CN113930388A - Chemical embryotoxicity prediction model and establishment method thereof - Google Patents
Chemical embryotoxicity prediction model and establishment method thereof Download PDFInfo
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Abstract
A chemical embryotoxicity prediction model includes a cell species; this cell type included differentiated cardiomyocytes induced by SP3 ES cells with karyotype XX. The establishment method comprises the steps of firstly, separating and culturing mouse embryo fibroblasts; secondly, in-vitro amplification culture of mouse ES; thirdly, karyotype analysis and sex identification of the mouse ES; fourthly, maintaining and identifying mouse ES pluripotency; fifthly, inducing and differentiating the mouse ES in vitro into myocardial cells; sixthly, immunochemical detection of the myocardial specific marker protein of mouse ES induced differentiated myocardial cells; seventhly, Real-time PCR detection of the expression profile of mouse ES-induced differentiated cardiomyocytes; eighthly, selecting a mode compound; ninth, detection of cytotoxicity of model compounds; tenthly, detecting the inhibitory effect of the model compound on the ES differentiation capacity; eleventh, evaluation of model compound embryotoxicity. Filling the blank of the embryonic stem cells of the female-free mice in the EST system.
Description
The present application is a divisional application of the following applications: application date: 8 months and 7 days in 2015; application No.: 201510478375.3, respectively; the invention discloses a model for predicting embryotoxicity of chemicals and an establishing method thereof.
Technical Field
The invention relates to a model for predicting embryotoxicity of chemicals and a method for establishing the model, in particular to a model for directionally inducing and differentiating mouse embryonic stem cells into myocardial cells with beating capacity, establishing a model for identifying and predicting embryotoxicity of chemicals on the obtained myocardial cells, and applying the model to the prediction of embryotoxicity of new chemicals.
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. From the principle of animal protection, Russell and Burch advocate the "3R" principle, namely Reduction (Reduction), optimization (optimization) and substitution (Replacement), and toxicology substitution is of great importance to the evaluation and management of the damage of exogenous chemicals, both from a scientific point of view and from an economic point of view.
Estimated by the european regulatory, assessment and restriction agency (REACH), over the next 15 years there will be 30000 chemicals to be tested for safety, and 7 million animals will be sacrificed according to existing testing 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 increases in 1000 per year, with more than 90% of these being free of safety assessment 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.
Since the european union implemented animal welfare guidelines in 1986, the european union has strongly supported the development and use of alternatives. Embryonic stem cell test (EST) is a type of experimental method for predicting potential embryotoxicity of compounds, which is verified by European Alternatives research center (EURL-ECVAM). The method fully utilizes the differentiation potential of the embryonic stem cells, sets an observation terminal point of differentiation, combines general toxicology data of the cells, and evaluates and classifies the potential embryotoxicity of the compound through a discrimination equation, thereby realizing the safety evaluation of the compound, and belonging to a substitute system of a test method. By means of EST, chemical exposure is carried out at different stages of induction or in different induction directions, the influence of the compound on the development of organisms in the reaction differentiation process can be simulated, and the method has unique advantages in toxicological researches such as toxicity screening, toxic target organ determination, toxic action mechanism and the like. With the continuous deepening of the international stem cell biological research, the embryonic stem cell model is further developed, namely, a target organ is searched by using the differentiation potential of the embryonic stem cell model or the expression of key genes in the differentiation process is monitored, the influence of a compound on organ tissues such as liver, muscle, blood vessel, bone and the like is found, and the potential toxicity of the compound on the corresponding organ tissues can be respectively evaluated by selecting a plurality of observation endpoints. The whole evaluation period is between 10 and 21 days, living animals are not needed in the whole experiment process, the labor, material, financial and time are saved, the repeatability is strong, the operation is easy, and the requirement of compound toxicity evaluation alternative research in the world is met, so that the wide attention is drawn.
EST is suitable for analyzing development process from cell level and judging whether a certain factor has embryo poison or teratogenicity potential. At present, EST multi-selection cardiomyocytes are used as a measurement index after differentiation and development, and the reason is that heart development starts at an early embryonic development stage, so that the heart development process in vitro by inducing embryonic stem cells (ES or ESC for short) to differentiate into cardiomyocyte mimics is easy to realize; secondly, the heart development is a highly specialized process, and the heart development can be abnormal due to slight interference on the development process, so that the sensitivity is high; third, since ES-induced cardiomyocytes have a beating ability, they have good visibility as an observation end point.
To verify the accuracy of EST, Scholz performed the following experiments: 1. the cytotoxic effect of the test agent on differentiated 3T3 fibroblasts; 2. the cytotoxic effect of the test agent on undifferentiated ES cells; 3. effect of the test substance on ES cell differentiation into cardiomyocytes. Corresponding in vivo test data are taken as the basis, among 10 tested substances, 100% of non-embryotoxic substances are accurately judged, 88.9% of weak embryotoxic substances are accurately judged, and 91.7% of strong embryotoxic substances are accurately judged. On the basis of the experimental data, a discriminant equation model is established by combining in-vivo experimental information of a tested object, Genschow and the like, and the embryotoxicity of the compound is subjected to three-stage prediction, namely the compound is judged to be non-embryotoxicity, low-embryotoxicity or high-embryotoxicity. EST has a function of screening compounds for embryotoxicity, and thus is used by various chemical manufacturers, pharmaceutical factories, and the like in the world for screening new materials or drugs and for safety testing of embryotoxicity.
Through a great deal of research, many scientists working internationally on optimizing EST propose that the following two points exist in the standardized operation flow of EST to be optimized: firstly, EST is established and popularized based on an ES cell line D3, and other ES cell lines are suggested to be adopted to establish a model at the same time, so that the accuracy of an evaluation result is ensured; second, the observation of the pulsation of the cardiomyocytes is greatly affected by subjective factors, so molecular biological indicators are suggested as the observation end points to improve accuracy.
At present, in other studies than the D3 cell line, ES cell lines such as J1, R1, E14TG2a, E14.1, DBA/1lacZ were used, and it was found that different cell lines had different sensitivities to induce differentiation and caused differences in the time of differentiation into the beating-competent myocardium. However, these used ES cell lines are all XY karyotypes, i.e., ES that are all "male". There is no precedent for establishing an embryotoxicity evaluation model and applying the model by using an ES cell line with a karyotype XX.
