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DNA methylation biomarkers for evaluating cardiac muscle cell damage and application thereof

A technology of methylation markers and cardiomyocytes, applied in the field of genetic engineering, to achieve accurate quantification, improved sensitivity and specificity, and easy detection

Inactive Publication Date: 2018-03-23
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of genomic DNA methylation to prompt cardiomyocyte damage. If damage-related DNA methylation can be screened out as a biomarker, it can quickly understand the degree of cardiomyocyte damage and provide a basis for prevention and treatment

Method used

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  • DNA methylation biomarkers for evaluating cardiac muscle cell damage and application thereof
  • DNA methylation biomarkers for evaluating cardiac muscle cell damage and application thereof
  • DNA methylation biomarkers for evaluating cardiac muscle cell damage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Single cell culture of embodiment 1 human embryonic stem cell line H9

[0034] The human embryonic stem cell line H9 was cultured by MEF-free method: the extracellular matrix Matrigel was diluted to a volume fraction of 1% with DMEM / F12 medium, and a 6-well cell culture plate was coated with the diluted extracellular matrix. H9 cells were inoculated into pre-coated culture plates, cultured in mTeSR1 medium supplemented with ROCK inhibitor Y27632 (final concentration: 5 μM), and replaced with fresh mTeSR1 medium the next day. Change the fresh culture medium every day, and cultivate for 3 to 4 days until the cell density reaches about 80%.

[0035] The extracellular matrix Matrigel was diluted to a volume fraction of 1% with DMEM / F12 medium, and a 12-well cell culture plate was coated with the diluted extracellular matrix. Digest the H9 cells cultured by the MEF-free method above into a single-cell suspension with Accutase digestion solution to make the concentration 2×1...

Embodiment 2

[0037] Example 2 Directed differentiation of human embryonic stem cell line H9 to cardiomyocytes

[0038] At the beginning of differentiation induction, culture was carried out with RPMI / B27 medium (without insulin) containing CHIR99021 (final concentration: 12 μM) for 24 hours, and then replaced with RPMI / B27 to continue the culture after 24 hours.

[0039] On the third day of induction of differentiation (172 hours after adding CHIR9902), replace with RPMI / B27 medium (without insulin) containing the Wnt inhibitor IWR-1 (5 mM) and culture for 48 hours without changing the medium.

[0040] On the 5th day after induction of differentiation, replace with RPMI / B27 medium (without insulin) to continue culturing. On the 7th day of differentiation induction, the medium was replaced with fresh RPMI / B27 medium, and the medium was changed every 3 days thereafter.

[0041] During the induction culture process, observe under the microscope every day whether there are spontaneously beati...

Embodiment 3

[0042] Example 3 Analysis of Environmental Chemicals Inducing Cardiomyocyte Damage

[0043] A representative environmental chemical, triclosan (TCS), was selected. During the differentiation of H9 into cardiomyocytes, TCS was added to induce embryonic stem cells to differentiate into cardiomyocytes.

[0044]Exposures were performed with TCS at a dose of 1 [mu]M. TCS exposure was started from the 0th day of differentiation induction, and the exposure was continued until the 20th day, and 0.1% DMSO was used as the solvent control.

[0045] Collect the morphology of the control group and the experimental group after chemical treatment, the results are as follows: image 3 .

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Abstract

The invention belongs to the field of the genetic engineering, and particularly provides a group of DNA methylation biomarkers for evaluating cardiac muscle cell damage and an application thereof. TheDNA methylation biomarker comprises 30 DNA methylation sites of 5 DMRs corresponding to 5 cardiac muscle differentiation key factors; and the five cardiac muscle differentiation key factors are GATA4, TNNT2, TNNI3, MEF2A and NKX2-5. Such type of the provided differential DNA methylation biomarkers is successfully developed to subvert the traditional biomarkers using protein as major means, a newsituation is created for the prevention and treatment of the cardiac muscle damage, and a reference is provided for the development of other disease biomarkers. The DNA methylation biomarkers are capable of evaluating the damage level of the cardiac muscle affected by environmental factors, and evaluating and forecasting the cardiotoxicity of environmental chemistry, and are the biomarkers with the prospect.

Description

field of invention [0001] The invention belongs to the field of genetic engineering, and in particular relates to a group of DNA methylation markers for evaluating myocardial cell damage and detecting cardiotoxicity and applications thereof. Background technique [0002] During the sensitive period of embryonic heart development, the heart is highly sensitive to changes in the embryonic development environment. Pregnant women are exposed to adverse environmental factors, such as environmental chemicals, which may affect the normal development of the fetus and cause birth defects such as cardiovascular malformations. In the daily life environment of human beings, there are a large number of various chemicals with a wide range of sources, which can directly or indirectly affect the health of the body through different action pathways and mechanisms, causing different degrees of damage, and often act in low doses and for a long time The cumulative effect is characteristic. At ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/154C12Q2600/142
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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