
The invention provides an in vitro detection method for carcinogenicity of a chemical based on FPR1 channel function, which comprises the steps of culturing FPR1-CHO cells until the cell fusion degree reaches more than 70%, adding a dye for incubation, exposing the incubated cells to a test solution containing the chemical, and carrying out high-throughput real-time fluorescence detection, wherein if the FPR1 channel of the cells is activated, the chemical can be determined to have carcinogenicity. The invention combines FPR1 target function detection and chemical carcinogenicity screening, finds that the abnormal activation of FPR1 and the change of a downstream signal conduction path can remarkably promote the malignant progress of tumors, can greatly improve the sensitivity of a chemical carcinogenicity prediction method, enlarges the detection range, shortens the test period, reduces interference factors, and has more stable and reliable results.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an in vitro detection method for carcinogenicity of a chemical based on FPR1 channel function.
Background
Tumors are the largest disease burden in most countries worldwide. 80% to 90% of human tumors are associated with environmental factors, the main of which are chemical factors. Heavy metals, biotoxins and the like are main environmental chemical carcinogenic factors, enter human bodies through atmosphere, water, soil, food and the like, and cause serious harm to health after accumulation and metabolism.
The carcinogenic process of chemicals is very lengthy and the growth phase of tumors is usually decades. The carcinogenic chemical has no obvious clinical manifestation on the body in the early period of exposure, and once clinical symptoms or imaging manifestations appear, the cells are predicted to be irreversibly malignant when entering the growth and development stage of the tumor, so that the treatment means is very limited, and irretrievable results are caused. Therefore, the detection of the chemical carcinogen has important significance for the prevention and control of the tumor.
The main bases for judging whether chemicals have carcinogenicity for a long time are mammal long-term carcinogenic tests (two years) and population epidemiological investigation, but the two methods are time-consuming and labor-consuming, have low detection efficiency and more interference factors, and are difficult to reflect carcinogenic mechanisms. Therefore, an in-vitro test system with good specificity and sensitivity and simple and convenient operation is established, the carcinogenic toxicity of the chemicals can be screened in a large batch, the detection efficiency is improved, and the carcinogenicity of the chemicals can be evaluated more scientifically.
Chemical carcinogens can be classified into genotoxic carcinogens and non-genotoxic carcinogens depending on whether DNA is directly targeted for action. The carcinogenic principle of the former is that chemicals directly act on DNA to form DNA adduct, induce chromosome damage, activate oncogene, inactivate cancer suppressor gene, mutate cell escape immune monitoring, balance of proliferation and apoptosis is broken, and tumor is generated. The non-genetic toxic carcinogen does not directly act on DNA, and induces tumor generation by inducing excessive oxidative stress and inflammatory reaction, methylation, acetylation, miRNA regulation and control abnormality and the like. The genetic end points of the traditional in vitro detection method for the genotoxic carcinogen are as follows: gene mutation, chromosome (genome) damage and DNA damage, but the existing in vitro detection method for non-genetic toxic carcinogenic substances has no mature detection system, and an improved genetic toxicity test method such as a Bhas 42 cell in vitro transformation test is mostly adopted. But the defects of the methods are obvious, and the method is only suitable for substances with definite carcinogenic mechanisms and has high false negative rate for unknown toxic chemicals; secondly, the detection flux is low, and the screening requirement of a large amount of new chemicals cannot be met. In the pathophysiological processes of tumors, carcinogenic chemicals directly or indirectly induce gene or protein expression or conformational changes in target cells or target tissues, which genes or proteins are time-ordered and tissue-specific. Therefore, the change of in vivo biological molecules can be used as a biological marker of carcinogen exposure, has better consistency and predictability than the existing in vitro detection method of carcinogens, and can provide a basis for carcinogenic evaluation of chemicals.