The expression of key specific genes in ES-induced cardiomyocytes detected by a molecular biology means can more accurately establish a dose-effect curve, and in the experiment of Seiler et al, the relevant data of the tested compound for inhibiting cardiac development obtained by a molecular observation endpoint is emphasized, so that a good dose-effect curve and the consistency with the traditional observation endpoint in the evaluation result are obtained. However, the existing EST only uses the gene Myh6 coding the heavy chain of the cardiac muscle protein as a molecular observation terminal, and whether the expression condition of other genes can be used as the molecular observation terminal for EST prediction of the embryotoxicity of chemicals is still to be confirmed.
Disclosure of Invention
In order to make up for the defects existing in the prior art, the invention discloses a chemical embryotoxicity prediction model, which comprises cell types; this cell type included differentiated cardiomyocytes induced by SP3 ES cells with karyotype XX.
Further, molecular observation of an end point is also included; the molecular observed endpoint included Myl 4.
Furthermore, the cell types also include cardiomyocytes induced by XY karyotype-induced differentiation of R1 ES cells.
Further, the molecular observed end point also includes cTnT and/or Myh 6.
Further, the upstream primer sequence of Myl4 is also included: AAGAAACCCGAGCCTAAGAAGG, and the downstream primer sequence of Myl 4: TGGGTCAAAGGCAGAGTCCT are provided.
Further, an upstream primer sequence of cTnT is also included: CAGAGGAGGCCAACGTAGAAG, cTnT downstream primer sequence: CTCCATCGGGGATCTTGGGT, upstream primer sequence of Myh 6: GCCCAGTACCTCCGAAAGTC and Myh 6: GCCTTAACATACTCCTCCTTGTC are provided.
The invention also discloses a method for establishing the chemical embryotoxicity prediction model, which is characterized by comprising the following steps of:
the method comprises the following steps: separating and culturing mouse embryo fibroblast;
step two: performing in-vitro amplification culture on the mouse ES;
step three: karyotyping and sex identifying mouse ES;
step four: maintenance and identification of mouse ES pluripotency;
step five: mouse ES is induced and differentiated into myocardial cells in vitro;
step six: immunochemical detection of myocardial specific marker proteins of mouse ES-induced differentiated cardiomyocytes;
step seven: real-time PCR detection of the expression profile of mouse ES-induced differentiated cardiomyocytes;
step eight: selection of model compounds: selecting 4 or more of the model compounds such that the set of model compounds comprises a teratogenic compound, and a teratogenic compound that differs in sex;
step nine: detection of cytotoxicity of model compounds: obtaining a half-lethal dose IC of each model compound on cells50；
Step ten: examination of inhibitory Effect of model Compounds on ES differentiation Capacity: half inhibitory dose ID of each model compound on ES cell differentiation was obtained50A value of (d);
step eleven: evaluation of model Compound embryotoxicity: IC obtained by step nine50And the ID obtained in step ten50Analyzing the influence degree of each model compound on the embryotoxicity, and establishing and obtaining a chemical embryotoxicity prediction model, thereby popularizing the prediction on the embryotoxicity of other chemicals;
the ES cells include SP3 ES cells.
Further, the method also comprises the step twelve: the molecular observation end point and the traditional observation end point are used for reflecting the correlation analysis of the model compound under the inhibition effect on the ES differentiation capacity, namely, the molecular observation end point and the traditional observation end point are used for measuring the correlation between half of the differentiation inhibition capacity by adopting the function of a matched scatter diagram of SPSS19.0 software, and meanwhile, a determining coefficient is calculated; when the determination coefficient between the molecular observation terminal point and the traditional observation terminal point is more than 0.9, the molecular observation terminal point and the traditional observation terminal point are highly correlated; therefore, the accuracy and the effectiveness of the chemical embryotoxicity prediction model are judged.
Further, the myocardial specific marker protein in the sixth step includes cTnT; the expression profile in step seven comprises Myh6, Myl4 and cTnT genes; the model compound in the step eight comprises 5-fluorouracil, aspirin, indomethacin, ascorbic acid, penicillin G; the fourth step comprises taking NIH 3T3 fibroblast as control; the ES also includes R1 ES cells.
Further, in the ninth step, half lethal dose IC of each model compound on 3T3 cells and ES cells was obtained503T3 and IC50ES; in the above step ten, half inhibitory dose ID of each model compound on ES cell differentiation was obtained50A value of (d); in the step eleven, the IC is processed503T3、IC50ES and ID50Data are substituted into the following discriminants:
(1)5.916lg(IC503T3)+3.500lg(IC50ES)－5.307[(IC503T3－ID50)/IC503T3]－15.27
(2)3.651lg(IC503T3)+2.394lg(IC50ES)－2.033[(IC503T3－ID50)/IC503T3]－6.85
(3)－0.125lg(IC503T3)－1.917lg(IC50ES)+1.500[(IC503T3－ID50)/IC503T3]－2.67
the embryo toxicity judgment standard is as follows:
if (1) > (2) and (1) > (3), judging the compound to be non-development toxicity;
if (2) > (1) and (2) > (3), determining the compound as weakly developmentally toxic;
if (3) > (1) and (3) > (2), the compound is judged to be highly developmentally toxic.
A preferred embodiment of the present invention is directed to the creation of a new, viable EST model using both sexed ES, using R1 ES and SP3 ES cell lines other than D3 ES, and focusing primarily on whether the newly created model has consistent results in the evaluation of chemicals. The invention firstly utilizes SP3 ES cells with the karyotype XX to establish an embryotoxicity evaluation model, and also utilizes R1 ES cells with the karyotype XY to establish the embryotoxicity evaluation model which is used as the contrast of SP3 ES and the embryotoxicity evaluation model; meanwhile, by comparing the two evaluation models and related data, whether sex difference exists in embryotoxicity of the tested chemicals can be distinguished.
One of the coding genes of the myocardial-specific myosin light chain, Myl4 and the troponin coding gene cTnT are introduced for the first time as molecular observation endpoints for observing the influence of the tested compound on the cardiac development, and the correlation between different molecular observation endpoints and the traditional observation endpoint is further verified so as to explore the feasibility of replacing the traditional observation endpoint with the molecular observation endpoint for EST embryo toxicity evaluation.
