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Pharmaceutical use of rictor/mTORC2 in heart development and disease therapy

A technology of cardiomyocytes and stem cells, applied in the field of pharmaceutical use of Rictor/mTORC2 in heart development and disease treatment

Inactive Publication Date: 2015-07-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the correlation between rictor / mTORC2 and embryonic cardiac differentiation and development and the occurrence of cardiac diseases

Method used

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  • Pharmaceutical use of rictor/mTORC2 in heart development and disease therapy
  • Pharmaceutical use of rictor/mTORC2 in heart development and disease therapy
  • Pharmaceutical use of rictor/mTORC2 in heart development and disease therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Effect of ES-D3 cells interfering with rictor on directional cardiomyocyte differentiation

[0018] 1. Interfering rictor gene in ES-D3 cells by lentiviral shRNA

[0019] Mouse ES-D3 cells were used, shRNA-rictor was added for transfection, and a control group was set. The shRNA-rictor interference sequence is as follows: 5'-CCGGGCCAGTAAGATGGGAATCATTCTCGAGAATGATTCCCATCTTACTGGCTTTTTG-3'; On the day of transfection, aspirate the cell supernatant, add the virus stock solution to the culture bottle according to the MOI value of 10 PFU / cell, and add normal culture solution to 2 ml, gently shake the Petri dish while adding to ensure even distribution and avoid local high concentration. Put the cells back into the incubator and incubate at 37°C for 72-96 h, then observe the fluorescence expression. The transfection efficiency is about 80%, and the cell differentiation experiment is carried out according to the hanging drop-suspension-attachment culture method. ...

Embodiment 2

[0026] Example 2 Western blot was used to detect the effect of ES-D3 cells interfering with rictor on the expression of myocardial specific proteins during cardiomyocyte differentiation.

[0027]The EB and cardiomyocyte clusters differentiated on d 3, d 5, d 5+1 and d 5+3 were collected to investigate the expression of related proteins. The protein was added to 5×SDS buffer, denatured by boiling for 5 min, and electrophoresed in a 10 % SDS-polyacrylamide gel for about 2 h; then the protein was transferred to a PVDF membrane with an electrotransfer apparatus for about 1.5 h; 5% skimmed milk was dissolved in room temperature for blocking for 1 h, rinsed with T-PBS (0.05% Tween) for 3 times (15 min, 5 min, 5 min); add goat anti-rictor (1:1000), rabbit anti-p-Akt (1 :1000), rabbit anti-p-Akt (1:1000), rabbit anti-brachyury (1:1000), rabbit anti-Nkx2.5, (1:1000), mouse anti-α-Actinin (1:1000), mouse anti-GAPDH (1:10000), incubate overnight at 4°C, rinse with T-PBS 3 times (15 mi...

Embodiment 3

[0029] Example 3: Quantitative detection of the effect of ES-D3 cells interfering with rictor on the number of cardiomyocytes by flow cytometry.

[0030] Collect ES-D3 cell-derived cardiomyocyte samples adherently differentiated for 3 days, digest into single cells with collagenase II, fix with 1% paraformaldehyde for 1 h, and then block with 3% BSA for 1 h, and apply primary antibody: cardiomyocytes Cell-specific protein Monoclonal mouse anti-α-Actinin, diluted 1:500, incubated overnight at 4°C. The next day, add the secondary antibody: Dylight549-Conjugatedgoat anti-mouse IgG, diluted 1:500, and incubate at 37°C for 1.5 h. On-board testing.

[0031] See results image 3 . The results showed that the number of cardiomyocytes significantly decreased after ES-D3 cells interfered with rictor. The number of cardiomyocytes in the interference rictor group was 5.8±1.7%, which was significantly reduced compared with 12.5±1.1% in the negative control group (** P <0.01).

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Abstract

The invention provides the application of rictor / mTORC2 in the differentiation of mouse embryonic stem cells into cardiomyocytes in vitro, which can be applied to the research of screening and evaluating cardiomyocyte differentiation-promoting agents targeting rictor / mTORC2. The present invention uses percoll reagent to purify cardiomyocytes derived from mouse embryonic stem cells in vitro directional differentiation, and transfects the plasmid encoded with rictor's histone acetyltransferase transcription coactivator p300 to increase the acetylation level of rictor in cardiomyocytes. Then promote the phosphorylation of Akt (ser473), the downstream effector of rictor, and increase the activity of mTORC2, thereby constructing a cardiomyocyte hypertrophy model, and further using this model to evaluate the anti-cardiac hypertrophy effect of resveratrol, which is conducive to the discovery of a new type of myocardial differentiation inducer.

Description

technical field [0001] The invention belongs to the fields of developmental biology, pharmacology and regenerative medicine, and relates to the field of research and application of a new protein function, in particular to rictor / mTORC2 (mammalian target of rapamycin complex 2, mammalian target of rapamycin complex 2) , mTORC2) have new functions in the in vitro differentiation of mouse embryonic stem cells into cardiomyocytes, which can be used to screen for cardiomyocyte differentiation agents targeting rictor / mTORC2. The present invention also confirms the correlation between rictor / mTORC2 and cardiac hypertrophy, and can be used in the development and research of drugs against cardiac hypertrophy. Background technique [0002] Mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation, growth, and differentiation. It forms two complexes, mTORC1 (mammalian target of rapamycin complex 1) and mTORC2 ( mammalian target of rapamycin complex 2). mTORC2 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735G01N33/68G01N15/14
Inventor 朱丹雁郑蓓王佳丹黄玉洁汤磊磊楼宜嘉
Owner ZHEJIANG UNIV
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