CN110208516B - Method for detecting developmental toxicity of chemicals - Google Patents
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides a method for detecting developmental toxicity of chemicals, which at least comprises the following steps: inducing the undifferentiated human embryonic stem cells to differentiate into the myocardial cells under the culture conditions of the chemicals to be detected and the chemicals without being detected respectively; respectively measuring the expression level of MYL4 in the myocardial cells under the culture conditions of the chemicals to be measured and the culture conditions of no chemicals to be measured; obtaining the expression quantity change of MYL4 in the cardiac muscle cells, namely ID, compared with the culture condition without the chemical to be detected under the culture condition with the chemical to be detected; and (4) according to the value of the ID, determining the developmental toxicity of the chemical to be detected by combining with a Fisher discriminant function equation. The invention firstly utilizes the human embryonic stem cell to establish a developmental toxicity test model, and can obtain the developmental toxicity information of the compound through in vitro tests in a short time; the invention can quickly detect and prompt whether a certain chemical has developmental toxicity information aiming at human.
Description
Technical Field
The invention relates to the field of chemical toxicity detection, in particular to a chemical developmental toxicity detection method.
Background
Currently, toxicity data of chemicals are mostly obtained from experimental animals, such as acute toxicity test, subacute toxicity test, chronic toxicity test, and the like. However, there are several non-negligible problems with the development of animal experiments: 1. 2, the experiment period is long, 3, a large number of experimental animals are needed, and 4, metabolism and mechanism research are difficult to carry out due to more influence factors in the body. The 3R principle, namely Reduction, optimization and Replacement (Reduction), is vigorously advocated for animal protection, and toxicological Replacement has very important significance for the hazard evaluation and management of exogenous chemicals from both a scientific perspective and an economic perspective.
Estimated from market research data, over the next 15 years 30000 chemicals will need to be tested for safety, and 7 million animals will be sacrificed according to current test guidelines, of which about 64% are for reproductive and developmental toxicity assessment of the chemicals; about 6 billion euros are spent for each 2000 new compounds tested for developmental toxicity. The number of new compounds has increased in 1000 per year, with more than 90% lacking in toxicity data. In the process, no matter the use condition of animals, or the consumption of manpower, material resources and financial resources in the test process, the huge number makes people feel eye surprise.
China is vast in breadth and large in population, so that the situation is more complicated than other countries when the health problem caused by compound exposure is evaluated. And because the types and the proportions of the compounds in various regions are different, the emphasis is different when epidemiological investigation is carried out, and the problems of novel pollutants and rapid diffusion thereof are brought together with rapid industrial development and rapid urbanization process. The application and popularization of the toxicological substitution method are also suitable for the national conditions of China.
Under the constraint of medical ethics, at present, the international safety evaluation test method mainly uses animals and cells, and almost no chemical substances can be allowed to obtain a human body test except newly developed medicines with safety guaranteed by previous clinical experiment results and great treatment potential. Therefore, from the perspective of toxicological substitution, how to establish a safety evaluation system and method capable of accurately reflecting human biological characteristics has great significance for guaranteeing human health.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a method for detecting developmental toxicity of chemicals.
To achieve the above and other related objects, a first aspect of the present invention provides a method for detecting developmental toxicity of a chemical, the method at least comprising the steps of:
(1) inducing the undifferentiated human embryonic stem cells to differentiate into the myocardial cells under the culture conditions of the chemicals to be detected and the chemicals without being detected respectively;
(2) respectively measuring the expression level of MYL4 in the myocardial cells under the culture conditions of the chemicals to be measured and the culture conditions of no chemicals to be measured; obtaining the expression quantity change of MYL4 in the cardiac muscle cells, namely ID, compared with the culture condition without the chemical to be detected under the culture condition with the chemical to be detected;
(3) and (4) according to the value of the ID, determining the developmental toxicity of the chemical to be detected by combining with a Fisher discriminant function equation.