The technical problem to be solved by the invention is as follows:
1. the pluripotent maintenance of mouse embryonic stem cells of two sexes in the in vitro culture process, and the application of inducing differentiation into myocardial cells on the basis;
2. the application of mouse embryonic stem cells of two sexes in an experimental model for evaluating the embryotoxicity of a compound in vitro;
3. the application of a compound embryotoxicity evaluation model established based on two sex mouse embryonic stem cells in the evaluation of the embryotoxicity of a new compound;
4. the application of a compound embryotoxicity evaluation model established based on two sex mouse embryonic stem cells in distinguishing whether the embryotoxicity of chemicals is different between sexes;
5. in order to facilitate the implementation of high-throughput screening, the molecular observation end point is used for replacing the traditional observation end point in a compound embryotoxicity evaluation model established based on the embryonic stem cells of mice with two sexes;
6. the invention relates to application of a compound embryotoxicity evaluation model established based on two sex mouse embryonic stem cells in screening, toxicity testing and toxicology research of medicines, chemical raw materials or new chemical substances.
The invention has the beneficial effects that:
1. establishing a new model which can be used for evaluating the embryotoxicity of chemicals;
2. the model can reflect whether sex difference exists in embryotoxicity caused by chemicals, and fills the blank of the embryonic stem cells of the female-free mice in an embryonic stem cell test system;
3. the model can utilize a molecular observation terminal to replace the traditional observation terminal to carry out evaluation, and is more beneficial to carrying out high-throughput screening on related toxicity of chemicals;
4. the model can be applied to screening, toxicity testing and toxicology research in medicines, chemical raw materials or new chemical substances.
The invention firstly utilizes SP3 ES cells with the karyotype XX to establish an embryotoxicity evaluation model, and also utilizes R1 ES cells with the karyotype XY to establish the embryotoxicity evaluation model which is used as the contrast of SP3 ES and the embryotoxicity evaluation model;
according to the invention, through comparing the two evaluation models and related data, whether sex difference exists in embryotoxicity of tested chemicals can be distinguished.
The invention also introduces a new molecular observation terminal for the first time, further verifies the correlation between different molecular observation terminals and the traditional observation terminal, and verifies the feasibility of replacing the traditional observation terminal with the molecular observation terminal for EST embryotoxicity evaluation.
Drawings
FIG. 1 is an electron micrograph of R1 ES cells cultured on trophoblast cells.
FIG. 2 is an electron micrograph of SP3 ES cells cultured on trophoblast cells.
FIG. 3 is a karyotype analysis chart of R1 ES cells.
FIG. 4 is a karyotype analysis chart of SP3 ES cells.
Fig. 5 is an electrophoretogram of PCR products of sex-specific markers Zfx and Sry for R1 ES cells and SP3 ES cells, respectively.
FIG. 6 is a graph showing the results of alkaline phosphatase staining of R1 ES cells.
FIG. 7 is a graph showing the results of alkaline phosphatase staining of SP3 ES cells.
FIG. 8 is a graph showing the results of alkaline phosphatase staining of NIH 3T3 fibroblasts.
FIG. 9 is an electrophoretogram of RT-PCR pluripotency gene products of R1 ES cells, SP3 ES cells, and NIH 3T3 fibroblasts.
FIG. 10 is an immunofluorescent staining pattern of R1 ES cell differentiated cardiomyocytes.
FIG. 11 is an immunofluorescent staining pattern of differentiated cardiomyocytes of SP3 ES cells.
FIG. 12 is a bar graph of the expression profiles of quantitative PCR detection of myocardial specific markers for R1 ES cells and SP3 ES cells.
FIG. 13 is a dose-response curve of R1 ES cells exposed to different concentrations of 5-fluorouracil.
Fig. 14 is a dose-response curve for exposure of R1 ES cells to different concentrations of aspirin.
Fig. 15 is a dose-response curve of R1 ES cells exposed to different concentrations of indomethacin.
FIG. 16 is a dose-response curve of R1 ES cells exposed to varying concentrations of ascorbic acid.
FIG. 17 is a dose-response curve of R1 ES cells exposed to different concentrations of penicillin G.
FIG. 18 is a dose-response curve of SP3 ES cells exposed to different concentrations of 5-fluorouracil.
Fig. 19 is a dose-response curve of SP3 ES cells exposed to different concentrations of aspirin.
FIG. 20 is a dose-response curve of SP3 ES cells exposed to different concentrations of indomethacin.
FIG. 21 is a dose-response curve of SP3 ES cells exposed to different concentrations of ascorbic acid.
FIG. 22 is a dose-response curve of SP3 ES cells exposed to different concentrations of penicillin G.
FIG. 23 is a schematic diagram of a model for predicting embryotoxicity of chemicals according to a preferred embodiment of the present invention.
FIG. 24 is a graph of correlation analysis between different observed endpoints.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments in conjunction with the accompanying drawings.
The cell material and reagents used in the present invention are described below:
1. mouse Embryonic stem cell (Embryonic stem cell, ES)
The ES R1 cell line used in the present invention was purchased from the institute of biochemistry and cell biology of Shanghai Life science institute of Chinese academy of sciences, and the SP3 cell line was offered as a gift to the Jinying researchers of Shanghai university of transportation medical institute/institute of health science of Chinese academy of sciences.
2. Culture solution and reagent
The culture solution used in the present invention is DMEM (Dubecco's Modified Eagle's Medium), KO DMEM (KnockOut Dubecco's Modified Eagle's Medium), and the formulations of these media are well known in the art, and not only are described in detail in general textbooks and test manuals, but also can be directly obtained commercially from companies in the form of finished products (e.g., Life Techoology).
The components that act as supplements may be 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.
Leukemia inhibitory factor (leukemia inhibitory factor) used for ES culture was purchased from Millipore.
The cell culture solution used in the present invention is as follows:
1) nutrient layer cell culture solution
89% DMEM medium + 10% newborn bovine serum (New-born bovine serum, NBS) + 1% Nonessential amino acids (NEAA).
2) ES cell culture solution
83% KO DMEM medium + 15% Fetal Bovine Serum (FBS) + 1% NEAA + 1% L-glutamine +105U LIF。
3) Differentiation culture solution
79% DMEM medium + 20% FBS + 1% NEAA.
3. Purchase of laboratory animals
Clean-grade Balb/c pregnant mice (pregnant for 11-13 days) were purchased from Shanghai Slek laboratory animals, Inc.
Example 1: separation and culture of mouse embryo fibroblast
In the present invention, Mouse Embryonic Fibroblasts (MEFs) are used as feeder cells for culturing ES.
1.1 isolation method of MEF primary cells:
(1) taking a beaker, filling 50ml of 75% ethanol, soaking surgical instruments such as scissors and forceps, roasting and sterilizing the soaked instruments by an alcohol lamp, and cooling for later use.