FPR1(Formyl Peptide Receptor 1) is mainly expressed in phagocytic leukocytes of mammals and is involved in pathophysiological processes including inflammation, virus defense and tumors. The existing research finds that FPR1 plays an important role in glioma, gastric cancer, lung cancer and other tumors. Compared with normal tissues, FPR1 shows abnormal high expression in tumor tissues, and tumor cells which are activated by the high expression FPR1 and FPR1 channels show obvious malignant phenotypes, including abnormal cell proliferation, abnormal reduction of apoptosis and high invasion. Animal experiments show that the FPR1 channel-activated mice show high tumorigenicity, and the malignant phenotype of tumors is obviously reduced after FPR1 is inhibited. The epidemiological investigation of the population shows that compared with the low expression patient, the prognosis of the tumor patient with the FPR1 high expression is worse, and the survival time is obviously shortened. Thus, FPR1 channel status is closely linked to tumorigenesis. Mechanistically, FPR1 can be involved in tumor development by modulating the chemotaxis of inflammatory cells. Research also finds that after the FPR1 channel is activated, the expression of angiogenesis factor VEGF can be promoted, the secretion of IL-8 and the like can be promoted, VEGF and IL-8 are both classical oncogenes, VEGF is a key molecule in a tumor angiogenesis signal pathway and is a mature target of an anti-tumor medicament. IL-8 is an inflammatory chemokine, and can remarkably stimulate cell proliferation and improve cell migration, invasiveness and metastasis.
FPR1 is a member of the G protein-coupled receptor (GPCR) family. GPCRs are the largest family of signal receptors in humans, and when exogenous chemicals act on GPCRs, the GPCRs change conformation, G-proteins are activated, triggering cellsA cellular cascade reaction. Therefore, the functional status of GPCRs is reflected by the detection of different biological products produced after activation of different G proteins, and existing methods for detecting GPCRs are: receptor binding assays, GTP γ s binding assays, cAMP assays, Radioactive IP3/IP1Detection, reporter gene systems, calcium flux detection, and the like. G.alpha.is the most studied class of G proteins, divided into four subclasses, G.alpha.s, G.alpha.i/o, G.alpha.q/11 and G.alpha. 12/13, where the G.alpha.q-coupled receptor can directly characterize GPCR function through changes in intracellular calcium ion concentration. FPR1 is a GPCR coupled with G alpha i, and the direct detection method is complex and tedious and has high requirements on experimental environment.
Disclosure of Invention
The invention aims to provide an in-vitro detection method for carcinogenicity of a chemical based on FPR1 channel function, which takes FPR1 as a detection target point and changes of intracellular calcium ion concentration as a core index to carry out early and preliminary detection on carcinogenicity chemicals. In studying the carcinogenic process of chemicals, the inventors discovered that carcinogenic chemicals can over-activate FPR1, affecting downstream signaling pathways whose dysfunction is linked to their carcinogenic mechanism. While the FPR1 target status can be monitored by intracellular calcium ion concentration. Therefore, the method for detecting the change of the intracellular calcium ion concentration can be applied to predict the carcinogenicity of the chemical.
In order to achieve the purpose, the invention provides the following technical scheme: an in vitro carcinogenic detection method of a chemical based on FPR1 channel function, which comprises the following steps of (1) cell inoculation culture: culturing FPR1-CHO cells until the cell fusion degree reaches more than 70% to obtain cells to be detected; (2) and (3) dye incubation: adding a dye into the cells to be detected for incubation to obtain stained cells; (3) treating cells with the test solution: exposing the stained cells to a test solution containing chemicals, wherein the chemicals comprise one or more of heavy metal substances and biotoxin substances; (4) high-throughput real-time fluorescence detection is performed, and if the FPR1 channel of the cell is activated, the chemical can be determined to be carcinogenic.
Further, in the step (3), the heavy metal substance includes one or more of cadmium, chromium and lead.
Further, in the step (3), the biotoxin substances comprise one or more of ochratoxin A, patulin and fumonisin.
Further, in the step (3), the solvent of the test solution comprises one or more of HBSS, DMSO and PBS.
Further, in the step (2), the dye incubation is carried out by staining with calcium ion dye Fluo-4 AM and incubating for 45-60 min at 37 ℃.
Further, in the step (4), the parameters of the high-flux real-time fluorescence detection are as follows, the range of the excitation wavelength is 470-495 nm, the range of the measurement wavelength is 515-575 nm, and the detection time is longer than 60 s.
Further, the detection time is 200s-1000s, wherein the test solution containing the chemical is added at the 60 th s.