The second aspect of the present invention provides a chemical developmental toxicity detection model, comprising:
I)-1.104+0.686×log10ID0+0.641×log10ID20；
II)-12.023+36.626×log10ID0+29.189×log10ID20；
III)-1.252+5.331×log10ID0+2.537×log10ID20；
if I) > II) and I) > III), judging that the compound has no developmental toxicity;
if II) > I) and II) > III), judging the compound to have weak developmental toxicity;
if III) > I) and III) > II), judging the strong developmental toxicity of the compound;
wherein, ID20The chemical concentration to be measured is IC20When the expression level of MYL4 changes, ID0The chemical concentration to be measured is IC0The expression level of the corresponding MYL4 is changed; IC (integrated circuit)20The concentration of the chemical to be detected in the inhibition of the cell viability of 20% of human embryonic stem cells; IC (integrated circuit)0＝IC20/100。
The third aspect of the invention provides a method for establishing a chemical developmental toxicity detection model, which comprises the following steps:
(1) inducing undifferentiated human embryonic stem cells to differentiate into cardiomyocytes under culture conditions containing and without each modeling chemical, the developmental toxicity classification of which is known;
(2) measuring the expression level of MYL4 in the cardiomyocytes under culture conditions comprising each modeling chemical and in the absence of said modeling chemical; obtaining ID, which is the change in the expression level of MYL4 in cardiomyocytes under the culture conditions with each modeling chemical as compared to the culture conditions without the modeling chemical;
(3) according to the value of the ID, a typical discriminant function equation is constructed;
(4) and obtaining a Fisher discriminant function equation according to the typical discriminant function equation.
The fourth aspect of the invention provides a chemical developmental toxicity detection device, which can realize the chemical developmental toxicity detection model.
A fifth aspect of the invention provides a computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements the aforementioned chemical developmental toxicity detection model.
A sixth aspect of the present invention provides a computer processing device, which comprises a processor and the aforementioned computer readable storage medium, wherein the processor executes a computer program on the computer readable storage medium, and can implement the aforementioned chemical development toxicity detection model.
A seventh aspect of the present invention provides an electronic terminal, comprising: a processor, a memory, and a communicator; the memory is used for storing a computer program, the communicator is used for being in communication connection with an external device, and the processor is used for executing the computer program stored by the memory so as to enable the terminal to execute the chemical development toxicity detection model.
As described above, the method for detecting developmental toxicity of chemicals according to the present invention has the following advantageous effects:
the invention firstly utilizes the human embryonic stem cell to establish a developmental toxicity test model, and can obtain the developmental toxicity information of the compound through in vitro tests in a short time;
the invention introduces a molecular index MYL4 as a developmental toxicity test index for the first time, and further verifies the effectiveness and reliability of the index.
The method is used for representing whether the chemicals have developmental toxicity information aiming at human or not by a mathematical modeling method for the first time;
the invention can quickly detect and prompt whether a certain chemical has developmental toxicity information aiming at human.
Drawings
FIG. 1 shows the culture and induced differentiation of human embryonic stem cells. hES and its differentiation-inducing cardiomyocytes identification. (A) hES in an undifferentiated state under light microscopy; (B) immunofluorescence staining of high expression pluripotency index SOX-2 in hES; (C) hES-induced differentiated cardiomyocytes; (D) hES induces immunofluorescence staining of the highly expressed cardiac-specific index troponin cTnT in differentiated cardiomyocytes. Scale bar 100 μm.
FIG. 2 shows the decision boundary of the linear discriminant function on the ability of compound to recognize developmental toxicity in example 8. N represents a non-developmentally toxic compound, W represents a weakly developmentally toxic compound, and S represents a strongly developmentally toxic compound.
Detailed Description
The method for detecting the developmental toxicity of the chemicals at least comprises the following steps:
(1) inducing the undifferentiated human embryonic stem cells to differentiate into the myocardial cells under the culture conditions of the chemicals to be detected and the chemicals without being detected respectively;
(2) respectively measuring the expression level of MYL4 in the myocardial cells under the culture conditions of the chemicals to be measured and the culture conditions of no chemicals to be measured; obtaining the expression quantity change of MYL4 in the cardiac muscle cells, namely ID, compared with the culture condition without the chemical to be detected under the culture condition with the chemical to be detected;
(3) and (4) according to the value of the ID, determining the developmental toxicity of the chemical to be detected by combining with a Fisher discriminant function equation.
Developmental toxicity refers to the deleterious effects that arise in the offspring before they reach the adult, caused by exposure of the offspring to exogenous physicochemical factors via the father and/or mother before birth, including: structural malformation, growth retardation, dysfunction and death.
In one embodiment, the developmental toxicity is selected from embryotoxicity. Embryotoxicity (embryotoxicity) refers to all toxicity of pregnancies before and after implantation and until the end of the organ formation stage caused by exogenous physicochemical factors, and is specifically characterized by organ deformity, growth retardation, implantation number reduction and the like caused by embryonic contamination.