(2) A plurality of bacterial culture dishes with the diameter of 60mm are prepared and respectively arranged as a No. 1 dish, a No. 2 dish and a No. 3 dish, and a proper amount of PBS sterilized at high temperature and high pressure is respectively filled in the bacterial culture dishes, and a proper amount of penicillin-streptomycin solution is added in the PBS. Wherein, the No. 1 dish is used for containing the uterus taken out when dissecting pregnant mice, the No. 2 dish is used for containing the embryo torn from the fetal membrane, and the No. 3 dish is used for containing the treated embryo trunk part.
(3) After anaesthetizing the pregnant rats, the skin was wiped with 75% ethanol, the skin was cut vertically, the abdominal cavity was opened, and the entire uterus was removed, taking care not to touch the skin and non-sterile areas. After the cervix and blood vessels were excised, they were placed in a No. 1 dish.
(4) The uterine tissue between the embryos was cut, the embryos were placed in No. 2 dishes, the embryos were squeezed out of the uterine sheets with forceps, maternal blood cells on the embryos were washed with PBS, the embryos were removed from the viscera, limbs and neck by forceps, and the embryos were placed in No. 3 dishes.
(5) Fully shearing the trunk part of each embryo body by using a sterilized surgical scissors in a super clean bench, digesting for 5 minutes by using a proper amount of preheated 0.25% trypsin, and adding a trophoblast culture solution according to a ratio of 1:2 to terminate digestion.
(6) The digested tissue mass and cells were transferred to a 15ml sterile centrifuge tube with a 10ml pipette, gently aspirated several times, placed in a tabletop centrifuge, and centrifuged at 21 ℃ for 5 minutes at 800 rpm.
(7) After centrifugation, the supernatant was discarded. Resuspending the cells in 10ml of culture medium, transferring to a 100mm diameter cell culture dish, labeled 0 passage, and placing at 37 deg.C with 5% CO2Culturing in an incubator.
1.2 passage and cryopreservation of MEF:
(1) cells were observed daily under a light microscope and passaged after plating out the dish (about 48 hours).
(2) The culture medium was aspirated, washed with autoclaved PBS and aspirated, then 3ml of pre-warmed 0.25% trypsin was added, placed in an incubator at 37 ℃, removed after 1 minute and 30 seconds, added in the culture medium at a ratio of 1:2 to terminate digestion, transferred into a 15ml centrifuge tube, and centrifuged at 21 ℃ for 5 minutes at 800 rpm.
(3) After centrifugation, the supernatant was discarded. Resuspend cells in 9ml of culture medium, 1: transferring 3 proportion into 3 cell culture dishes with diameter of 100mm, supplementing culture solution until the culture solution is submerged at the bottom of the culture dish, marking as 1 generation, and culturing in a 5% CO2 incubator at 37 ℃.
(4) The cells were passaged by repeating the first 3 steps and labeled 2 passages.
(5) Because the 3 rd to 5 th generations of MEF cells are suitable for being used as trophoblast cells of embryonic stem cells, partial cells needing to be cultured are subjected to passage and subsequent operation, the rest cells are subjected to cryopreservation operation, and then are recovered when the cells need to be used later.
(6) Digesting and resuspending cells according to the steps, counting the cells, adjusting the cell density to be 2x106 cells/ml, centrifuging, discarding the supernatant, adding an equal amount of cryopreservation liquid (the formula of the cryopreservation liquid is 99.25% FBS + 0.75% DMSO), flicking, uniformly mixing, adding 1ml of cell suspension into each cryopreservation tube, covering, marking information such as cell types, algebra numbers, dates, name of a cryopreserving person and the like on the outer wall of each tube, wrapping the cryopreservation tubes with paper, placing the tubes in a refrigerator at minus 80 ℃ for overnight, and then placing the tubes into a liquid nitrogen tank for cryopreservation.
Example 2: in vitro amplification culture of mouse ES
2.1 preparation of trophoblast cells
(1) Selecting MEF cells with proper algebra and density, adding mitomycin C into a cell culture solution according to the working concentration of 10 mu g/ml, uniformly mixing, and placing in an incubator for continuous culture.
(2) After 2 hours, the culture medium containing mitomycin C was aspirated and washed 5 times with autoclaved PBS, each for 5 minutes.
(3) After washing, the culture medium is replaced with fresh one, and the ES cells are recovered or the culture is continued. The treated trophoblast cells supported both growth and maintenance of the undifferentiated state of mouse ES cells within 5 days.
2.2 Resuscitation of ES cells
(1) Taking out the ES cell freezing tube from the liquid nitrogen, putting the ES cell freezing tube into a water bath kettle at 37 ℃ for dissolving, taking out the ES cell freezing tube when the ES cell freezing tube is dissolved to be in a small ice crystal state, and putting the ES cell freezing tube into a super clean bench after sterilization treatment.
(2) After opening the cryopreservation tube, pressing 1: adding the cell culture solution according to the proportion of 1, completely melting the added frozen stock solution, slightly blowing the culture solution, transferring the culture solution into a 15ml centrifuge tube, centrifuging at the temperature of 21 ℃ for 5 minutes at 800 r/min, removing the supernatant, and adding 5ml of ES culture solution to resuspend the suspension into single cell suspension. Care should be taken to avoid the generation of bubbles during the blowing process.
(3) The medium in MEF trophoblast cells was aspirated, and 5ml of ES medium was added after the ES cell suspension was transferred to trophoblast.
(4) Observing the formation condition of embryonic stem cell clone under a light microscope every day, and changing the liquid half a day; and (5) carrying out passage when the length reaches about 70%.
(5) This procedure revives ES R1 cells and ES SP3 cells between 20 and 25 passages, respectively, for subsequent experiments.
2.3 removal of trophoblasts
When the ES cells are proliferated to a required amount in a cloned manner, the trophoblast cells can be removed by a differential adherence method, so that the purity of the ES cells for subsequent experiments is ensured. The principle of the differential adherence method is to utilize the difference of adherence performance of mixed type cells and obtain target cells from the mixed type cells by controlling adherence time. The method comprises the following steps:
(1) pre-spreading 0.1% gelatin in a cell culture dish with a diameter of 100mm, placing in a cell culture box for 30min or more, taking out, and sucking out the residual liquid for later use.
(2) When ES cells were grown to a suitable density on the feeder layer, they were digested into single cells with 3ml of 0.25% trypsin preheated at 37 ℃ and then resuspended in 6ml of culture medium at a ratio of 1: 3, transferring the mixture into a cell culture dish coated by gelatin, properly supplementing a culture solution until the mixture just submerges the bottom of the culture dish, and putting the culture dish into a cell culture box for continuous culture.
