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Establishment and application for embryonic stem cell in vitro differentiation traditional Chinese medicine active substance pharmacodynamic screening model

An embryonic stem cell and cell technology, applied in the field of regenerative medicine, can solve the problems of low differentiation success rate of ES cells, etc., and achieve the effects of intuitive observation and evaluation indicators, large amount of biological information, and strong specificity.

Inactive Publication Date: 2013-04-17
北京清美联创干细胞科技有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

Foreign countries began to study the directed differentiation of ES cells in vitro at the beginning of this century. There have been reports of directed differentiation of ES cells into cardiomyocytes, nerve cells, endothelial cells, etc., but the success rate of differentiation of ES cells is far lower than that of drug-induced differentiation.

Method used

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  • Establishment and application for embryonic stem cell in vitro differentiation traditional Chinese medicine active substance pharmacodynamic screening model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Study on the Directed Differentiation of ES Cells into Cardiomyocytes Induced by Ginkgo Flavonoids, a Chinese Medicine Monomer

[0020] 1. Support culture of ES cells:

[0021] ES-D3 cells in the conditioned medium (containing DMEM medium, fetal bovine serum), press 2x10 7 ~5x10 7 Cells / ml were inoculated on trophoblast cells (mouse fibroblasts or STO cell lines), and subcultured according to conventional methods.

[0022] 2. Drug-induced directed differentiation of ES cells into cardiomyocytes:

[0023] ① ES-D3 cells were digested with trypsin to make a single-cell suspension, inoculated on the inner surface of the lid of the culture dish at 20-30uL, added 10mL ES-Hanks solution into the culture dish, turned the lid over to form a hanging drop, and put it in the incubator Hanging drop culture for 2 days. Thereafter, the hanging drop was transferred to a petri dish containing 10 mL of ES cell differentiation medium (DMEM medium, fetal calf serum), and ES ...

Embodiment 2

[0027] Example 2: Research on the expression of special genes dependent on myocardial development

[0028] If the drug can induce the directional differentiation of ES cells into cardiomyocytes, the following studies are carried out before drug co-cultivation, when the embryoid body is formed and when the embryoid body is beating after drug co-culture:

[0029] (1) DNA library cloning → extraction of phage DNA as a template after cultivation, PCR reaction amplification → sequencing of amplified products;

[0030] (2) Semi-quantitative reverse transcription-polymerase chain reaction identification → function prediction research on some genes;

[0031] (3) RT→PCR semi-quantitative identification of differentially expressed genes→function prediction of some genes.

Embodiment 3

[0032] Example 3: Research on the expression of myocardial structural proteins

[0033] (1) Electron microscopy shows the characteristic map of myocardial structural proteins, and the drug effect is shown by the presence or absence and the degree of maturity.

[0034] (2) Protein electrophoresis, western blot, evaluation of expression differences, post-expression modification, etc. When necessary, the electrical signals of the beating embryoid bodies were collected and analyzed.

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Abstract

The invention discloses a pharmacodynamic screening model established by using an embryonic stem cell, which is used for preliminary screening and evaluation of pharmacological functions. Under drug induction, the embryonic stem cell is directionally differentiated into special-type cells so as to establish an efficient drug screening model which is used for preliminary screening and evaluation of pharmacological functions. The pharmacological functions of the drug can be evaluated preliminarily as long as the drug can take effect in one or a few stages. The model has an efficient drug screening characteristic, has direct and accurate evaluation index, and can evaluate the pharmacological functions of a plurality of natural drugs rapidly. Furthermore, the pharmacodynamic screening model takes less reagent, time and cost, has a wealth of cell differentiation information, has high efficiency and stimulation capability, and has both the advantages of pharmacological functions and mechanism research.

Description

technical field [0001] The invention belongs to the field of regenerative medicine, and relates to the establishment and application of a drug efficacy screening model, mainly relating to the establishment and application of a drug efficacy screening model for the in vitro differentiation of embryonic stem cells in Chinese medicines, which can quickly and efficiently assess the efficacy of traditional Chinese medicines. Screening and Evaluation. Background technique [0002] The classic drug efficacy screening models include (1) overall animal level drug efficacy screening model: its advantage is that it can intuitively reflect the therapeutic effect of drugs from the overall level; the disadvantage is that it requires a large number of animals, the experimental period is long, and a large number of test substances are required. Not suitable for preliminary large-scale screening of drug efficacy; (2) Drug efficacy screening model at the level of tissues and organs (such as i...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/0793C12N5/071C12Q1/02
Inventor 田杰
Owner 北京清美联创干细胞科技有限公司