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Group of novel molecular markers of one group of human myocardial cells, and applications of novel molecular markers

A technology of cardiomyocytes and markers, applied in the field of new molecular markers, can solve problems such as side effects, high cost of surgical treatment, high incidence of heart disease and mortality

Active Publication Date: 2013-12-18
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No matter in developed or developing countries, the morbidity and mortality of heart disease remain high. However, facing such a severe test, the current treatment of heart disease is not optimistic. For example, bypass surgery is expensive and has certain risks and possible side effects. Interventional therapy is still in the exploratory stage, and there are few treatments that treat both symptoms and root causes.

Method used

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  • Group of novel molecular markers of one group of human myocardial cells, and applications of novel molecular markers
  • Group of novel molecular markers of one group of human myocardial cells, and applications of novel molecular markers
  • Group of novel molecular markers of one group of human myocardial cells, and applications of novel molecular markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, the cultivation of human embryonic pluripotent stem cells

[0062] The human embryonic pluripotent stem cell line H9 was cultured using the following method:

[0063] 1. Inoculate H9 cells into a culture plate pre-cultured with mitomycin-inactivated mouse embryonic fibroblasts, and use human pluripotent stem cell medium (CDF12 medium) to co-culture with mouse embryonic fibroblasts .

[0064] 2. Dilute the extracellular matrix to a volume fraction of 1% with DMEM / F12 medium, and coat the culture plate with the diluted extracellular matrix.

[0065] 3. Inoculate the H9 cells cultured in step 1 into a culture plate pre-coated with 1% extracellular matrix by volume fraction, and culture in mTeSR medium.

Embodiment 2

[0066] Example 2. Differentiation of human embryonic pluripotent stem cells into human cardiomyocytes

[0067] The differentiation protocol of human embryonic pluripotent stem cell line H9 into human cardiomyocytes is based on the Palecek protocol (Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, Raval KK, Zhang J, Kamp TJ, Palecek SP.2012.Robust Cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.Proceedings of the National Academy of Sciences of the United States of America109:E1848-1857.) was improved, and the improved protocol further improved the purity of human cardiomyocytes, The specific steps of the program are as follows:

[0068] 1. Dilute the extracellular matrix with DMEM / F12 medium to a concentration of 1% by volume, and coat the culture plate with the diluted extracellular matrix.

[0069] 2. Digest the human embryonic pluripotent stem cell line H9 into a single cell suspension, and 5 cel...

Embodiment 3

[0076] Embodiment 3, identification of human cardiomyocytes

[0077] Human cardiomyocytes derived from human embryonic pluripotent stem cells not only exhibit cardiomyocyte-specific contractile function in morphology, but also can further identify cardiomyocyte-specific biological markers by immunofluorescence and flow cytometry.

[0078] 1. Immunofluorescence identification

[0079] (1) The human cardiomyocytes obtained in Example 2 were divided into four groups, and each group was fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 / PBS. Then the four groups were incubated overnight at 4°C with four primary antibodies (mouse anti-cTnT, mouse anti-MF20, rabbit anti-MLC2v, mouse anti-α-Actinin). After the primary antibody was washed, the secondary antibody was incubated at room temperature for 1 hour. The secondary antibody used in the first group was Alex Fluor488goat anti-mouse IgG, the secondary antibody used in the second group was Alex Fluor488goat ant...

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Abstract

The invention discloses a group of novel molecular markers of human myocardial cells, and applications of the novel molecular markers. The group of markers comprises the following 29 genes: POPDC2, ALPK2, TRIM55, ASPH, FILIP1, HSPB7, NPPA, KBTBD10, MYL4, CRYAB, LRRN4, MYOZ2, OBSCN, SMPX, NRK, ABLIM1, SYNPO2L, ANKRD1, TNNI1, MYH6, ABRA, TPM1, C15orf52, MYOM1, MASP1, MYBPC3, TNS1, FLNC and F2RL2. The markers play an important role in screening and identifying the myocardial cells, and have potential effect and huge significance on the early development research of human hearts, the construction of heart disease models, treatment of heart diseases, and the development of heart disease medicament targets or in research and development of medicaments.

Description

technical field [0001] The present invention relates to a group of novel molecular markers of human cardiomyocytes and applications thereof. Background technique [0002] Heart disease is a killer that threatens human health. Every year, tens of millions of people lose their lives due to various heart diseases, which brings great pressure and burden to families and public health. No matter in developed or developing countries, the morbidity and mortality of heart disease remain high. However, facing such a severe test, the current treatment of heart disease is not optimistic. For example, bypass surgery is expensive and has certain risks and possible side effects. Interventional therapy is still in the exploratory stage, and there are few therapeutic methods that treat both symptoms and root causes. The reason is largely due to the lack of human understanding of the heart and heart diseases. The etiology of heart disease can be traced back to the loss or dysfunction of car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/12
Inventor 刘光慧顾颖胡安·卡洛斯·伊斯毕华·贝尔蒙特曲静杨济平张维琦任若通
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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