Mesenchymal stem cell model with loss of RAP1 function and preparation method and application thereof

A cell model and pluripotent stem cell technology, applied in the field of human mesenchymal stem cell model and its preparation, can solve problems such as over-activation of telomerase and carcinogenesis, and achieve the effects of delaying the aging process, improving cell proliferation, and prolonging telomere length.

Active Publication Date: 2019-05-17
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the easiest way to extend telomeres is to overexpress telomerase, but overactivation of telomerase has the potential risk of canceration, so finding other safer methods to regulate telomere length has become an important issue

Method used

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  • Mesenchymal stem cell model with loss of RAP1 function and preparation method and application thereof
  • Mesenchymal stem cell model with loss of RAP1 function and preparation method and application thereof
  • Mesenchymal stem cell model with loss of RAP1 function and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1. Construction and Identification of Embryonic Stem Cell Lines with Loss of RAP1 Function

[0084] This example relates to the targeted inactivation of the human RAP1 gene in human embryonic stem cells (genome sequence is GenBank: NC_000016.10 at position 75,647,737-75,657,442, updated on PRI 12-JUL-2017; cDNA sequence is GenBank: NM_018975.3, updated on PRI 10-JUL-2017), the function of RAP1 protein was lost, and human embryonic stem cells with loss of RAP1 function were obtained.

[0085] The present invention firstly designs and obtains homology arm sequences targeting both sides of exon 2 of the RAP1 gene in the human genome through a molecular cloning method, and then constructs them on the pCR2.1-neo vector to obtain the RAP1 homology arm vector. Then construct a gRNA vector targeting exon 2 of the RAP1 gene (product of Addgene Company, trade name and catalog number are gRNA Cloning Vector, Plasmid #41824). Finally, the RAP1 homology arm vector, gRNA exp...

Embodiment 2

[0137] Example 2, RAP1 - / - Directed differentiation of hESC in vitro to produce RAP1 - / - hMSC and RAP1 - / - hMSC phenotyping

[0138] In this embodiment, the RAP1 obtained in Example 1 - / - hESC were further differentiated into mesenchymal stem cells in vitro (RAP1 - / - hMSC), and found that RAP1 - / - hMSCs can delay the aging phenotype to a certain extent.

[0139] 1. RAP1 - / - Directed differentiation of hESC in vitro to produce RAP1 - / - hMSC

[0140] RAP1 - / - The hESCs were differentiated into embryoid bodies (EBs) and differentiated for 48-72 hours to obtain embryoid bodies (EBs). Then, the EBs were inoculated into 6-well plates coated with Matrigel (Invitrogen Company) for culture, and continued to culture for 2 weeks until fibrous cells appeared. After another passaging, the cell populations in which CD73, CD90 and CD105 were all positive were sorted by flow cytometry ( figure 2 Middle A), that is, loss of RAP1 function (RAP1 - / - ) human mesenchymal stem cells (d...

Embodiment 3

[0177] Example 3, RAP1 - / - Detection of hMSC Telomere Length

[0178] 1. Use Southern blotting to analyze the length of telomere restriction fragments

[0179] To investigate whether RAP1 loss-of-function affects changes in telomere length, RAP1 + / + hMSC (wild type) and RAP1 - / - hMSC cells (passages P2 and P9) were the test cells. They were normally cultured and digested to collect the cells. The complete genomic DNA was extracted and the relative length of telomere restriction fragments was analyzed by Southern blotting. Specific steps are as follows:

[0180] 1) Digest and collect cells, centrifuge at 1000 rpm for 5 minutes;

[0181] 2) Wash once with PBS, use the salting-out method DNA extraction kit (product of Meiji Company, article number: D3313-01) to extract the genomic DNA of the test cells, and obtain a large amount of complete genomic DNA;

[0182] 3) Use HinfⅠ (product of NEB Company, product number: R0155) and RsaⅠ (product of NEB Company, product number: R01...

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Abstract

The invention discloses a mesenchymal stem cell model with loss of the RAP1 function and a preparation method and application thereof. The preparation method comprises the steps of reducing the content and / or the activity of RAP1 in pluripotent stem cells or inhibiting the expression of a RAP1 encoding gene in the pluripotent stem cells to obtain the pluripotent stem cells with loss of the RAP1 function; inducing the pluripotent stem cells with loss of the RAP1 function to obtain the mesenchymal stem cells with loss of the RAP1 function; the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. The mesenchymal stem cell model with loss of the RAP1 function shows improvement of cell reproductive capacity, low degeneration speed, improvement of retention capacityin vivo and telomere length increase, and can be utilized to study the relationship between telomere length change and cell senescence, screen toxic molecules that accelerate aging of cells and serveas a cellular material for proliferation and enhancement of the retention capacity in vivo for cell therapy.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a RAP1 function-losing mesenchymal stem cell model and its preparation method and application, in particular to a RAP1 function-losing human embryonic stem cell model and a RAP1 function-losing human mesenchymal stem cell model and its preparation method and application application. Background technique [0002] Telomere DNA is the DNA repeating sequence at the end of eukaryotic linear chromosomes. It forms a high-level structure together with the complex protein components it recruits, and maintains the stability of chromosome ends, which has important physiological significance. The abnormality of telomeres will lead to the instability of cell chromosomes, causing DNA damage and repair reactions, which in turn will lead to abnormal chromosome recombination and end bonding. Along with DNA replication and cell division, the "Aging Theory" believes that the excessive shorteni...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N15/85C12N5/10C12Q1/02A61K35/28A61P39/06
Inventor 刘光慧曲静张钘刘尊鹏王思
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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