(3) After 1 hour, the culture dish was taken out, the supernatant was collected, centrifuged, discarded, resuspended in ES culture medium, and transferred to a gelatin-coated culture dish for culture. The part of cells are high-purity ES cells.
The trophoblast cells and the R1 ES cells were cultured as shown in FIG. 1. The trophoblast cells and SP3 ES cells were cultured as shown in FIG. 2.
Example 3: karyotyping and sex determination of mouse ES
3.1 karyotyping
(1) Culturing the ES cells on a culture dish coated by gelatin, and performing subsequent operation after the cells cover more than 80% of the area of the culture dish;
(2) according to the result of the growth curve described in 2.4, 50ng/ml colchicine was added in the logarithmic growth phase and the culture was continued for 2 hours.
(3) After the culture solution is completely sucked, EDTA-pancreatin digestive juice is added, the mixture is placed in a constant temperature incubator at 37 ℃ for 5 minutes, then a pipette is used for gently blowing the mixture, and then the cell suspension is collected into a graduated centrifuge tube and centrifuged for 5 minutes at 800 rpm at room temperature.
(4) The supernatant was removed by pipette, approximately 0.1ml was left, and then a hypotonic solution 0.56% w/v KCl was added to make up to 1ml and placed in a water bath for 5 minutes.
(5) Adding methanol: glacial acetic acid ═ 3: 1(v/v), after fixing the hypotonic cells at room temperature for 40 minutes, 800 rpm, centrifuging for 5 minutes, removing the supernatant, and carrying out secondary fixation for 20 minutes, followed by 800 rpm, centrifuging for 5 minutes, and removing the supernatant.
(6) Adding fresh fixing solution 0.5ml, resuspending, sucking the suspension with a pipette, dropping at a distance of 50cm, and baking in a constant temperature oven at 65 deg.C overnight.
(7) 1ml of Giemsa stock solution was added to 9ml of PBS buffer solution to prepare a staining solution.
(8) And (4) taking out the glass slide from the oven, adding a staining solution for staining for 10min, washing and drying.
(9) Slide reading, analyze under microscope and record 20 split phases.
The results show that 2 n-40 is the normal karyotype of the mouse embryonic stem cells, and the normal karyotype rate of R1 ES and SP3 ES used in the experiment is more than 50%. The results of karyotyping R1 ES cells are shown in FIG. 3. The results of karyotyping SP3 ES cells are shown in FIG. 4.
3.2 sex determination
ES cells were cultured on gelatin-coated dishes and 1X 10 ES cells were harvested for each of the two sexes6Cells were treated with Qiagen FlexiGene DNA drawer kit to extract genomic DNA. Sex identification is carried out on the two ES cell lines by respectively utilizing the X chromosome specific gene Zfx and the Y chromosome specific gene Sry, wherein the forward sequence of Zfx is AAGAGAGTCCATTCAAGTGTGA, the reverse sequence is GCTACCTTTGTTGCCGAAAT, the annealing temperature is 58 ℃, the annealing time is 35 seconds, the cycle number is 31, and the size of an amplification product is 399 bp; the forward sequence of Sry is CTTTTTCCAGGAGGCACAGA, the reverse sequence is GACAGGCTGCCAATAAAAGC, the annealing temperature is 60 ℃, the annealing time is 35 seconds, the cycle number is 31, and the size of an amplification product is 250 bp.
The PCR results are shown in FIG. 5. The PCR product of R1 ES cells included two bands, X chromosome-specific gene Zfx and Y chromosome-specific gene Sry, and was therefore XY, i.e., male; the PCR product of SP3 ES cells contained only one band of the X chromosome-specific gene Zfx and was therefore XX, female.
Example 4: maintenance and characterization of mouse ES pluripotency
4.1 morphological Observation
The growth characteristics and morphological characteristics of the ES cells were observed by phase contrast microscopy, as shown in FIGS. 1 and 2.
4.2 alkaline phosphatase (AKP) staining
(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;
(2) sucking up 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 PBST and can be observed by a microscope.
The results of alkaline phosphatase staining of R1 ES cells are shown in FIG. 6. The results of alkaline phosphatase staining of SP3 ES cells are shown in FIG. 7. The results of alkaline phosphatase staining of NIH 3T3 fibroblasts as controls are shown in FIG. 8. R1 ES cells, staining positive; SP3 ES cells, staining positive; NIH 3T3 fibroblasts showed negative staining.
4.3 detection of ES cell pluripotency molecular markers
4.3.1 extraction of total RNA from ES cells
(1) Before harvesting ES cells, completely sucking up the culture solution, and washing the cells with PBS sterilized at high temperature and high pressure for 3 times at 5 min;
(2) after the PBS was aspirated, 1ml Trizol reagent was added to the cell culture dish, mixed well and transferred to a 1.5ml EP tube (without RNase) with the following protocol chloroform: adding chloroform into the Triozl (1:5), shaking up forcibly, and placing on ice for 10 minutes;
(3) centrifugation at 12000 Xg for 15 min at 4 ℃ transferred the supernatant to a RNase-free 1.5ml centrifuge tube, mixed with isopropanol: adding isopropanol into the supernatant (1:1), shaking up, and standing for 15 minutes on ice;
(4) centrifuge at 12000 Xg for 10min at 4 ℃ and discard the supernatant. Adding 1ml of 75% ethanol prepared by DEPC water into each tube, shaking until RNA is suspended in ethanol, and standing for 5 minutes on ice;
(5) centrifuging at 4 ℃ and 7500 Xg for 5 minutes, completely sucking out 75% ethanol remained in the centrifuge tube in a super clean bench, and completely drying RNA precipitate;
(6) the RNA pellet was dissolved in DEPC water and stored at-80 ℃.
4.3.2 determination of Total RNA concentration
Mu.l of RNA was diluted 100-fold with DEPC water and the concentration and A260nm/A280nm ratio were determined spectrophotometrically. When the ratio of A260/A280nm is between 1.8 and 2.0, the RNA quality is judged to be qualified and can be used for reverse transcription to synthesize cDNA.
4.3.3 Synthesis of cDNA by reverse transcription
Reverse transcription kit
The RT reagent Kit is purchased from Dalibao bioengineering Co., Ltd and carries out reverse transcription operation on RNA according to the Kit instruction.