The invention has the beneficial effects that:
the invention takes FPR1 as a target point for carcinogenicity screening, takes the concentration of calcium ions in cells as a detection index, and combines a high-throughput technology to predict the carcinogenicity of chemicals. By adopting the method of co-expressing the multifunctional G protein G alpha 15/16, the FPR1 target is activated, the FPR1 is abnormally activated, the receptor signal transduction pathway is converted into the calcium ion signal pathway for detection, and the high-flux real-time fluorescence detection system is combined, so that the screening of the carcinogenic toxicity of a large batch of chemicals becomes possible, the detection time is obviously shortened, the interference factors are reduced, the sensitivity is improved, the result is objective and quantifiable, a novel in-vitro method for detecting the carcinogenic toxicity of the chemicals is initiated, and a foundation is laid for the safety evaluation and mechanism research of the chemicals.
Drawings
Fig. 1 is a schematic diagram illustrating the effect of different continuous detection times on the activation of the FPR1 channel in embodiment 1 of the present invention.
FIG. 2 is a diagram showing the OTA activation of FPR1 by ochratoxin at different concentrations in example 2 of the present invention. Wherein A is a time-effect curve graph of intracellular calcium inflow caused by OTA with different concentrations; b is the calcium influx effect value caused by OTA with different concentrations.
FIG. 3 is a schematic diagram of the different concentrations of patulin-activated FPR1 in example 3 of the present invention. Wherein A is a time-effect curve chart of calcium influx in cells caused by patulin with different concentrations; b is the calcium influx effect value caused by different concentrations of patulin.
FIG. 4 is a schematic representation of different concentrations of fumonisin FPR1 in example 4 of the present invention. Wherein A is a time-effect curve graph of intracellular calcium influx caused by different concentrations of fumonisin; b is the calcium influx effect value caused by different concentrations of fumonisins.
FIG. 5 is a diagram of the activation of the FPR1 by cadmium at different concentrations in example 5 of the present invention. Wherein A is a time-effect curve graph of calcium influx in cells caused by cadmium with different concentrations; b is the calcium influx effect value caused by different concentrations of cadmium.
FIG. 6 is a graph showing the activation of FPR1 at different concentrations of Cr in example 6 of the present invention. Wherein A is a time-effect curve graph of intracellular calcium influx caused by chromium with different concentrations; b is the calcium influx effect value caused by chromium with different concentrations.
FIG. 7 is a graph showing the activation of FPR1 by lead of various concentrations in example 7 of the present invention. Wherein A is a time-effect curve graph of calcium influx in cells caused by lead with different concentrations; b is the calcium influx effect value caused by lead with different concentrations.
FIG. 8 is a graphical representation of the inability of different concentrations of caprolactam to activate FPR1 in comparative example 1 of the present invention. Wherein A is a time-effect curve graph of intracellular calcium inflow caused by caprolactam with different concentrations; b is the calcium influx effect value caused by different concentrations of caprolactam.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
The reagent of the invention is prepared as follows:
(1) cell culture medium: 90% Ham's F12 culture solution, 10% fetal bovine serum, 200 μ g/mL Zeocin, 100 μ g/mL Hygromycin B;
(2) phosphate Buffered Saline (PBS): 8g NaCl, 0.2g KCl, 1.44g Na2HPO4，0.24g KH2PO4Dissolving in 900mL double distilled water, adjusting pH to 7.4, diluting to 1L, and autoclaving at 115 deg.C for 15 min;
(3) cell digestive juice: 0.25g of trypsin, 0.02g of EDTA, 70mL of PBS solution for dissolution, pH value adjustment to 7.2-7.4, volume constant to 100mL, filtration and sterilization;
(4) hank's Balanced Salt Solution (HBSS): 8.0g NaCl, 0.4g KCl, 0.1g MgSO4·7H2O，0.1g MgCl2·6H2O，0.06g Na2HPO4·2H2O，0.06g KH2PO41.0g glucose, 0.14g CaCl2, 0.35g NaHCO3Adding 700mL of double distilled water, fully and uniformly mixing, adjusting the pH value to 7.2-7.4, and fixing the volume to 1000 mL;
(5) calcium ion dye: 1mg Fluo-4 AM, dissolved in 227.9. mu.L DMSO to make 4mM stock solution, protected from light at-20 ℃. Before use, 10. mu.L of Fluo-4 AM stock solution was added with 9mL of HBSS buffer and 1mL of 5% BSA, and pre-heated at 37 ℃ for use.
