Chemical embryotoxicity prediction model and establishing method thereof

A technology of embryotoxicity and predictive models, applied in the field of prediction of embryotoxicity of new chemicals, can solve problems such as research, difficult metabolism and mechanism, and many influencing factors

Inactive Publication Date: 2015-10-21
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are several problems that cannot be ignored in carrying out animal experiments: 1. It requires a lot of financial resources; 2. The experiment cycle is long; 3. It requires a large number of experimental animals; 4. It is difficult to carry out metabolism and mechanism research due to many influencing factors in the body
However, currently existing ESTs only use the gene Myh6, which encodes the heavy chain of myocardin, as the molecular endpoint. Whether the expression of other genes can also be used as molecular endpoints for EST to predict the embryotoxicity of chemicals remains to be confirmed.

Method used

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  • Chemical embryotoxicity prediction model and establishing method thereof
  • Chemical embryotoxicity prediction model and establishing method thereof
  • Chemical embryotoxicity prediction model and establishing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: Isolation and culture of mouse embryonic fibroblasts

[0099] In the present invention, mouse embryonic fibroblasts (MEF) are used as trophoblast cells for culturing ES.

[0100] 1.1 Isolation method of MEF primary cells:

[0101] (1) Take a beaker filled with 50ml of 75% ethanol, soak scissors, tweezers and other surgical instruments, the soaked instruments are roasted and sterilized by an alcohol lamp, then cooled and set aside.

[0102] (2) Prepare several bacterial culture dishes with a diameter of 60 mm, which are respectively numbered as No. 1 dish, No. 2 dish, and No. 3 dish, and put in appropriate amount of PBS sterilized by high temperature and high pressure, and add appropriate amount of penicillin-streptomycin solution into it. Among them, dish No. 1 contained the uterus taken out during the dissection of pregnant mice, dish No. 2 contained embryos with torn membranes, and dish No. 3 contained embryo trunks after treatment.

[0103] (3) After ane...

Embodiment 2

[0115] Example 2: In vitro expansion and culture of mouse ES

[0116] 2.1 Preparation of trophoblast cells

[0117] (1) Select MEF cells with appropriate algebra and density, add mitomycin C to the cell culture medium at a working concentration of 10 μg / ml, mix well and place in an incubator to continue culturing.

[0118] (2) After 2 hours, the culture medium containing mitomycin C was aspirated, and washed with PBS sterilized by high temperature and high pressure for 5 times, 5 minutes each time.

[0119] (3) After washing, replace with fresh culture medium to recover ES cells or continue culturing. The treated trophoblast cells supported the growth and maintenance of the undifferentiated state of mouse ES cells for up to 5 days.

[0120] 2.2 Recovery of ES cells

[0121] (1) Take out the ES cell cryopreservation tube from the liquid nitrogen, put it in a 37°C water bath to dissolve, take it out when it dissolves to the state of small ice crystals, and put it into the ult...

Embodiment 3

[0132] Example 3: Karyotype analysis and gender identification of mouse ES

[0133] 3.1 Karyotype analysis

[0134] (1) ES cells are cultured on a gelatin-coated petri dish, and the follow-up operations are carried out after the cells cover more than 80% of the petri dish;

[0135] (2) According to the results of the growth curve described in 2.4, add 50 ng / ml colchicine in the logarithmic growth phase, and continue culturing for 2 hours.

[0136] (3) Add EDTA-trypsin digestion solution after sucking up the culture solution, place it in a constant temperature incubator at 37°C for 5 minutes, blow gently with a pipette, then collect the cell suspension into a graduated centrifuge tube, and store at room temperature Centrifuge at 800 rpm for 5 minutes.

[0137] (4) Use a pipette to remove the supernatant, leaving about 0.1ml, then add hypotonic solution 0.56% w / v KCl to make up to 1ml, and put it in a water bath for 5 minutes.

[0138] (5) Add methanol: glacial acetic acid = ...

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Abstract

The invention provides a chemical embryotoxicity prediction model. The chemical embryotoxicity prediction model comprises a cell type. The cell type comprises myocardial cells induced and differentiated from SP3ES cells with the karyotype as XX. An establishing method of the chemical embryotoxicity prediction model comprises the steps that 1, mouse embryo fibroblasts are separated and cultured; 2, in vitro multiplication culturing of the mouse ESs is conducted; thirdly, the karyotype analysis of the mouse ESs and gender determination are conducted; fourthly, the pluripotency of the mouse ESs is maintained and evaluated; fifthly, the mouse ESs are induced and differentiated to be myocardial cells in a in-vitro mode; sixthly, chemoimmunology detection of a cardiac-specific marker protein of the myocardial cells induced and differentiated from the mouse ESs is conducted; seventhly, real-time PCR detection of the expression profile of the myocardial cells induced and differentiated from the mouse ESs is conducted; eighthly, a pattern chemical is selected; ninthly, the cytotoxicity of the pattern chemical is detected; tenthly, the restraining effect of the pattern chemical on the ES differentiative capacity is detected; eleventhly, the embryotoxicity of the pattern chemical is evaluated. The chemical embryotoxicity prediction model fills up the blank that female mouse embryonic stem cells do not exist in an EST system.

Description

technical field [0001] The present invention relates to a chemical embryotoxicity prediction model and its establishment method, in particular to a kind of mouse embryonic stem cell direction induction differentiation into cardiomyocytes with beating ability, and establishment of chemical embryotoxicity recognition on the obtained cardiomyocytes and prediction models, and applied to the prediction of embryotoxicity of new chemicals. Background technique [0002] At present, the toxicity data of chemicals are mostly obtained in experimental animals, such as acute toxicity test, subacute toxicity test, chronic toxicity test and so on. However, there are several problems that cannot be ignored in carrying out animal experiments: 1. It requires a lot of financial resources; 2. The experiment period is long; 3. It requires a large number of experimental animals; 4. It is difficult to carry out metabolism and mechanism research due to many influencing factors in the body. Out of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12Q1/02
Inventor 王艳程薇胡庆亮张星
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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