(1) Preparing a reaction solution:
(2) and (3) uniformly mixing the reaction solution, putting the reaction solution into a PCR instrument, and reacting according to the following conditions:
(3) RT-PCR reaction system:
(4) RT-PCR reaction conditions:
4.3.4 RT-PCR
the following primers were synthesized by Shanghai Sangni BioLimited:
wherein the annealing temperature and the annealing time of each primer are shown in the following table:
(5) electrophoresis of RT-PCR reaction products
Prepare 2% agarose gel, put into electrophoresis tank. And (3) uniformly mixing a product obtained by the PCR reaction with 6x loading buffer in proportion, adding the mixture into a loading hole of agarose gel, and adding a DNA marker at the same time. After 30 minutes of electrophoresis, the gel was placed in a gel imager and recorded by photographing.
The RT-PCR results are shown in FIG. 9. Wherein Oct4 and Ssea-1 are highly expressed in R1 ES and SP3 ES cells, and are not expressed in NIH 3T3 fibroblasts.
Example 5: mouse ES in vitro induced differentiation into cardiomyocytes
After the ES cells were digested with 0.25% trypsin, they were resuspended in differentiation medium (formula: 79% DMEM medium + 20% FBS + 1% NEAA) in one drop per 20. mu.l and dropped on the lid of a petri dish. The R1 and SP3 cell lines are respectively provided with 750 cells/20 mu l, 1000 cells/20 mu l and 1250 cells/20 mu l for comparing differentiation rates so as to optimize differentiation operation. 10ml PBS was added to the culture dish to ensure the culture environment was moist. The cover of the culture dish was inverted over the culture dish and placed in a 5% CO2 incubator at 37 ℃ for culture. After 72 hours, one Embryoid Body (EB) was visually observed in each hanging drop. Then, EB was transferred to a bacterial culture dish, a differentiation medium was added, after 2 days of suspension culture, adherent culture was performed, and the medium was changed every 2 days until a pulsating area could be observed under a light microscope.
Example 6: immunochemical detection of mouse ES-induced differentiated cardiomyocytes (detection of myocardial-specific marker protein)
(1) Differentiated ES cells were cultured in advance on a cell slide, and the slide was spread on a slide glass after being taken out and placed in a wet box with a cover. Washing the slices with PBS for 3 times, each time for 5 min;
(2) preparing 0.1% Triton solution with PBS, adding onto cell slide, washing with PBS for 5min for 3 times, each for 5 min;
(3) absorbing excessive water with filter paper, adding 2% BSA dropwise to block nonspecific antigen, and incubating at room temperature for 1 hr;
(4) BSA was aspirated, primary antibody solution (mouse anti-cTnT antibody) was added dropwise, and incubated overnight at 4 ℃;
(5) re-warming for 30min at room temperature;
(6) washing with PBS for 3 times, each for 10 min;
(7) absorbing excessive water by using filter paper, dropwise adding a secondary antibody solution (Alexa 488-labeled donkey anti-mouse fluorescent antibody), incubating for 1h at room temperature, and keeping out of the sun;
(8) washing with PBS for 3 times each for 10min in dark;
(9) absorbing excessive water by using filter paper, dropwise adding a DAPI solution, incubating at room temperature for 10min, and keeping out of the sun;
(10) washing with PBS for 3 times each for 10min in dark;
(11) sealing the film with an anti-fluorescence attenuator, and observing under a fluorescence upright microscope.
The immunofluorescence observation of the cardiomyocytes differentiated from the R1 ES cells is shown in FIG. 10. Immunofluorescence observations of differentiated cardiomyocytes of SP3 ES cells are shown in fig. 11. Green fluorescence is a cell positive for expression of the cardiac muscle specific marker cTnT. Both the cardiomyocytes differentiated from the R1 ES cells and the cardiomyocytes differentiated from the SP3 ES cells have green fluorescence, and are positive cells expressing the cardiac muscle specific marker cTnT.
Example 7: detection of expression Profile of mouse ES-induced differentiated cardiomyocytes (Real-time PCR)
The gene expression profile of ES-induced differentiated cardiomyocytes was examined by selecting a myocardium-specific gene and examining the expression level of the gene.
The operation method of real-time quantitative PCR is as follows:
(1) real-time quantitative PCR reaction system:
(2) real-time quantitative reaction conditions:
the results of quantitative PCR of the expression profile of mouse ES-induced differentiated cardiomyocytes are shown in FIG. 12. It can be seen that in the induced differentiated cardiomyocytes, the expression levels of the pluripotency genes Oct4 and Nanog are extremely low in R1 ES and SP3 ES, while the expression levels of the myocardial specific genes Bmp4, Nkx2.5, Myh6, Myl4 and cTnT are higher.
Example 8: selection of model Compounds
Compounds of known background were selected for model building and half-lethal dose IC of each compound on 3T3 cells and ES cells was obtained503T3，IC50ES, and half inhibitory dose ID on ES cell differentiation50The value of (c).
The information on the model compounds is shown in Table 1.
TABLE 1 background information for model Compounds
Wherein, the aspirin has different effects on mice of different sexes, and the female has higher aspirin tolerance degree than the male.
Example 9: detection of cytotoxicity of model Compounds
Alamar Blue reagent used for detecting cytotoxicity was purchased from Life technology.
After digesting the 3T3 cells and ES cells into single cells, the cells were resuspended, and seeded at a density of 1000 cells/200. mu.l and 500 cells/200. mu.l into each well of a 96-well plate, and each model compound was added at different concentrations, and a negative control group was set. The concentrations of the model compounds are shown in the following table.
Each group was set with 3 duplicate wells, the culture medium was changed every 2 days, by day 7, after washing the cells with PBS, the culture medium without serum and model compound was added, according to the kit instructions, cell activity was detected with Alamar Blue reagent, dose-response curves were drawn, and half lethal doses IC of model compound on 3T3 cells and ES cells were calculated, respectively503T3 and IC50ES。
TABLE 2 concentration gradient of model Compound (μ g/ml)
The dose-response curves are shown in the "diamond" and "circle" labeled curves of FIGS. 13-22, i.e., the AB assoay-R1 ESC curves of FIGS. 13-17, the AB assoay-SP 3 ESC curves of FIGS. 18-22, and the AB assoay-3T 3 curves of FIGS. 13-22. The AB assay-R1 ESC curve reflects the viability of R1 ES cells as the dose of the compound is increased, and IC can be obtained50Data of R1 ES; the AB assay-SP3 ESC curve reflects the activity of SP3 ES cells as the dose of the compound is increased, and IC can be obtained50Data of SP3 ES; the AB assay-3T3 curve reflects the change in viability of 3T3 cells with increasing compound dose, and IC can be obtained50 3T 3.