The invention utilizes the differentiation of the embryonic stem cells into the myocardial cells to simulate the development of the early heart of the embryo, and not only can represent the deformity but also can indirectly represent the risk of embryonic death due to the normality of the structure and the function of the heart.
In one embodiment, the culture conditions without the test chemical are blank controls of culture conditions with the test chemical.
In one embodiment, in step (1), the chemical concentration to be measured is IC20The concentration of the chemical to be measured is IC0And inducing undifferentiated human embryonic stem cells to differentiate into cardiomyocytes under the culture condition without the chemicals to be tested, wherein IC20The concentration of the chemical to be detected in the inhibition of the cell viability of 20% of human embryonic stem cells; IC (integrated circuit)0＝IC20/100。
At one endIn one embodiment, the IC20The determination method comprises the following steps: culturing undifferentiated human embryonic stem cells under the culture conditions of chemicals to be tested with different concentrations, and determining the concentration of the chemicals to be tested at which 20% of the cells of undifferentiated human embryonic stem cells are inhibited by activity, namely IC20。
In one embodiment, in step (2), the ID includes an ID20And ID0Wherein ID20The chemical concentration to be measured is IC20When the expression level of MYL4 changes, ID0The chemical concentration to be measured is IC0The expression level of the corresponding MYL4 was varied.
The chemical is selected from the group consisting of an element, a compound, or a mixture.
In one embodiment, the developmental toxicity classification of the test chemical comprises no developmental toxicity, weak developmental toxicity, strong developmental toxicity.
In one embodiment, the Fisher discriminant function equation is:
I)-1.104+0.686×log10ID0+0.641×log10ID20；
II)-12.023+36.626×log10ID0+29.189×log10ID20；
III)-1.252+5.331×log10ID0+2.537×log10ID20；
in one embodiment, the method for determining the developmental toxicity of the chemical to be tested comprises:
if I) > II) and I) > III), judging that the compound has no developmental toxicity;
if II) > I) and II) > III), judging the compound to have weak developmental toxicity;
if III) > I) and III) > II), the compound is judged to be highly developmentally toxic.
The invention provides a chemical developmental toxicity detection model, which comprises the following components:
I)-1.104+0.686×log10ID0+0.641×log10ID20；
II)-12.023+36.626×log10ID0+29.189×log10ID20；
III)-1.252+5.331×log10ID0+2.537×log10ID20；
if I) > II) and I) > III), judging that the compound has no developmental toxicity;
if II) > I) and II) > III), judging the compound to have weak developmental toxicity;
if III) > I) and III) > II), judging the strong developmental toxicity of the compound;
wherein, ID20The chemical concentration to be measured is IC20When the expression level of MYL4 changes, ID0The chemical concentration to be measured is IC0The expression level of the corresponding MYL4 is changed; IC (integrated circuit)20The concentration of the chemical to be detected in the inhibition of the cell viability of 20% of human embryonic stem cells; IC (integrated circuit)0＝IC20/100。
The invention provides a method for establishing a chemical developmental toxicity detection model, which comprises the following steps:
(1) inducing undifferentiated human embryonic stem cells to differentiate into cardiomyocytes under culture conditions containing and without each modeling chemical, the developmental toxicity classification of which is known;
(2) measuring the expression level of MYL4 in the cardiomyocytes under culture conditions comprising each modeling chemical and in the absence of said modeling chemical; obtaining ID, which is the change in the expression level of MYL4 in cardiomyocytes under the culture conditions with each modeling chemical as compared to the culture conditions without the modeling chemical;
(3) according to the value of the ID, a typical discriminant function equation is constructed;
(4) and obtaining a Fisher discriminant function equation according to the typical discriminant function equation.
In one embodiment, the culture conditions without the modeling chemical are a blank of culture conditions containing each modeling chemical.
In one embodiment, in step (1), the modeling chemicals are concentrated separatelyDegree is IC20And the concentration of the chemical for modeling is IC0And inducing the undifferentiated human embryonic stem cells to differentiate into cardiomyocytes under culture conditions without a modeling chemical, wherein IC is20To model the concentration of chemicals at which cell viability of 20% human embryonic stem cells was inhibited; IC (integrated circuit)0＝IC20/100。
The IC20The determination method comprises the following steps: culturing undifferentiated human embryonic stem cells under the culture conditions of different concentrations of the chemical for modeling, and determining the concentration of the chemical for modeling at which 20% of the undifferentiated human embryonic stem cells are inhibited from cell viability, i.e., IC20。
In one embodiment, in step (2), the ID includes an ID20And ID0Wherein ID20Chemical concentrations for modeling as IC20When the expression level of MYL4 changes, ID0Chemical concentrations for modeling as IC0The expression level of the corresponding MYL4 was varied.
