Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Pharmaceutical application of Rictor/mTORC2 in heart development and disease treatment

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

Inactive Publication Date: 2013-12-11
ZHEJIANG UNIV
View PDF5 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Pharmaceutical application of Rictor/mTORC2 in heart development and disease treatment
  • Pharmaceutical application of Rictor/mTORC2 in heart development and disease treatment
  • Pharmaceutical application of Rictor/mTORC2 in heart development and disease treatment

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).

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an application of rictor / mTORC2 (mammalian target of rapamycin complex 2) in in-vitro differentiation of mouse embryonic stem cells into myocardial cells, namely a research applicable to screening and evaluation of a myocardial cell differentiation promoter taking rictor / mTORC2 as a target. According to the application, the myocardial cells from in-vitro directional differentiation of the mouse embryonic stem cells are purified by a percoll reagent, and plasmids of a histone acetyltransferase transcriptional coactivator p300 coded with rictor are transfected, so that a rictor acetylation level in the myocardial cells is raised, phosphorylation of a rictor downstream effector Akt (ser473) is further promoted, the activity of mTORC2 is improved, and a cardiomyocyte hypertrophy model is constructed; and furthermore, the cardiomyocyte hypertrophy model is utilized, an effect of resveratrol on resisting myocardial hypertrophy is evaluated, and the discovery of a novel myocardial differentiation inducer is facilitated.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N33/68G01N15/14
Inventor 朱丹雁郑蓓黄玉洁汤磊磊楼宜嘉
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com