An in vitro carcinogenic detection method for a chemical based on FPR1 channel function comprises the following steps:
(1) cell plating: logarithmic growth phase FPR1-CHO cells (available from Kingsler Biotech Co., Ltd.) were digested with cell digests at 2X 104The density of each well is cultured in a 96-well black transparent flat-bottom cell plate overnight, and the cells are observed under a microscope the next day, and the cells are fused to reach 70-80% of the bottom wall of the culture flask and have normal morphology.
(2) And (3) dye incubation: removing all culture solution in the cell hole, adding 60 mu L of calcium ion dye into each hole, and incubating at 37 ℃ for 45-60 min. After incubation, 140 μ L HBSS buffer was added to each well. And (3) sucking and removing 150 mu L of the solution in the hole, then gently and slowly adding 150 mu L of HBSS buffer solution to avoid blowing up adherent cells, and repeating the step for 5-6 times. After the final removal of 150. mu.L of the liquid in the wells, 125. mu.L of HBSS buffer was added to each well and allowed to stand at room temperature for 5 min.
(3) Treating the chemical to be detected: and preparing the chemical to be detected into a sample solution to be detected with a certain concentration by using DMSO, HBSS or PBS, wherein the concentration of the sample solution is 1000 times of the final concentration of the detection. And adding 372 mu L of HBSS buffer solution into 3 mu L of sample solution, fully and uniformly mixing, and adding 96-hole transparent sharp-bottom enzyme label plates with the volume of 60 mu L per hole. Buffer control wells were filled with 60. mu.L of HBSS buffer per well, and negative control wells were filled with 60. mu.L of HBSS buffer containing the same volume of solvent as the sample solution per well (taking 3. mu.L of DMSO as an example, 372. mu.L of HBSS buffer was added to each well, and 60. mu.L of HBSS buffer was added to each well after mixing well).
(4) High-flux real-time fluorescence detection: respectively placing a cell plate and a sample adding plate at corresponding positions of a high-flux real-time fluorescence detection analysis system, setting the range of excitation wavelength to be 470-495 nm, the range of determination wavelength to be 515-575 nm, the interval of fluorescence signal acquisition to be 1s and the sample adding volume to be 25 muL. After the start of the test, the fluorescence intensity of the baseline was measured without applying the sample for the first 60s, and the mean value of the fluorescence signals at the first 10 time points was used as the basic fluorescence intensity value (F)0). After sample addition, the fluorescence intensity value (F) was continuously measured for 900s with time as the abscissa and fluorescence intensity F as the ordinate, and a time-effect curve for each reaction well was generated by a high-throughput real-time fluorescence detection analysis system.
Image analysis by using image processing software graphics, calculating time-
Area Under the Curve (AUC) from 60s to 300s, the AUC value was used as a measure of the intracellular calcium ion concentration.
In the specific embodiment of the invention, experiments were designed for at least 3 replicates, analyzed using SPSS 22.0 statistical software, and group-to-group differences were examined using One-way ANOVA. Statistical significance was considered when p < 0.05.
"increased calcium ion", "increased calcium flux", "increased calcium ion concentration" refers to a statistically significant increase in fluorescence intensity value relative to a negative control.
Example 1 an in vitro carcinogenic assay of a chemical based on the FPR1 channel, optimization of high throughput real-time fluorescence detection conditions;
(1) cell plate
Taking FPR1-CHO cells in logarithmic growth phase at 5 × 1031 x 10 per hole42 x 10 pieces/hole44 x 10 pieces/hole4The density of cells/well was seeded in 96-well black transparent flat-bottomed cell plates. The next day, the degree of cell fusion was observed under an inverted microscope.
(2) Dye incubation
Calcium ion dye Fluo-4 AM was added in a volume of 60. mu.L per well and incubated at 37 ℃ for 45min to 60 min. After the incubation was completed, the cells were washed 3-4 times with HBSS buffer to remove the dye that did not enter the cells. After washing, HBSS buffer was supplemented to a final volume of 175. mu.L.
