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A method for improving the osteogenic differentiation ability of human mesenchymal stem cells

A technology of osteogenic differentiation and mesenchymal stem cells, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems such as carcinogenesis, interfere with normal gene expression, and affect genome stability, so as to improve differentiation ability and technology Simple, easy-to-operate effect

Active Publication Date: 2016-08-24
深圳市雄聚投资发展有限公司
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  • Summary
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  • Application Information

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Problems solved by technology

However, the use of vectors to introduce foreign genes will affect the stability of the genome, and the integration site of foreign genes may also interfere with the normal expression of cancer-related genes, causing the risk of cancer

Method used

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  • A method for improving the osteogenic differentiation ability of human mesenchymal stem cells
  • A method for improving the osteogenic differentiation ability of human mesenchymal stem cells
  • A method for improving the osteogenic differentiation ability of human mesenchymal stem cells

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specific Embodiment approach 1

[0017] Embodiment 1: The method for improving the differentiation ability of human mesenchymal stem cells in this embodiment is carried out according to the following steps:

[0018] 1. Optimizing the synthesis of the hNanog gene sequence: without changing the encoded amino acid sequence of the hNanog gene, optimizing the codons of the hNanog gene to obtain the optimized hNanog gene;

[0019] 2. Carrier connection: use EcoRI and NdeI to double-digest the optimized hNanog gene to obtain the target gene fragment, then use EcoRI and NdeI to double-digest the pET28b plasmid to obtain the carrier fragment, and combine the target gene fragment and the carrier fragment at 16 ℃ for 24 hours to obtain the pET28b-nanog recombinant expression vector;

[0020] 3. Low temperature induction of competent Escherichia coli and optimized hNanog recombinant protein expression: Transform the pET28b-nanog recombinant expression vector into Escherichia coli competent cells, and after 12 hours of in...

specific Embodiment approach 2

[0024] Specific embodiment two: the difference between this embodiment and specific embodiment one is: the system of double enzyme digestion of hNanog gene in step two is as follows:

[0025] Element

[0026] Enzyme digestion reaction conditions: 37°C water bath, 4h. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0027] Specific embodiment three: the difference between this embodiment and specific embodiment one or two is: the system of pET28b plasmid double-enzyme digestion in step two is as follows:

[0028]

[0029]

[0030] Enzyme digestion reaction conditions: 37°C water bath, 4h. Others are the same as in the first or second embodiment.

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Abstract

A method for improving the osteogenic differentiation ability of human mesenchymal stem cells relates to a method for improving the differentiation ability of human mesenchymal stem cells. The invention provides a method for improving the differentiation ability of human mesenchymal stem cells. There is no danger of changing the genome of cells, which fundamentally solves the problem of possible canceration. Methods: 1. Optimization of hNanog gene sequence synthesis; 2. Vector connection; 3. Competent Escherichia coli low temperature induction of optimized hNanog recombinant protein expression; 4. Extraction and purification of recombinant protein; 5. BMSCs osteogenesis experiment induced by recombinant protein. The method of the invention is more convenient and easy to operate, and reduces the difficulty of promoting MSCs differentiation in vitro. It is used to improve the differentiation ability of human mesenchymal stem cells.

Description

technical field [0001] The invention relates to a method for improving the differentiation ability of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are adult stem cells that can self-renew and have strong multidirectional differentiation potential. Under appropriate induction conditions, they can differentiate into cartilage, osteogenesis, muscle, fat, nerve, etc. cells of various tissues. MSCs are widely found in spinal cord, fat, umbilical cord blood and other tissues, and have sufficient sources. They have become ideal candidate seed cells for cell and tissue regeneration and repair, and are widely used in clinical research and treatment of bone, cartilage, nervous system, and cardiovascular diseases. Clinical transplantation requires a considerable number of MSCs. The number of MSCs in any tissue in the human body is limited, and the use of a large number of MSCs will seriously affect human health. Therefore, it must be cultured in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N15/12C12N15/70C07K14/47
Inventor 顾宁岳磊胡海龙
Owner 深圳市雄聚投资发展有限公司
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