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Application of glutamic acid receptor 5 in directionally differentiating embryonic stem cells to cardiac muscle cells

A glutamate receptor, embryonic stem cell technology, applied in the field of glutamate receptor 5 in the directed differentiation of embryonic stem cells into cardiomyocytes

Inactive Publication Date: 2010-11-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on the expression and functional regulation of mGluR5, which may be involved in important cardiac physiological functions, during myocardial development using the ES cell in vitro cardiogenic system at home and abroad.

Method used

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  • Application of glutamic acid receptor 5 in directionally differentiating embryonic stem cells to cardiac muscle cells
  • Application of glutamic acid receptor 5 in directionally differentiating embryonic stem cells to cardiac muscle cells
  • Application of glutamic acid receptor 5 in directionally differentiating embryonic stem cells to cardiac muscle cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Semi-quantitative RT-PCR detection of mGluR5 gene expression during the differentiation of ES cells into cardiomyocytes.

[0022] ES cells formed embryoid body (embryoid body, EB) by hanging drop culture method, and EB adherent culture was differentiated into cardiomyocytes, DMSO was added during adherent culture as a negative control, and retinoic acid (RA) was used as a positive control. Collect ES cells, EBs, cardiomyocytes differentiated from ES cells at different times (d 5+3, d 5+7, d 5+11), and collect differentiation-inducing agent 1×10 -8 Total mRNA was extracted from cardiomyocytes contracted at the same time point under the action of mol / L RA, and mouse mature heart tissue (heart, H) according to the instructions of Trizol reagent. After extraction, the mRNA was precipitated according to the instructions, and then dissolved in 0.1% diethylpyrocarbonate (DEPC) water. RT reaction: 5 μg total RNA, 0.5 μg oligo(dT) 6 Primers, 5 μL deionized water, 65...

Embodiment 2

[0026] Example 2. Detection of mGluR5 protein expression in ES cell cardiogenic system by Western blot.

[0027] Collect ES cells, EBs, cardiomyocytes differentiated from ES cells at different times (d 5+3, d 5+7, d 5+11, d 5+13), or collect differentiation-inducing agent 1×10 -8Cardiomyocytes contracted at the same time point under the action of mol / L RA, mouse mature heart tissue (heart, H), lysis buffer (PH 7.5 Tris-hydrochloric acid 20mmol / L, NaCl 150mmol / L, EDTA 1mmol / L, Triton X-1001%, sodium deoxycholate 0.5%, PMSF 1mmol / L, leupeptin 10μg / ml, aprotitin 30μg / ml), pipetting repeatedly on ice for 30min, centrifuging at 13000×g for 30min at 4°C The obtained supernatant was the total protein, and the protein concentration was quantified by the Lowry kit method (DC protein detection kit, Bio-Rad, CA, US), and then frozen for future use. SDS-PAGE, western blotting and film densitometric analysis methods were performed routinely. The amount of protein loaded was 100 μg / lane. ...

Embodiment 3

[0029] Example 3. Identification of mGluR5 protein expression in ES cell cardiogenic system by immunofluorescence double staining

[0030] Take samples of beating cardiomyocytes on the 11th day of EB adherent differentiation (d 5+11), wash them twice with phosphate buffered solution (PBS), fix with 2-4% formaldehyde at room temperature for 15 minutes, and then fix with pure cold methanol 10 min, and then treated with PBS / TritonX-100 containing 5% fetal bovine serum for 1 h, then added primary antibody: rabbit monoclonal anti-mGluR5, diluted 1:100, diluted 1:100, and incubated overnight. On the second day, wash three times with PBS, add secondary antibody: TRITC-goat anti-rabbit IgG, dilute 1:200, incubate at 37°C for 1.5h, wash with PBS, stain with DAPI, observe with a fluorescent microscope, and take pictures for recording.

[0031] See results Figure 4 , showing that ES cells were differentiated into cardiomyocytes, mGluR5 was positively expressed, and mGluR5 was co-expres...

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Abstract

The invention provides application of metabolic glutamic acid receptor 5 as a target spot in screening and evaluating induced differentiators or regulators for promoting the differentiation of stem cells to cardiac muscle cells through establishing a screening and evaluating model using mGluR 5 as a regulating target spot by directionally differentiating the embryonic stem cells to a cardiac muscle cell model. The invention defines the expression characteristics of the mGluR 5 in the process of differentiating the embryonic stem cells to the cardiac muscle cells and the close relation of the regulation and control of the mGluR 5 and the differentiation and development of the cardiac muscle cells. The invention develops new application of the mGluR 5 as the regulating target spot in differentiating the embryonic stem cells to the cardiac muscle cell model and can be used for evaluating cardiac muscle differentiation-promoting inducers or metabolic glutamic acid receptor regulators using the mGluR 5 as the target spot.

Description

technical field [0001] The invention belongs to the fields of developmental biology, molecular biology and pharmacology, and relates to the expression and function research of type 5 metabotropic glutamate receptors related to the directional differentiation of embryonic stem cells into cardiomyocytes. As a target for interfering with the differentiation of stem cells into cardiomyocytes, thereby regulating myocardial differentiation and development, it is used in the screening and evaluation of cardiomyocyte differentiation inducers or metabotropic glutamate receptor modulators. Background technique [0002] Metabotropic glutamate receptors (mGluRs) are a new family of G protein-coupled receptors. At present, studies on mGluRs-mediated physiological / pathological processes, pharmacology and related molecular levels are mainly focused on the central nervous system. A small number of studies have reported that mGluRs not only play an important role in the central nervous syst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02G01N33/53G01N21/64G01N15/10
Inventor 朱丹雁周立民崔荣张莹莹楼宜嘉张翔南梁星光
Owner ZHEJIANG UNIV
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