(3) Treatment of chemicals to be tested
The FPR1 agonist fMLP was dissolved in DMSO as a 100mM sample solution. The wells were diluted with HBSS buffer at a ratio of 1:125, and a 96-well transparent sharp-bottomed microplate was added in a volume of 60. mu.L per well.
(4) High-throughput real-time fluorescence detection
Respectively placing a cell plate and a sample adding plate at corresponding positions of a high-flux real-time fluorescence detection analysis system, wherein the excitation wavelength range is 470-495 nm, the determination wavelength range is 515-575 nm, the fluorescence signal acquisition interval is 1s, and the sample adding volume is 25 mu L. After the test started, the average of the fluorescence signals at the previous 10 time points was used as the basic fluorescence intensity value (F) at the sample loading time of 60s0). After sample addition, fluorescence intensity values (F) are continuously detected, the continuous detection time is respectively set as 100s, 500s and 900s, the time is taken as an abscissa, the fluorescence intensity F is taken as an ordinate, and a time-effect curve of each reaction well is generated by a high-throughput real-time fluorescence detection analysis system.
(5) Results
As a result, as shown in Table 1, the cell fusion degree was different in the next day for different cell plate densities, and the response intensity value to fluorescence was also different. Wherein, 5 is multiplied by 103A/hole and 1 x 104The fusion degree of the density of the seed plates per hole on the next day is 40% -60%, the fusion degree is low, the basic fluorescence intensity is 100-200, the response is too low, and the fluorescence detection analysis of the sample is not facilitated. 4X 104The density of the plates per well was 90 confluency the day after% cell density affects the proliferation state of cells and accelerates cell aging. 2X 104Under the density of the plate per hole, the cell fusion degree of the next day reaches 80%, and the basic fluorescence intensity is 505, so that the requirement of subsequent high-throughput real-time fluorescence detection is met. Therefore, the cell plate density is preferably set to 2X 104Per well.
TABLE 1 Condition optimization of different board densities
| Plate density (per hole) | Degree of fusion in the next day (%) | Basal fluorescence intensity F0 |
| 5×103 | ～40 | 120 |
| 1×104 | ～55 | 180 |
| 2×104 | ～80 | 505 |
| 4×104 | ～90 | 516 |
The optimized result of the detection time is shown in fig. 1, when the continuous detection time is 100s, the fluorescence intensity can be seen from the time-effect curve to be not restored to a constant level, which indicates that the reaction process of calcium inflow is not completely finished; when the detection time is continued to be 500s, the fluorescence intensity just returns to a constant level; the detection time was continued for 900s and the fluorescence intensity was completely returned to a constant level. In summary, to fully record the reaction process of calcium influx, the detection duration was set to 900 s.
Example 2
An FPR1 channel-based in vitro assay for carcinogenicity of chemicals, which evaluates the carcinogenicity of ochracin A (OTA);
(1) cell plate
Taking FPR1-CHO cells in logarithmic growth phase at 2 × 104The density of cells/well was seeded in 96-well black transparent flat-bottomed cell plates. The next day, the degree of cell fusion was observed under a microscope and dye incubation was allowed to occur up to 70% or more.
(2) Dye incubation
Calcium ion dye Fluo-4 AM was added in a volume of 60. mu.L per well and incubated at 37 ℃ for 45min to 60 min. After the incubation was completed, the cells were washed 3-4 times with HBSS buffer to remove the dye that did not enter the cells. After washing, HBSS buffer was supplemented to a final volume of 175. mu.L.
(3) Treatment of chemicals to be tested
OTA was first dissolved in DMSO to form 20mM, 10mM, 5mM sample solutions. The cells were diluted individually with HBSS buffer at a ratio of 1:125, and a 96-well clear sharp-bottomed microplate was added in a volume of 60. mu.L per well, and the cells were exposed to OTA in DMSO solution.
