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Environmental chemical myocardium development toxicity evaluation technical method

An environmental chemistry, myocardial technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of staying and lack of whole genome exploration, and achieve the effect of promoting children's health and improving the quality of the population

Pending Publication Date: 2018-05-18
NANJING MEDICAL UNIV
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  • Claims
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AI Technical Summary

Problems solved by technology

[0005] Exposure to exogenous chemicals can cause changes in related genes during myocardial differentiation, but research in this area is still mainly at the level of single genes, lacking a genome-wide discussion

Method used

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  • Environmental chemical myocardium development toxicity evaluation technical method
  • Environmental chemical myocardium development toxicity evaluation technical method
  • Environmental chemical myocardium development toxicity evaluation technical method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 The method of human embryonic stem cell line H9 directed cardiomyocyte differentiation

[0032] Human embryonic stem cell line H9 cells (purchased from the Stem Cell Bank of the Chinese Academy of Sciences) were resuscitated in a six-well culture plate coated with extracellular matrix Matrigel (volume fraction: 1%), and mTeSR1 medium containing 5 μM ROCK inhibitor Y27632 was used for resuscitation Cultured at 37°C, 5% CO 2 cultivated under conditions. On the second day of recovery, the culture medium was replaced with mTeSR1 medium, and the cells grew to about 80%. Digestive enzymes were added to each well to digest them into single cells for subsequent differentiation.

[0033] Count 0.5×10 6 Cells were seeded into Matrigel-coated 12-well culture plates, and cultured in mTeSR1 medium containing 5 μM ROCK inhibitor Y27632 for 24 hours. Continue to culture in mTeSR1 medium for 72 hours, and change the medium every day. On the 0th day of differentiation, ...

Embodiment 2

[0034] Example 2 Effects of chemical exposure on the differentiation of stem cells into cardiomyocytes

[0035] During the differentiation of human embryonic stem cell line H9 into cardiomyocytes, the environmental chemical TCS was added. TCS acts on the whole cardiomyocyte differentiation process, the treatment group is 1 μ M TCS, diluted with DMSO, wherein the DMSO concentration is 0.1%; the control group: 0.1% DMSO treatment. During the whole process of chemical exposure, pictures were taken under the microscope every day, and the cell morphology of the treatment group and the control group were compared and observed. On the 21st day of cardiomyocyte differentiation (the last day of chemical exposure), immunofluorescence was used to detect the expression of myocardial marker proteins α-actinin and cTnT. Compared with the control group, the differentiation rate of cardiomyocytes in the TCS treatment group decreased, and the morphology of the differentiated myocardial cells ...

Embodiment 3

[0036] Example 3 Cardiomyocyte Morphology Observation and Beat Frequency Counting

[0037] The culture plate was observed daily under a light microscope for the appearance of spontaneously beating cell clusters, and the appearance of rhythmically beating cell clusters was considered a functional marker of successfully differentiated cardiomyocytes. The number of spontaneous and rhythmic beating cardiomyocyte clusters was counted as the index of cardiomyocyte differentiation efficiency, and the cell clusters at the beating site were photographed microscopically, and the beating frequency and beating duration were recorded. Beat rates per minute were calculated and compared for the chemical-treated and control groups.

[0038] On day 12, the control group began to show beating clumps of cardiomyocytes. Take the 12-well plates of the control group and the experimental group under the microscope, take pictures and record them for at least 1 minute, and record at least 3 fields of...

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Abstract

The invention belongs to the technical field of environmental chemical toxicity evaluation and specifically provides an evaluation technical method based on a transcriptome sequencing technology to analyze environmental chemicals to directionally induce and differentiate human embryonic stem cells into myocardial cell toxicity. The scheme for directionally inducing and differentiating the human embryonic stem cells into myocardial cells disclosed by the invention can be applied to screening myocardium development toxicity of environmental chemicals under environmental exposure dosage or crowdexposure dosage; by means of a human embryonic stem cell directional myocardial cell differentiating model optimized by the technical method disclosed by the invention, a risk evaluation system for embryo myocardium development toxicity of environmental chemical exposure can be established; the technical scheme can be popularized and applied to myocardium toxicity forecasting and evaluation of environmental chemicals, medicine and the like, so that theoretical basis is provided for early preventing CHD (Coronary Heart Disease), and contributions are made to promoting child health and improvingpopulation quality. Furthermore, transcriptome sequencing utilized by the technical method disclosed by the invention can define embryo myocardium development toxicity caused by environmental chemical exposure for the whole genome level.

Description

technical field [0001] The invention belongs to the technical field of environmental chemical toxicity evaluation, and specifically relates to a method for evaluating the toxicity of environmental chemicals to human embryonic stem cells induced to differentiate into cardiomyocytes based on transcriptome sequencing technology. Background technique [0002] Congenital heart disease (CHD), referred to as congenital heart disease, is the most common birth defect. The incidence rate of newborns in my country is 0.6%-0.8%, accounting for the first place in various birth defects. More than 200,000 children with CHD are born in my country every year, which brings a heavy burden to society and families. CHD is an abnormality in the structure and function of the heart and blood vessels caused by the obstacles in the embryonic development process, which can seriously endanger the health of infants and young children, lead to intrauterine abortion, stillbirth, etc., and not only affect ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N5/077
CPCC12N5/0657C12Q1/6883C12Q2600/142C12Q2600/158
Inventor 杜桂珍秦玉峰陆春城宋玲夏彦恺王心如
Owner NANJING MEDICAL UNIV
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