In one embodiment, the modeling chemical is selected from one or more of penicillin G, 6-aminonicotinamide, hydroxyurea, 5-fluorouracil, sodium saccharin, genistein, aspirin, all-trans retinoic acid, dexamethasone, digoxin, imipramine, doxorubicin, lithium chloride, and caffeine.
The chemical developmental toxicity detection device provided by the invention can realize the chemical developmental toxicity detection model.
The present invention provides a computer-readable storage medium having stored thereon a computer program which, when executed by a processor, implements the aforementioned chemical developmental toxicity detection model.
The computer processing device provided by the invention comprises a processor and the computer readable storage medium, wherein the processor executes a computer program on the computer readable storage medium, and can realize the chemical development toxicity detection model.
The invention provides an electronic terminal, comprising: a processor, a memory, and a communicator; the memory is used for storing a computer program, the communicator is used for being in communication connection with an external device, and the processor is used for executing the computer program stored by the memory so as to enable the terminal to execute the chemical development toxicity detection model.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
The cell material and reagents used in the present invention are described below:
1. human Embryonic stem cells (human Embryonic stem cell, hES)
The hES cell line H1(WA01) used in the present invention WAs purchased from the institute of biochemistry and cell biology, Shanghai Living sciences, China academy of sciences.
2. Culture solution and reagent
The culture Medium used in the present invention is DMEM/F12 (Dubecco's Modified Eagle's Medium), KO DMEM (KnockOut Dubecco's Modified Eagle's Medium), the formulations of which are well known in the art, and are not only described in detail in common textbooks and test manuals, but also commercially available from companies directly in the form of finished products (e.g., ThermoFisher company, USA).
The components that are supplements are any components that maintain or promote cell growth, for example, they may include, but are not limited to: amino acids, vitamins, proteins, trace elements, sugars, lipids, and the like.
The cytokines BMP-4 recombinant human protein, Activin A recombinant human protein and bFGF recombinant human protein used for hES culture and induced differentiation were purchased from ThermoFisher, USA.
The cell culture solution used in the present invention is as follows:
1) cell separation liquid a:
10mg/ml collagenase IV + 0.01% pancreatin.
2) Culture solution # 1: for routine culture of hES, the composition is as follows.
83% DMEM/F12 medium + 10% Fetal Bovine Serum (FBS) + 1% NEAA + 1% L-glutamine +50ng/ml basic fibroblast growth factor bFGF.
3) Culture solution # 2: the first phase for hES induces differentiation as follows.
87% DMEM medium + 10% FBS + 2% B27 supplement + 1% non-essential amino acids +40ng/ml activin A +250 μ g/ml ascorbic acid.
4) Culture solution # 3: for the second phase of hES induction differentiation, the composition is as follows.
87% RPMI1640 medium + 10% FBS + 2% B27 supplement + 1% non-essential amino acids +50ng/ml human insulin +60ng/ml bone morphogenic protein 4.
5) Culture solution # 4: the third stage for hES induced differentiation, with the following composition.
82% RPMI1640 medium + 15% FBS + 2% B27 supplement + 1% non-essential amino acids +10ng/ml human insulin +20ng/ml bone morphogenic protein 4.
EXAMPLE 1 culture of human embryonic Stem cells
hES will be provided in a state of normal growth after recovery, and when the cell density reaches above 70%, the cells will be seeded in pellet form into Matrigel pre-coated (self-contained, 1:20 diluted) culture dishes.
(1) Passage of hES: cells were passaged using cell isolate a.
On days 4-5 after the hES inoculation, the stem cells proliferated to a certain extent, and the formation of clones with larger diameter and higher density (about 70%) was observed in the culture dish, at which time the passaging was possible.
The hES medium to be passaged was aspirated and washed 2 times with D-PBS.