(4) High-throughput real-time fluorescence detection
Respectively placing a cell plate and a sample adding plate at corresponding positions of a high-flux real-time fluorescence detection analysis system, wherein the excitation wavelength range is 470-495 nm, the determination wavelength range is 515-575 nm, the fluorescence signal acquisition interval is 1s, and the sample adding volume is 25 mu L. After the start of the test, the fluorescence intensity of the baseline was measured without applying the sample for the first 60s, and the mean value of the fluorescence signals at the first 10 time points was used as the basic fluorescence intensity value (F)0). After sample application, continuously detecting fluorescence intensity value (F) with detection time of 900s, time as abscissa and fluorescence intensity F as ordinate by high-throughput real-time fluorescenceThe detection and analysis system generates a time-effect curve for each reaction well.
(5) Results
As shown in FIG. 2, the final concentrations of 5. mu.M, 10. mu.M and 20. mu.M of OTA acted on FPR1-CHO cells, and the intracellular calcium concentration was significantly increased (p < 0.001), while the negative control group showed no significant change in calcium concentration, indicating that OTA could activate FPR 1.
The international agency for research on cancer IARC classifies OTA as a class 2B carcinogen, a possible human carcinogen, and animal experiments and epidemiological investigations have shown that OTA is associated with digestive and urinary tumors. In the method result, compared with a negative control, the intracellular calcium ion concentration of the OTA is remarkably increased under the dosage of 5-20 muM, which represents that the FPR1 channel is activated, and the detection of the carcinogen OTA through the FPR1 channel state is reflected, so that the method can be used for predicting the carcinogenicity of the biotoxin.
Example 3
An FPR1 channel-based in vitro assay for carcinogenicity of chemicals, assessing carcinogenicity of patulin;
(1) cell plate
The cell plating procedure was the same as in example 2.
(2) Dye incubation
The dye incubation step was the same as in example 2.
(3) Treatment of chemicals to be tested
Patulin was dissolved in DMSO to a concentration of 20mM, 10mM, 5mM sample solution. The wells were diluted with HBSS buffer at a ratio of 1:125, and a 96-well transparent sharp-bottomed microplate was added in a volume of 60. mu.L per well.
(4) High-throughput real-time fluorescence detection
High throughput real-time fluorescence detection was as in example 2.
(5) Results
As shown in FIG. 3, when patulin was applied to FPR1-CHO cells at final concentrations of 5. mu.M, 10. mu.M, and 20. mu.M, as compared to the negative control, the intracellular calcium ion concentration was significantly increased (p < 0.01), indicating that patulin could activate FPR 1.
Patulin is a class 3 carcinogen, classified by the carcinogenic factors of IARC. In the method result of the invention, the concentration of calcium ions in cells is obviously increased under the dosage of 5-20 mu M, the FPR1 channel is opened, which reflects that the patulin can induce the FPR1 channel to open and cause calcium inflow. Thus, the method of the present invention can be used to detect the carcinogen patulin.
Example 4
An in vitro assay for carcinogenicity of a chemical based on the FPR1 channel, for assessing carcinogenicity of fumonisin;
(1) cell plate
The cell plating procedure was the same as in example 2.
(2) Dye incubation
The dye incubation step was the same as in example 2.
(3) Treatment of chemicals to be tested
Fumonisin B1 was dissolved in DMSO to a sample solution with a concentration of 20mM, 10mM, 5 mM. The wells were diluted with HBSS buffer at a ratio of 1:125, and a 96-well transparent sharp-bottomed microplate was added in a volume of 60. mu.L per well.
(4) High-throughput real-time fluorescence detection
High throughput real-time fluorescence detection was as in example 2.
(5) Results
As shown in FIG. 4, fumonisin B1 at a final concentration of 20. mu.M, 10. mu.M, and 5. mu.M significantly induced intracellular calcium influx (p < 0.001) compared to the negative control.
According to the IARC carcinogenic classification, fumonisin B1 is a class 2B carcinogen. In the method result of the invention, the fumonisin B1 can effectively activate an FPR1 channel, which reflects that the method can be used for detecting the carcinogenicity of fumonisin B1.
Example 5
An in vitro carcinogenic assay for FPR1 channel-based chemicals to assess the carcinogenicity of cadmium;
(1) cell plate
The cell plating procedure was the same as in example 2.
(2) Dye incubation
The dye incubation step was the same as in example 2.
