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Method for improving efficiency of differentiating rat bone marrow mesenchymal stem cells into myocardial cells

A bone marrow-derived mesenchymal and stem cell differentiation technology, applied in bone/connective tissue cells, biochemical equipment and methods, cells modified by the introduction of foreign genetic material, etc., can solve problems such as limiting the potential clinical application of cardiac repair, and increase Induction efficiency, rigorous and careful experimental process, and reasonable and accurate verification results

Inactive Publication Date: 2021-03-12
YANGZHOU UNIV
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Problems solved by technology

However, only a very small number of mesenchymal stem cells are positive for cardiomyocyte-specific proteins during differentiation, which significantly limits their potential clinical application in cardiac repair.

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  • Method for improving efficiency of differentiating rat bone marrow mesenchymal stem cells into myocardial cells
  • Method for improving efficiency of differentiating rat bone marrow mesenchymal stem cells into myocardial cells

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Embodiment Construction

[0017] The following embodiments further describe the present invention in conjunction with the accompanying drawings.

[0018] (1) Design interference targets according to the CDS region sequence of G9a provided by NCBI. After the upstream and downstream sequences were synthesized, they were annealed to form oligonucleotide double strands, and the pGMLV-SC5 RNAi vector was ligated by BamHI and EcoRI double digestion to obtain recombinant interference plasmids, respectively Sh-G9a. Then the recombinant interference plasmid was sequenced for further verification. After successful sequencing, the vector was subjected to lentiviral packaging.

[0019] (2) FuGENE (Promega) liposomes were used to mediate G9a-shRNA lentiviral recombinant plasmids to transfect rat bone marrow mesenchymal cell lines. 24 hours before transfection, the rat bone marrow mesenchymal cells were divided into 1 × 10 5 Each well was inoculated in a 24-well plate and cultured with 10% FBS-containing DMEM med...

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Abstract

The invention relates to a method for improving the efficiency of differentiating rat bone marrow mesenchymal stem cells into myocardial cells, which comprises the following steps: (1) constructing alentiviral interference vector G9a-shRNA, and transfecting the G9a-shRNA to the rat bone marrow mesenchymal stem cells; and (2) inducing the bone marrow mesenchymal stem cells transfected with the interference vector through 5-azacytidine, and detecting the positive rate of troponin positive cells by flow cytometry on the 21st day. According to the invention, in the process of inducing the bone marrow mesenchymal stem cells to differentiate into the myocardial cells by the 5-azacytidine, the H3K9me2 methylase G9a is knocked down, and the final induction efficiency is observed, so that a convenient and rapid way is expected to be found in the process of differentiating the bone marrow mesenchymal stem cells into the myocardial cells. According to the method, the rat bone marrow mesenchymalstem cells are transfected by adopting FuGENE (Promega) lipidosome mediated G9a-shRNA lentivirus recombinant plasmids to reduce the expression of H3K9me2, so that the induction efficiency is improved,the verification result is reasonable and accurate, and the experimental process is rigorous and rigorous.

Description

technical field [0001] The invention relates to a method for improving the differentiation efficiency of rat bone marrow mesenchymal stem cells into cardiomyocytes, and belongs to the field of stem cell differentiation and cultivation. Background technique [0002] Biological pacemakers offer a new option for the treatment of bradyarrhythmias, and bone marrow mesenchymal stem cells are one of the main sources of heart repair and regenerative cell therapy because these cells are readily available and virtually immune to any adverse immune response Not inherently immunogenic. However, only a very small number of mesenchymal stem cells are positive for cardiomyocyte-specific proteins during differentiation, which obviously limits their potential clinical application in cardiac repair. Contents of the invention [0003] The purpose of the present invention is to address the above problems and provide a method for improving the differentiation efficiency of rat bone marrow mes...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/10C12N5/0775C12N15/867
CPCC12N5/0657C12N5/0663C12N9/1007C12N15/86C12N2506/1353C12N2500/40C12N2740/15043
Inventor 孙小林顾翔徐佩李红校朱业左其生李碧春
Owner YANGZHOU UNIV
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