Adding 2ml of cell separation liquid A into a culture dish, placing the culture dish in a 37 ℃ and 5% CO2 incubator for incubation, taking out the culture dish after 20-25 minutes, observing that the clone structure is obviously loose, adding 6ml of culture liquid # 1 to stop digestion when the outer edge of the clone is curled, transferring the obtained cell mass into a centrifuge tube, rotating at 800 rpm, and centrifuging for 5 minutes at room temperature.
The supernatant was aspirated off, passaged according to the cell mass at a ratio of 1:4 to 1:8 to a petrigel-precoated dish, and cultured with an appropriate amount of hES medium.
Example 2 identification of pluripotency of human embryonic stem cells
In this example, the identification of pluripotency of human embryonic stem cells was carried out by using a rapid alkaline phosphatase (AKP) staining method (the kit was purchased separately).
(1) Preparing 5ml of 100mmol/L Tris-HCl, adjusting the pH value to 8.2, adding a staining reagent into the Tris-HCl according to the kit specification, and uniformly mixing the mixture for later use; sucking out the culture solution in the ES cell culture dish, and washing with PBS sterilized at high temperature and high pressure for 3 times at a time of 5 minutes to remove the influence of the culture solution on the dyeing effect;
(3) adding a proper amount of staining reagent into a culture dish, and incubating for 20 minutes in an incubator at 37 ℃ in a dark place;
(4) after being washed by PBS, 4 percent paraformaldehyde is added, and after 2 minutes, the mixture is washed by TBST and can be observed by a microscope.
EXAMPLE 3 induced differentiation of human embryonic Stem cells into cardiomyocytes
When 70% of the cells passaged with cell isolation solution A had grown, 10ml of culture solution # 2 was used per 10cm dish, and the temperature was set at 37 ℃ and 5% CO2The incubator continues to culture cells.
On day 3 of differentiation, the cells were further cultured in an amount of 10ml of culture solution #3 per 10cm dish.
On day 6 of differentiation, cells were further cultured in an amount of 10ml of culture medium # 4 per 10cm dish, and the culture medium was replaced once on each of day 9, day 11 and day 15 of differentiation.
Cells from day 15 to day 20 of differentiation were used for compound developmental toxicity testing.
Example 4 identification of cardiomyocytes derived from human embryonic Stem cells
The cells used in this example were derived from example 3.
(1) After the cell culture was completed, 4% paraformaldehyde was added, and the mixture was left at room temperature for 20 minutes, and washed with PBS for 3 times for 5 minutes each.
(2) After completion of washing, an identification antibody (FITC-labeled fluorescent antibody MYL4) was added, and after incubation at 37 ℃ for 2 hours, the cells were washed 3 times with PBS for 5 minutes each, and finally observed under a fluorescent microscope. The green fluorescence labeled cells are the myocardial cells.
Example 5 selection of developmental toxicity reference Compounds
The developmental toxicity reference compound of this example was selected with reference to the pregnancy medication safety classification issued by the U.S. food and drug administration. The safety of drug administration during pregnancy was classified into 5 classes, wherein class A and class B correspond to no developmental toxicity class in example 5, class C and class D correspond to weak developmental toxicity class in example 5, and class X corresponds to weak developmental toxicity class in example 5. Each compound was randomly divided into a building block and a validation block. The modeling group is used for building a typical rule discriminant function equation, and the verification group is used for verifying the stability of the typical rule discriminant function.
Specific information is shown in table 1:
TABLE 1
Example 6 detection of cytotoxicity of developmentally toxic reference Compounds on hES
(1) Undifferentiated hES cells were seeded into each well of a 96-well plate, 1-5 clones per well, and different concentrations of each developmentally toxic reference compound (see example 5) were added, while negative controls were set up, 5 replicate wells were set up for each well.
(2) On day 3 of culture, after washing the cells with PBS, the culture medium without serum and reference compound was added and the ratio of 1: alamar Blue reagent was added at 10 deg.C, 5% CO at 37 deg.C2Incubate in incubator for 2 h.
(3) After the 96-well plate is taken out, the plate is placed on a microplate reader, and the absorbance of each well is measured by using a double wavelength of 570nm/600 nm. Cell viability was calculated in each well according to Alamar Blue reagent instructions.
(4) The culture medium in the 96-well plate was aspirated and washed 3 times with PBS for 5 minutes each.
(5) To each well, 80. mu.l of RIPA protein lysate was added, incubated on ice for 10 minutes, and the protein content in each well was determined using the BCA protein assay kit.
(6) Cell viability was corrected for protein content and dose-response curves were plotted.