(3) Treatment of chemicals to be tested
Cadmium acetate was dissolved in DMSO to a concentration of 20mM, 10mM, 5mM of sample solution. The wells were diluted with HBSS buffer at a ratio of 1:125, and a 96-well transparent sharp-bottomed microplate was added in a volume of 60. mu.L per well.
(4) High-throughput real-time fluorescence detection
High throughput real-time fluorescence detection was as in example 2.
(5) Results
As shown in FIG. 5, cadmium acetate significantly induced an increase in intracellular calcium ion of FPR1-CHO (p < 0.01) at concentrations of 5. mu.M, 10. mu.M, and 20. mu.M, in a dose-response relationship, compared to the negative control group, indicating that cadmium acetate is carcinogenic.
IARC ranks cadmium and inorganic cadmium compounds as class 1 carcinogens. The method of the invention can reflect the opening condition of the FPR1 channel by measuring the concentration of the calcium ions in the cells. The results show that the method of the invention can be used for detecting the carcinogenicity of cadmium.
Example 6
An FPR1 channel-based in vitro assay for carcinogenicity of chemicals, for assessing carcinogenicity of chromium;
(1) cell plate
The cell plating procedure was the same as in example 2.
(2) Dye incubation
The dye incubation step was the same as in example 2.
(3) Treatment of chemicals to be tested
Potassium dichromate was dissolved in DMSO to a concentration of 10mM, 5mM, 1mM of sample solution. The wells were diluted with HBSS buffer at a ratio of 1:125, and a 96-well transparent sharp-bottomed microplate was added in a volume of 60. mu.L per well.
(4) High-throughput real-time fluorescence detection
High throughput real-time fluorescence detection was as in example 2.
(5) Results
As shown in FIG. 6, potassium dichromate at 1. mu.M, 5. mu.M and 10. mu.M showed an increase in intracellular calcium concentration (p < 0.01) on FPR1-CHO cells in a dose-response relationship, compared to the negative control group, indicating that potassium dichromate is carcinogenic.
Chromium in the hexavalent state (cr (vi)), (vi) is a class 1 carcinogen as identified by IARC. The results show that the method of the invention finds that Cr (VI) can activate FPR1 channel to cause calcium influx, and the method of the invention can be used for detecting the carcinogenicity of chromium.
Example 7
An in vitro carcinogenic assay for chemicals based on the FPR1 channel to assess the carcinogenicity of lead;
(1) cell plate
The cell plating procedure was the same as in example 2.
(2) And (3) dye incubation:
the dye incubation step was the same as in example 2.
(3) Treatment of chemicals to be tested
Lead acetate was dissolved in DMSO to a sample solution of 10mM, 5mM, 1mM concentration. The wells were diluted with HBSS buffer at a ratio of 1:125, and a 96-well transparent sharp-bottomed microplate was added in a volume of 60. mu.L per well.
(4) High-throughput real-time fluorescence detection
High throughput real-time fluorescence detection was as in example 2.
(5) Results
As shown in FIG. 7, 1. mu.M to 10. mu.M of lead acetate showed an increase in intracellular calcium concentration (p < 0.01) on FPR1-CHO cells in a dose-response relationship, indicating that lead acetate was carcinogenic, as compared with the negative control group.
IARC classifies lead as a class 2B carcinogen. The results show that the method of the invention finds that lead can effectively activate FPR1 channel to cause calcium influx, and the method of the invention can be used for detecting the carcinogenicity of lead.
Comparative example 1 caprolactam failed to activate FPR1 to induce calcium influx
(1) Cell plate
The cell plating procedure was the same as in example 2.
(2) Dye incubation
The dye incubation step was the same as in example 2.
(3) Treatment of chemicals to be tested
Caprolactam was prepared as a sample solution at 50mg/L, 25mg/L, 12.5 mg/L. The wells were diluted with HBSS buffer at a ratio of 1:125, and a 96-well transparent sharp-bottomed microplate was added in a volume of 60. mu.L per well.
(4) High-throughput real-time fluorescence detection
High throughput real-time fluorescence detection was as in example 2.