Example 10: detection of inhibitory Effect of model Compounds on ES differentiation potency
See example 5 for methods for inducing mouse ES to differentiate into cardiomyocytes in vitro. Differentiated cells are exposed to different concentrations of the compound during culture.
At the end of the experiment, total RNA of each compound, each dose group of cells was extracted separately with Trizol reagent and the RNA was inverted to cDNA, as detailed in example 4. By using
Premix Ex Taq TM II kit, according to the kit instructions, detecting the expression levels of Myh6, Myl4 and cTnT relative to Gapdh at the observation end points on an ABI 7900HT rapid real-time quantitative PCR instrument, comparing the expression level of the observation end point of each exposure dose group with the expression level of the observation end point of a control group, and calculating the ID of each model compound50And dose-response curves were plotted.
See example 6 for the procedure of real-time quantitative PCR.
Dose-response curves are shown in FIGS. 13-22 labeled "squares", "triangles", "x symbols" and "meter symbols", i.e., the Beting curve, Myh6 curve, Myl4 curve and cTnT curve in FIGS. 13-22. The Beting curve reflects the change of the end-point Beating capacity with the increase of the compound dosage in ES differentiated myocardial cells, and ID can be obtained50Data on pulsatility; the Myh6 curve is reflected in ESC differentiated cardiomyocytes, and the ID can be obtained by the curve that the expression level of Myh6 at the molecular observation end point changes with the increase of the compound dosage50Data for Myh 6; the Myl4 curve reflects that in ESC differentiated cardiomyocytes, and the ID can be obtained by the curve that the expression level of Myl4 at the molecular observation end point changes with the increase of the compound dosage50Data for Myl 4; the cTnT curve is reflected in ESC differentiated cardiac muscle cells, and the curve of the expression level of cTnT at the molecular observation end point changing with the increase of the compound dosage can be used for obtaining ID50cTnT.
Example 11: evaluation of model Compound embryotoxicity
The ICs obtained in example 9 and example 10 were combined50And ID50Data are substituted into the following discriminants:
(1)5.916lg(IC503T3)+3.500lg(IC50ES)－5.307[(IC503T3－ID50)/IC503T3]－15.27
(2)3.651lg(IC503T3)+2.394lg(IC50ES)－2.033[(IC503T3－ID50)/IC503T3]－6.85
(3)－0.125lg(IC503T3)－1.917lg(IC50ES)+1.500[(IC503T3－ID50)/IC503T3]－2.67
the embryo toxicity judgment standard is as follows:
if (1) > (2) and (1) > (3), judging the compound to be non-development toxicity;
if (2) > (1) and (2) > (3), determining the compound as weakly developmentally toxic;
if (3) > (1) and (3) > (2), the compound is judged to be highly developmentally toxic.
IC of each mode Compound50See Table 3, ID50See table 4 for values and embryotoxicity determinations.
TABLE 3 IC of model Compounds on different cells50Value of
TABLE 4 ID of model Compounds on different cells50Determination of embryotoxicity of value and model Compounds
FIG. 23 is a schematic diagram of a preferred embodiment of the model for predicting embryotoxicity of chemicals according to the present invention, which is combined with examples 1-11, showing the main steps therein: 3T3 cells, R1 ES cells and SP3 ES cells were exposed to the compounds, and the corresponding IC was determined50(ii) a Exposure to compounds during differentiation of R1 ES cells and SP3 ES cells, determinedCorresponding ID50. Through IC50And ID50And analyzing to obtain the embryotoxicity prediction result of the corresponding compound.
Example 12: molecular observed endpoint and traditionally observed endpoint were used to reflect correlation analysis under the inhibitory effect of model compounds on ES differentiation potency
ES cells can be differentiated into cardiomyocytes having a beating ability after directional induction, and when the inhibitory effect of a compound on the ES differentiation ability is examined, the change in the beating ability under exposure to model compounds of different concentrations is usually observed under a microscope, so as to calculate half the amount of the differentiation inhibitor. This method is a traditional observed endpoint. However, in the observation process, subjective errors among individuals and large workload make the evaluation model based on the traditional observation endpoint unfavorable for the development of high-throughput screening, so that the molecular biological endpoint is of great application significance to replace the traditional observation endpoint.
In this example, the correlation between the molecular observed end point and the conventional observed end point for measuring the half-differentiation inhibitory ability was analyzed by using the paired scattergram function of the SPSS19.0 software, and the determination coefficient was calculated. It is generally considered that a coefficient (abbreviated as R) is determined between two indexes2) If the ratio is more than 0.9, the high correlation between the two indexes can be proved. As can be seen from fig. 24, in both R1 ES cells and SP3 ES cells, there is a high correlation between the determinant coefficients between the conventionally observed endpoint and the 3-molecule observed endpoints Myh6, Myl4 and cTnT of 0.944, 0.986, 0.987 and 0.937, 0.902, 0.965, respectively.
In summary, among the selected model compounds, the results of the judgment of embryotoxicity of 5-fluorouracil, indomethacin, ascorbic acid, penicillin G based on the model of male R1 ES cells and the model of female SP3 ES cells were consistent with the background information of each model compound; the embryotoxicity judgment results of aspirin are different between a model based on male R1 ES cells and a model based on female SP3 ES cells, and the 'male' model judges that the compound is a weak embryotoxicity compound and is consistent with the background information of the model compound; the "female" model judges the compound as a non-embryotoxic compound, and although the judgment result is different from that of the "male" model, the result is consistent with that of the compound in a female experimental animal. As can be seen from examples 11 and 12, the results of embryotoxicity evaluations conducted using the molecular observation endpoints were consistent with those conducted using the conventional observation endpoints, and therefore, it was possible to use the molecular observation endpoints instead of the conventional observation endpoints.