(7) Selection of 20% cell viability inhibitory dose (IC) for each reference Compound20) As a reference compound dose in the subsequent differentiation of hES into cardiomyocytes.
Example 7 detection of the MYL4 marker when a developmentally toxic reference compound acts on hES-derived cardiomyocytes
(1) hES in vitro inductionSee example 3 for methods of differentiation into cardiomyocytes. Separately exposing differentiated cells to the same Compound IC as in example 7 during culture20Toxicity at concentration test in the culture broth.
(2) On day 20 of differentiation, the cells were digested with pancreatin (0.25% containing 4U/ml DNase) preheated at 37 ℃ for 5min, after termination of the digestion with serum-containing medium, the centrifuge was set at 1000 rpm, centrifuged for 5min and the supernatant discarded.
(3) 1ml of 2% paraformaldehyde fixing solution was added to each tube, and after fixing at room temperature for 30min, the tubes were washed with PBS for 5 minutes each for 3 times.
(4) The membrane was broken with PBS containing 0.2% saponin for 10min, and then washed with PBS 3 times for 5min each.
(5) Blocking was incubated with blocking solution containing 0.2% saponin, 1% BSA and 10% goat serum to reduce non-specific binding.
(6) The FITC-labeled fluorescent antibody MYL4 was diluted with blocking solution at a ratio of 1:400 and incubated on ice for 2 hours in the absence of light.
(7) Washed 3 times with pre-cooled PBS for 5 minutes each time, centrifuged and the supernatant discarded.
(8) Resuspending the cells with 50. mu.l of pre-chilled PBS and detecting by flow cytometry, the yield was identical to the compound IC in example 720The ratio of MYL 4-labeled cells under the action of the compound at the concentration is compared with that of a control group to obtain a relative value of the influence of the compound on the differentiation process of the myocardial cells, namely an index ID (inhibition of differentiation) reflecting the degree of inhibition of differentiation.
Expression levels of MYL4 at the time of detection of the resulting ID0 and ID20 are shown in Table 2:
TABLE 2
| Compound (I) | ID0 | ID20 |
| PG | 1.04 | 1.02 |
| 6AM | 0.96 | 1.49 |
| HU | 1.29 | 0.95 |
| AS | 1.94 | 2.85 |
| RA | 1.21 | 0.43 |
| DEX | 0.70 | 7.55 |
| DG | 1.01 | 4.86 |
| IM | 2.22 | 2.67 |
| DOX | 0.87 | 1.99 |
| SA | 0.96 | 1.05 |
| FU | 1.51 | 0.74 |
| GE | 1.08 | 1.23 |
| LC | 1.35 | 2.12 |
| CF | 1.65 | 4.78 |
Example 8 establishment and validation of a mathematical model based on developmental toxicity reference Compounds and MYL4 detection indices
The data used to build the mathematical model in this example was derived from example 7. The classification is according to see example 5.
Three classes of developmental toxic compounds (no developmental toxicity, weak developmental toxicity, strong developmental toxicity) were classified by linear discriminant analysis and 3 Fisher discriminant function equations were constructed.
In order to realize the visualization of the discrimination result, the obtained linear discrimination function equation is displayed in the form of 2 canonical discriminant functions, and is respectively used as a horizontal coordinate and a vertical coordinate to draw a scatter diagram and a decision boundary (see the attached figure 2). The classification result of the developmental toxicity prediction model constructed based on the linear discriminant analysis is verified by a model self-identification method, and the stability of 2 typical discriminant functions is verified by a leave-one-out cross validation (LOOCV).
The mathematical modeling and the chart drawing in the embodiment are completed in an open source software R language environment and are realized through lda algorithm of MASS package. The complete program code is shown in the appendix.
The linear discriminant function equation constructed in this embodiment has the discriminant function LD in the form:
LD1＝1.061×log10ID0+1.334×log10ID20
LD2＝0.838×log10ID0–0.221×log10ID20
the characteristic value (Eigenvalue) of the discriminant function is used for reflecting the discriminative power of the established function, and the larger the value is, the stronger the discriminative power of the discriminant function is. The characteristic values of the LD1 and the LD2 are 5.787 and 0.15 respectively, which indicates that the discriminant function has the capability of distinguishing 3 types of test compounds; typical Correlation coefficients (Canonical Correlation) of LD1 and LD2 are 0.923 and 0.121, respectively, which suggests that the discriminant function LD1 has a high degree of linear Correlation and LD2 has a low degree of linear Correlation.