(5) Results
As shown in FIG. 8, no significant change in intracellular calcium ion was observed at a concentration of 50. mu.g/L, 25. mu.g/L, 12.5. mu.g/L caprolactam (p > 0.05), indicating that caprolactam failed to activate FPR1, indicating that it was not carcinogenic.
Caprolactam is the only human non-carcinogen (class 4) identified by IARC, and the method of the invention finds that caprolactam can not activate the FPR1 target spot, and the intracellular calcium flow has no obvious change, thus showing that the method of the invention can screen out non-carcinogenic chemicals.
Carcinogenicity is an important toxic end point for the safety evaluation of chemicals. The existing chemical carcinogenicity detection method is mainly an in-vivo mammal long-term carcinogenesis test and has the defects of low flux, long period, low sensitivity and high cost. The in vitro method is mainly a genetic toxicity test method, is only suitable for detecting genetic toxicity carcinogens, and has the characteristics of low flux, high false negative rate and the like. By means of a toxicological genomics technology, the carcinogenic effect and the action mechanism of exogenous stimulation on an organism are systematically and comprehensively revealed by observing the dynamic change rule of genes in tissues or cells under the action of the exogenous stimulation. The traditional carcinogenicity in-vitro detection technology uses a toxic phenotype as a detection object, a biomarker method surrounds a carcinogenic target, and the carcinogenicity of a chemical is judged by detecting the function of the carcinogenic target. The method of the invention takes FPR1 as a biomarker in the carcinogenic process, develops the carcinogenic screening technology based on the FPR1 target function, monitors the change of calcium current in cells by applying real-time high-flux fluorescence imaging analysis to reflect the change of the target function, has low cost, wide detection range and high sensitivity compared with the existing genetic toxicity in-vitro detection method and the long-term carcinogenic test of mammals, is beneficial to the subsequent mechanism research, can be used for screening carcinogenicity of a large quantity of chemicals, and provides a basis for exposure evaluation of the carcinogenic chemicals, monitoring of tumorigenesis development and prevention and control. The invention discloses an in vitro carcinogenic detection method of a chemical based on an FPR1 channel, which is not reported in the literature.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (7)
1. An in vitro detection method for carcinogenicity of a chemical based on FPR1 channel function is characterized in that: the method comprises the following steps:
(1) inoculating and culturing cells: culturing FPR1-CHO cells until the cell fusion degree reaches more than 70% to obtain cells to be detected;
(2) and (3) dye incubation: adding a dye into the cells to be detected for incubation to obtain stained cells;
(3) treating cells with the test solution: exposing the stained cells to a test solution containing chemicals, wherein the chemicals comprise one or more of heavy metal substances and biotoxin substances;
(4) high-throughput real-time fluorescence detection is performed, and if the FPR1 channel of the cell is activated, the chemical can be determined to be carcinogenic.
2. The in vitro method for detecting carcinogenicity of a chemical based on the function of FPR1 channel according to claim 1, wherein: in the step (3), the heavy metal substances comprise one or more of cadmium, chromium and lead.
3. The in vitro method for detecting carcinogenicity of a chemical based on the function of FPR1 channel according to claim 1, wherein: in the step (3), the biotoxin substances comprise one or more of ochratoxin A, patulin and fumonisin.
4. The method for the in vitro detection of carcinogenicity of a chemical based on the function of FPR1 channel according to any one of claims 1 to 3, wherein: in the step (3), the solvent of the test solution comprises one or more of HBSS, DMSO and PBS.
5. The method for the in vitro detection of carcinogenicity of a chemical based on the function of FPR1 channel according to any one of claims 1 to 3, wherein: in the step (2), the dye incubation is carried out by staining with calcium ion dye Fluo-4 AM and incubating for 45-60 min at 37 ℃.
6. The method for the in vitro detection of carcinogenicity of a chemical based on the function of FPR1 channel according to any one of claims 1 to 3, wherein: in the step (4), the parameters of the high-flux real-time fluorescence detection are as follows,
the range of the excitation wavelength is 470-495 nm, the range of the measurement wavelength is 515-575 nm, and the detection time is more than 60 s.
7. The in vitro cancer-causing assay method for a chemical based on the FPR1 channel function of claim 6, wherein: the detection time is 200s-1000s, wherein the test solution containing chemicals is added at the 60 th s.
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