The sequences and symbols involved in the present invention are listed in Table 5
TABLE 5 sequence listing
| Sequence name | Sequence of | Description of sequences |
| Zfx forward sequence | aagagagtccattcaagtgtga | SEQ ID NO.1 |
| Zfx reverse sequence | gctacctttgttgccgaaat | SEQ ID NO.2 |
| Forward sequence of Sry | ctttttccaggaggcacaga | SEQ ID NO.3 |
| Reverse sequence of Sry | gacaggctgccaataaaagc | SEQ ID NO.4 |
| Oct4 upstream primer sequence | cggaagagaaagcgaactagc | SEQ ID NO.5 |
| Oct4 downstream primer sequence | attggcgatgtgagtgatctg | SEQ ID NO.6 |
| Ssea1 upstream primer sequence | acggataaggcgctggtacta | SEQ ID NO.7 |
| Sequence of downstream primer of Ssea1 | ggaagccatagggcacgaa | SEQ ID NO.8 |
| Upstream primer sequence of Gapdh | aggtcggtgtgaacggatttg | SEQ ID NO.9 |
| Downstream primer sequence of Gapdh | tgtagaccatgtagttgaggtca | SEQ ID NO.10 |
| Upstream primer sequence of Bmp4 | ttcctggtaaccgaatgctga | SEQ ID NO.11 |
| Downstream primer sequence of Bmp4 | cctgaatctcggcgacttttt | SEQ ID NO.12 |
| Upstream primer sequence of Nkx2.5 | gacaaagccgagacggatgg | SEQ ID NO.13 |
| Downstream primer sequence of Nkx2.5 | ctgtcgcttgcacttgtagc | SEQ ID NO.14 |
| Upstream primer sequence of Myh6 | gcccagtacctccgaaagtc | SEQ ID NO.15 |
| Downstream primer sequence of Myh6 | gccttaacatactcctccttgtc | SEQ ID NO.16 |
| Upstream primer sequence of Myl4 | aagaaacccgagcctaagaagg | SEQ ID NO.17 |
| Downstream primer sequence of Myl4 | tgggtcaaaggcagagtcct | SEQ ID NO.18 |
| Sequence of forward primer of cTnT | cagaggaggccaacgtagaag | SEQ ID NO.19 |
| Downstream primer sequence of cTnT | ctccatcggggatcttgggt | SEQ ID NO.20 |
| Upstream primer sequence of Gapdh | aggtcggtgtgaacggatttg | SEQ ID NO.21 |
| Downstream primer sequence of Gapdh | tgtagaccatgtagttgaggtca | SEQ ID NO.22 |
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
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Claims (7)
1. Use of a chemical embryotoxicity prediction model for distinguishing sex differences in embryotoxicity of a chemical, wherein the chemical embryotoxicity prediction model comprises a cell type including cardiomyocytes derived from SP3 ES cells with a karyotype of XX and cardiomyocytes derived from R1 ES cells with a karyotype of XY and a molecular observation endpoint; the molecular observed endpoints include Myl4, cTnT, and Myh 6.
2. The use of claim 1, wherein the method of establishing the chemical embryotoxicity prediction model comprises the steps of:
the method comprises the following steps: separating and culturing mouse embryo fibroblast;
step two: in vitro expansion culture of mouse ES, including SP3 ES cells and R1 ES cells;
step three: karyotyping and sex identifying mouse ES;
step four: maintenance and identification of mouse ES pluripotency;
step five: mouse ES is induced and differentiated into myocardial cells in vitro;
step six: immunochemical detection of myocardial specific marker proteins of mouse ES-induced differentiated cardiomyocytes, said myocardial specific marker proteins comprising cTnT;
step seven: real-time PCR detection of expression profiles of mouse ES-induced differentiated cardiomyocytes, including Myh6, Myl4, and cTnT genes;
step eight: selection of model compounds: selecting 4 or more of the model compounds such that the set of model compounds comprises a teratogenic compound, and a teratogenic compound that differs in sex;
step nine: model compound cellsAnd (3) detection of toxicity: obtaining a half-lethal dose IC of each model compound on cells50；
Step ten: examination of inhibitory Effect of model Compounds on ES differentiation Capacity: half inhibitory dose ID of each model compound on ES cell differentiation was obtained50A value of (d);
step eleven: evaluation of model Compound embryotoxicity: IC obtained by step nine50And the ID obtained in step ten50Analyzing the influence degree of each model compound on the embryotoxicity, and establishing and obtaining the chemical embryotoxicity prediction model, thereby popularizing the prediction on the embryotoxicity of other chemicals.
3. The use of claim 2, wherein the method of establishing a model for predicting chemical embryotoxicity further comprises the steps of twelve: the molecular observation end point and the traditional observation end point are used for reflecting the correlation analysis of the model compound under the inhibition effect on the ES differentiation capacity, namely, the molecular observation end point and the traditional observation end point are used for measuring the correlation between half of the differentiation inhibition capacity by adopting the function of a matched scatter diagram of SPSS19.0 software, and meanwhile, a determining coefficient is calculated; when the determination coefficient between the molecular observation terminal point and the traditional observation terminal point is more than 0.9, the molecular observation terminal point and the traditional observation terminal point are highly correlated; therefore, the accuracy and the effectiveness of the chemical embryotoxicity prediction model are judged.
4. The use of claim 2, wherein the model compounds in step eight comprise 5-fluorouracil, aspirin, indomethacin, ascorbic acid, penicillin G; the fourth step included NIH 3T3 fibroblasts as controls.
5. The use of claim 2, wherein in step nine a half-lethal dose IC of each model compound on 3T3 cells and ES cells is obtained503T3 and IC50ES; in the above step ten, half inhibitory dose ID of each model compound on ES cell differentiation was obtained50A value of (d); said step (c) isEleven by combining the IC503T3、IC50ES and ID50Data are substituted into the following discriminants:
(1)5.916lg(IC503T3)+3.500lg(IC50ES)－5.307[(IC503T3－ID50)/IC503T3]－15.27
(2)3.651lg(IC503T3)+2.394lg(IC50ES)－2.033[(IC503T3－ID50)/IC503T3]－6.85
(3)－0.125lg(IC503T3)－1.917lg(IC50ES)+1.500[(IC503T3－ID50)/IC503T3]－2.67
the embryo toxicity judgment standard is as follows:
if (1) > (2) and (1) > (3), judging the compound to be non-development toxicity;
if (2) > (1) and (2) > (3), determining the compound as weakly developmentally toxic;
if (3) > (1) and (3) > (2), the compound is judged to be highly developmentally toxic.
6. The use of claim 1, wherein the model for predicting chemical embryotoxicity further comprises the upstream primer sequence of Myl 4: AAGAAACCCGAGCCTAAGAAGG, and the downstream primer sequence of Myl 4: TGGGTCAAAGGCAGAGTCCT are provided.
7. The use of claim 1, wherein the chemical embryotoxicity prediction model further comprises an upstream primer sequence of cTnT: CAGAGGAGGCCAACGTAGAAG, cTnT downstream primer sequence: CTCCATCGGGGATCTTGGGT, upstream primer sequence of Myh 6: GCCCAGTACCTCCGAAAGTC and Myh 6: GCCTTAACATACTCCTCCTTGTC are provided.
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