And (3) respectively taking values of the 2 dictionary judgment function formulas as horizontal and vertical coordinates, and drawing a scatter diagram and a decision boundary, which are shown in the attached figure 2. It can be seen that LD1 mainly contributes to the differentiation of non-developmental toxic, weakly developmental toxic and strongly developmental toxic compounds, with high eigenvalues and strong discriminative power; LD2 contributes primarily to the discrimination between non-developmentally toxic compounds and developmentally toxic compounds, with lower characteristic values and weaker discriminatory power due to the far fewer number of non-developmentally toxic compounds than with developmentally toxic compounds.
The Wilks' lambda value is an index for checking whether the established discrimination model is significant or not, reflects the ratio of the square sum in the group to the total square sum, and is between 0 and 1; when the value of Wilks 'lambda is 1, it means that the means of all groups are equal, and when the value of Wilks' lambda is 0, it means that the means of all groups are not equal. The Wilks' lambda values of LD1 and LD2 constructed in this example were 0.145 and 0.985, respectively, and the p values were both less than 0.05, suggesting that the constructed canonical discriminant function formula contributes to different groups 3 of compounds, and the compound effects of no developmental toxicity, weak developmental toxicity and strong developmental toxicity can be effectively distinguished.
The classification result of the developmental toxicity prediction mathematical model constructed based on the linear discriminant analysis is firstly verified by a model self-identification method, the stability of 2 canonical discriminant functions is verified by an LOOCV method, and the result shows that the average error rate of the established discriminant functions for distinguishing the compounds without developmental toxicity, weak developmental toxicity and strong developmental toxicity is 14.29 percent and is lower than the standard of 20 percent, so that the constructed mathematical model is considered to have excellent stability.
For convenience of use, the LD function constructed in this example is set forth and applied with the Fisher discriminant function equation:
I)-1.104+0.686×log10ID0+0.641×log10ID20；
II)-12.023+36.626×log10ID0+29.189×log10ID20；
III)-1.252+5.331×log10ID0+2.537×log10ID20。
the criteria for the developmental toxicity of the compounds are:
if I) > II) and I) > III), judging that the compound has no developmental toxicity;
if II) > I) and II) > III), judging the compound to have weak developmental toxicity;
if III) > I) and III) > II), the compound is judged to be highly developmentally toxic.
After the mathematical model is established, the total accuracy of the model is analyzed by using the compounds of the verification group.
First, the Fisher discriminant function equation established in this example is used to calculate the data of each compound, and the results are shown in table 2:
TABLE 3
| Grouping | I | II | III | Predictive classification | Actual classification | |
| PG | Building module | -1.09 | -11.18 | -1.14 | Is free of | Is free of |
| 6AM | Building module | -1.01 | -7.61 | -0.90 | High strength | High strength |
| HU | Building module | -1.04 | -8.64 | -0.72 | High strength | High strength |
| AS | Building module | -0.62 | 11.79 | 1.44 | Weak (weak) | Weak (weak) |
| RA | Building module | -1.28 | -19.73 | -1.75 | Is free of | High strength |
| DEX | Building module | -0.65 | 7.83 | 0.14 | Weak (weak) | Weak (weak) |
| DG | Building module | -0.66 | 8.14 | 0.51 | Weak (weak) | Weak (weak) |
| IM | Building module | -0.59 | 13.14 | 1.68 | Weak (weak) | Weak (weak) |
| DOX | Building module | -0.95 | -5.53 | -0.82 | High strength | High strength |
| SA | Building module | -1.10 | -11.94 | -1.28 | Is free of | Is free of |
| FU | Authentication group | -1.07 | -9.28 | -0.63 | High strength | High strength |
| GE | Building module | -1.02 | -8.17 | -0.85 | High strength | High strength |
| LC | Authentication group | -0.81 | 2.28 | 0.27 | Weak (weak) | Weak (weak) |
| CF | Authentication group | -0.52 | 15.77 | 1.63 | Weak (weak) | Weak (weak) |
Based on the Fisher discriminant function constructed in this example, three function values of the reference compound were calculated, and the accuracy of the predicted classification result and the actual classification result were compared, and the results are shown in table 4:
TABLE 4
In the above examples, examples 1 to 7 are experimental procedures required for compound testing. In the embodiment 8, the developmental toxicity prediction equation is required to be obtained, and the actual operation and the use do not need to be repeated, and the developmental toxicity prediction equation is directly used for calculation.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (8)
1. A method for detecting developmental toxicity of chemicals is characterized by at least comprising the following steps:
(1) inducing the undifferentiated human embryonic stem cells to differentiate into the myocardial cells under the culture conditions of the chemicals to be detected and the chemicals without being detected respectively;
(2) respectively measuring the expression level of MYL4 in the myocardial cells under the culture conditions of the chemicals to be measured and the culture conditions of no chemicals to be measured; obtaining the expression quantity change of MYL4 in the cardiac muscle cells, namely ID, compared with the culture condition without the chemical to be detected under the culture condition with the chemical to be detected;
(3) according to the value of the ID, the development toxicity of the chemical to be detected is judged by combining a Fisher discriminant function equation;
in the step (1), the concentrations of the chemicals to be measured are IC20The concentration of the chemical to be measured is IC0And inducing undifferentiated human embryonic stem cells to differentiate into cardiomyocytes under the culture condition without the chemicals to be tested, wherein IC20The concentration of the chemical to be detected in the inhibition of the cell viability of 20% of human embryonic stem cells; IC (integrated circuit)0＝IC20/100；
In step (2), the ID includes an ID20And ID0Wherein ID20The chemical concentration to be measured is IC20When the expression level of MYL4 changes, ID0The chemical concentration to be measured is IC0The expression level of the corresponding MYL4 is changed;
the developmental toxicity classification of the chemical to be detected comprises no developmental toxicity, weak developmental toxicity and strong developmental toxicity;
the Fisher discriminant function equation is as follows:
I)-1.104+0.686×log10 ID0+0.641×log10 ID20；
II)-12.023+36.626×log10 ID0+29.189×log10 ID20；
III)-1.252+5.331×log10 ID0+2.537×log10 ID20；
the method for judging the developmental toxicity of the chemical to be detected comprises the following steps:
if I) > II) and I) > III), judging that the compound has no developmental toxicity;
if II) > I) and II) > III), judging the compound to have weak developmental toxicity;
if III) > I) and III) > II), the compound is judged to be highly developmentally toxic.
2. The method of detecting developmental toxicity of a chemical of claim 1, wherein the IC is20The determination method comprises the following steps: culturing undifferentiated human embryonic stem cells under the culture conditions of chemicals to be tested with different concentrations, and determining the concentration of the chemicals to be tested at which 20% of the cells of undifferentiated human embryonic stem cells are inhibited by activity, namely IC20。
3. The method for detecting developmental toxicity of a chemical of claim 1 wherein the chemical is selected from the group consisting of simple substances, compounds and mixtures.
4. A method for detecting developmental toxicity of a chemical, the method comprising the steps of: the developmental toxicity of the chemical to be tested is judged by using the following conditions:
I)-1.104+0.686×log10 ID0+0.641×log10 ID20；
II)-12.023+36.626×log10 ID0+29.189×log10 ID20；
III)-1.252+5.331×log10 ID0+2.537×log10 ID20；
if I) > II) and I) > III), judging that the compound has no developmental toxicity;
if II) > I) and II) > III), judging the compound to have weak developmental toxicity;
if III) > I) and III) > II), judging the strong developmental toxicity of the compound;
wherein, ID20The chemical concentration to be measured is IC20When the expression level of MYL4 changes, ID0The chemical concentration to be measured is IC0The expression level of the corresponding MYL4 is changed; IC (integrated circuit)20To be testedConcentration of chemical at which cell viability of 20% human embryonic stem cells is inhibited; IC (integrated circuit)0＝IC20/100。
5. A chemical developmental toxicity detection apparatus capable of implementing the method for detecting chemical developmental toxicity according to claim 4.
6. A computer-readable storage medium, on which a computer program is stored which, when being executed by a processor, carries out the method for detecting developmental toxicity of chemicals according to claim 4.
7. A computer processing device comprising a processor and the aforementioned computer readable storage medium, the processor executing a computer program on the computer readable storage medium, capable of implementing the method of detecting developmental toxicity of a chemical of claim 4.
8. An electronic terminal, comprising: a processor, a memory, and a communicator; the memory is used for storing a computer program, the communicator is used for being in communication connection with an external device, and the processor is used for executing the computer program stored by the memory so as to enable the terminal to execute the method for detecting the developmental toxicity of chemicals in claim 4.
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