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Human mesenchymal stem cell capable of delaying cell senescence and resisting malignant transformation and preparation method and application thereof

A technology of pluripotent stem cells and pluripotent stem cells, applied in the field of human mesenchymal stem cells and their preparation, can solve the problems of weakened application value, increased risk of gene mutation, and worrying safety, and achieves great application and promotion value and optimal tissue repair function. , the effect of good ability

Active Publication Date: 2019-03-01
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a series of preclinical studies have proved the feasibility of improving the inherent stress resistance of cells, gene regulation mediated by integrated viral vectors inevitably involves random integration of the genome, which increases the risk of gene mutation and worries about safety, which greatly weakens its potential. Clinical application value

Method used

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  • Human mesenchymal stem cell capable of delaying cell senescence and resisting malignant transformation and preparation method and application thereof
  • Human mesenchymal stem cell capable of delaying cell senescence and resisting malignant transformation and preparation method and application thereof
  • Human mesenchymal stem cell capable of delaying cell senescence and resisting malignant transformation and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1, Preparation of FOXO3 Enhanced Pluripotent Stem Cells

[0053] 1. Construction of recombinant plasmids

[0054] Insert the DNA molecule shown in Sequence 1 of the sequence listing between the SalI and SpeI restriction sites of the pAMHDAdGT8-4 vector to obtain a recombinant plasmid.

[0055] 2. Preparation of recombinant adenovirus

[0056] 1. Take the recombinant plasmid obtained in step 1, and digest it with PI-SceI to obtain a linearized plasmid.

[0057] 2. Introduce the linearized plasmid obtained in step 1 and the virus AdHPBGF35 into 116 cells, package the recombinant adenovirus, and then obtain recombinant adenovirus particles by ultra-high speed centrifugation.

[0058] 3. Infect target cells with recombinant adenovirus particles

[0059] Infect H9 cells (1×10 7 cells: 15btu virus), on the 2nd-4th day after infection, add G418 (25~400g / ml, product of Invitrogen Company) for positive selection, and on the 10th-13th day after infection, add 4μM Ganc...

Embodiment 2

[0070] Example 2. Preparation of FOXO3-enhanced mesenchymal stem cells and their functional testing

[0071] 1. Preparation of FOXO3-enhanced mesenchymal stem cells

[0072] 1, with the FOXO3 that embodiment 1 obtains 2SA / 2SA hESC cells were differentiated into embryoid bodies to obtain embryoid bodies (EBs).

[0073] 2. Inoculate the embryoid body obtained in step 1 in a matrigel-coated 6-well plate and culture (using mesenchymal stem cell differentiation medium) until fibrous cells appear (about 2 weeks).

[0074] 3. After step 2 is completed, the cells are collected, digested with trypsin, and then inoculated into a new mesenchymal stem cell differentiation medium, and cultured for 2 weeks.

[0075] 4. After completing step 3, use flow cytometry to sort cells that are positive for CD73, CD90, and CD105, which are FOXO3-enhanced mesenchymal stem cells, also known as FOXO3 2SA / 2SA MSC cells.

[0076] 2. Preparation of control mesenchymal stem cells

[0077] Substitution ...

Embodiment 3

[0144] Example 3, Application of FOXO3 Enhanced Mesenchymal Stem Cells in Cell Transplantation Therapy

[0145] Test cells: FOXO3 prepared in Example 2 2SA / 2SA MSC cells or FOXO3 + / + MSC cells.

[0146] A lentiviral vector that overexpresses luciferase luciferase, referred to as luc lentiviral vector. Reference: SIRT6safeguards human mesenchymal stem cells from oxidative stress by coactivatingNRF2.Pan et.al,.Cell research.(2016)26:190-205; that is, "luciferase(Control)-expressed vector" in the literature.

[0147] Lentiviral packaging plasmid psPAX, referred to as plasmid psPAX, Adgene, Cat. No. 12260. Lentiviral packaging plasmid pMD2.G, referred to as plasmid pMD2.G, Adgene, Cat. No. 12259. 293T cells (human embryonic kidney cells): ATCC, CRL-3216.

[0148] In this example, the tested cells were used for cell transplantation therapy.

[0149] 1. Preparation of luciferase-labeled test cells.

[0150] 1. Use Lipofectamine 3000 kit (Invitrogen) to co-transfect 15 μg luc ...

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Abstract

The invention discloses a human mesenchymal stem cell capable of delaying cell senescence and resisting malignant transformation, and a preparation method and application thereof. The invention protects a recombinant pluripotent stem cell. The preparation method of the stem cell comprises the following step that the genome of the pluripotent stem cell is subjected to two mutations: a codon of theamino acid residue at position 253 of encoded FOXO3 protein is mutated from a codon of encoded serine to a codon of encoded alanine, and a codon of the amino acid residue at position 315 of the encoded FOXO3 protein is mutated from a codon of encoded serine to a codon of encoded alanine. The invention also protects a recombinant mesenchymal stem cell prepared from the recombinant pluripotent stemcell, as well as application of the recombinant cells in the prepared product. The product is used for cell transplantation therapy. The two key and bottleneck problems about the effectiveness and safety of cell transplantation therapy are solved simultaneously, and the human mesenchymal stem cell has great application and promotion value in the field of cell transplantation therapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a human mesenchymal stem cell capable of delaying cell aging and resisting malignant transformation, a preparation method and application thereof. Background technique [0002] Cell transplantation therapy is the most promising and potential development direction in the field of regenerative medicine in recent years. It is a method to transplant specific cells into the lesion in the body to promote tissue regeneration and cure the disease. Due to their self-renewal ability and pluripotency, stem cells can not only maintain a stable number of cells through cell proliferation, but also differentiate into other cells for tissue repair, and become the first choice for cell therapy. Although cell transplantation therapy has shown excellent therapeutic effects in the treatment of leukemia, such as bone marrow stem cell transplantation, its wider application still faces many challenges. At p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K14/00C12N15/11
CPCC07K14/00C12N5/0606C12N5/0662C12N2510/02
Inventor 曲静刘光慧颜鹏泽王丽霞雷敬辉任若通
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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