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Method of growing myocardial cells

A cardiomyocyte and cell technology, applied in animal cells, vertebrate cells, genetically modified cells, etc., can solve problems such as unclear ubiquitin-protease system

Inactive Publication Date: 2006-12-20
DAIICHI SANKYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little is known about how the ubiquitin-protease system is involved in the regulation of proliferation in cardiomyocytes

Method used

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  • Method of growing myocardial cells
  • Method of growing myocardial cells
  • Method of growing myocardial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0242] [Example 1: Preparation of recombinant adenovirus]

[0243] The adenoviral vector containing the CDK4 gene, the cyclin D1 (D1NLS) gene added with the nucleotide sequence encoding the nuclear localization signal (NLS), or the Skp2 gene was prepared using a recombinant adenovirus production kit (Adenovirus Expression Vector Kit; Takara Bio) make.

[0244] That is, when preparing Ad-CDK4, an adenoviral vector containing the CDK4 gene, plasmid pCMV-CDK4 (obtained from Dr. Sander van den Heuvel [Massachusetts General Hospital Cancer Center; USA]; van den Heuvel et al., Science 262: 2050, 1993) was cut with BamHI to prepare mouse CDK4 cDNA fragment, and after using T4 DNA polymerase to blunt the end, it was inserted into the SwaI site of cosmid pAxCAwt according to the operation instructions attached to the above recombinant adenovirus production kit to make cosmid pAd -CDK4. Then, recombinant adenovirus Ad-CDK4 was produced by transfecting the cosmid and restriction enzyme...

Embodiment 2

[0256] [Example 2: p27 Kip1 Protein accumulation in DNLS / CDK-treated cardiomyocytes]

[0257] Cardiomyocytes were isolated from 2-4 day old rats (Sprague-Dawley strain), and the cardiomyocyte fraction was recovered by Percol concentration gradient centrifugation (Tamamori et al., Am. J. Physiol. 275: H2036, 1998). 95% or more of the cells thus obtained were confirmed to be cardiomyocytes by immunostaining using an anti-sarcomeric actin antibody. The neonatal rat cardiomyocytes were suspended in Eagle basal medium (Flow Laboratories) supplemented with 5% fetal bovine serum (FBS; Flow Laboratories), inoculated on a culture dish, and cultured in a carbon dioxide incubator at 37°C 24 hours. The next day, the culture solution was replaced with serum-free Eagle basal medium, and after cultivating for 24 hours, the recombinant virus Ad-D1NLS (moi=10~100) and Ad-CDK4 (moi=100) prepared in Example 1 were Added to the culture medium and cultured for 48 hours. Hereinafter, the recomb...

Embodiment 3

[0268] 〔Example 3: The forced expression of Skp2 leads to the accumulation of p27 in cardiomyocytes Kip1 protein reduction]

[0269] It is known that in normal proliferating cells, p27 Kip1 Protein is contained as F-box protein Skp2 ubiquitin ligase or SCF Skp2 The complex is ubiquitinated and broken down by the proteasome (Carrano et al., Nature Cell Biol. 1:193, 1999; Tsvetkov et al., Curr. Biol. 9:661, 1999). Therefore, the expression of Skp2 protein in cardiomyocytes was investigated by immunoblotting. The preparation of cardiomyocytes, the transfection method of Ad-D1NLS and Ad-CDK4, the preparation of nuclear protein, and Western blot analysis were the same as the above methods, and the above-mentioned anti-Skp2 antibody was used to detect the Skp2 protein.

[0270] The result is as Figure 5shown. In fibroblasts (REF52 cells) used as control cells, the expression of Skp2 protein was significantly increased by FBS stimulation and D1NLS+CDK4 stimulation to promote pr...

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Abstract

The growth of myocardial cells is induced by expressing cycline and CDK in the myocardial cells and regulating the function or action of a Cip / Kip protein, or by inhibiting the production of a Cip / Kip protein. Among the Cip / Kip family proteins, it is preferable to regulate the function of p27<KIP1> or inhibiting the production thereof. As a recombinant vector to be used therefor, it is intended to provide a vector having: (1) a cyclin gene; (2) a cyclin-dependent kinase gene and (3) one or more members selected from the group consisting of a gene encoding a factor inhibiting the function or action of a Cip / Kip protein and nucleic acid sequences inhibiting the production of Cip / Kip family proteins.

Description

technical field [0001] The present invention relates to methods for proliferating mammalian cardiomyocytes. Background technique [0002] Because the cardiomyocytes of mature individuals lose the ability of cell division, if they are exposed to various stresses such as ischemia and myocarditis, the cardiomyocytes will be necrotic and lost, and the cardiomyocytes cannot be replenished. As a result, although the remaining cardiomyocytes compensatory hypertrophy to maintain heart function, once this state continues, once the allowable range of the myocardial tissue is exceeded, it goes without saying that the cardiomyocytes will be exhausted and die, and eventually the myocardial function will be low or short. heart failure. [0003] Heart disease, including heart failure, has become the second leading cause of death in Japan. In addition, the prognosis of patients with heart disease is very poor, and the 5-year survival rate is only about 50%. Therefore, the development of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N5/10A61K31/7088A61K35/34A61K35/76A61K48/00A61P9/00A61P9/04A61P9/10A61K35/12A61P43/00C07K14/47C12N5/077C12N9/12C12N15/113
CPCC12N9/1205C07K14/4738C12N2510/00A61K31/7088A61K35/12A61K48/00C12N5/0657A61P43/00A61P9/00A61P9/04A61P9/10C12N15/09
Inventor 安达三美中山敬一北岛繁孝高木弘光
Owner DAIICHI SANKYO CO LTD
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