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Method for restraining mesenchymal stem cells from differentiating into fat cells

A technology of mesenchymal stem cells and adipocytes, applied in the field of genetic engineering, can solve problems that have not been reported, and achieve the effects of simple operation, strong in vitro proliferation, and high efficiency

Inactive Publication Date: 2013-05-15
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on whether ICAM-1 regulates the differentiation ability of MSCs and its mechanism of action

Method used

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  • Method for restraining mesenchymal stem cells from differentiating into fat cells
  • Method for restraining mesenchymal stem cells from differentiating into fat cells
  • Method for restraining mesenchymal stem cells from differentiating into fat cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of MIGR1-ICAM-1 Retroviral Vector

[0031] In this experiment, a retroviral vector (MIGR1) was purchased from Clonetech.

[0032] The raw materials used in this experiment are as follows: α-MEM medium (Gibco,), fetal bovine serum (Hyclone), mice (Experimental Animal Center, Academy of Military Medical Sciences), Trizol (Invitrogen), PrimeScript reverse transcription kit (Invitrogen) , TaqDNA polymerase (Invitrogen), mouse ICAM-1 primer (Shanghai Sangong), agarose gel DNA recovery kit (Tiangen Biochemical), pMD19-T Simple Vector (Dalian Bao Biology), Escherichia coli competent bacteria DH5α (Invitrogen).

[0033] 1. Amplification of ICAM-1 full-length DNA fragment

[0034] The mouse spleen was aseptically isolated, and the mononuclear cells were obtained by grinding and lysing red blood cells. Take 1×10 7 The mouse spleen mononuclear cells were placed in a 1.5ml EP tube, and the total RNA was extracted by the Trizol method. According to the instructions ...

Embodiment 2

[0043] Identification of Expression Efficiency of Recombinant Retroviral Expression Plasmid MIGR1-ICAM-1 and Empty Plasmid MIGR1 Transfected Packaging Cell 293T and Mesenchymal Stem Cells (C3H10T 1 / 2) and ICAM-1

[0044] In this experiment, packaging cells (293T) and mesenchymal stem cells (C3H10T 1 / 2) were purchased from ATCC.

[0045] The raw materials used in this experiment are as follows: packaging plasmid ECOS (Clonetech), α-MEM medium (Gibco), fetal bovine serum (Hyclone), LipofectamineTM 2000 (Invitrogen), Polybrene (Sigma), SYBR Green JumpStartTM Taq ReadyMixTM kit (Sigma), PE-labeled anti-mouse ICAM-1 (anti-mouse ICAM-1 ) and the corresponding isotype control antibody (IgG2a PE) (ebioscience).

[0046] 1. Recombinant retrovirus expression plasmid MIGR1-ICAM-1 and empty plasmid MIGR1 transfected 293T cells

[0047] 24h before transfection, digest 293T cells and count them with 1.5×10 6 Seed in 6-well plates, so that the cell fusion rate reaches 80%~90% when transfec...

Embodiment 3

[0059] Detection of the function of mesenchymal stem cells highly expressing ICAM-1 in differentiating into adipocytes

[0060] The raw materials used in this experiment are as follows: dexamethasone (Sigma), IBMX (Sigma), insulin (Sigma), high glucose DMEM medium (Gibco), fetal bovine serum (Hyclone), penicillin (Sigma), streptomycin (Sigma), oil red O (Sigma), mouse C / EBPα and PPARγ primers (Shanghai Sangong).

[0061] Adipogenic differentiation was detected according to the method previously published by our laboratory (Zhu H, Guo Z K, Jiang X X, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc, 2010, 5(3) : 550-560.).

[0062] Harvest the cell C3H10T 1 / 2-MIGR1-ICAM-1 / MSC overexpressing ICAM-1 and the control cell C3H10T 1 / 2- MIGR1 / MSC, respectively, according to 2×10 4 / hole and 1.0×10 6 Each well was planted in a 48-well culture plate and a 6-well culture plate, and the culture system of the self-differentiation ...

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Abstract

The invention belongs to the technical field of gene engineering and particularly relates to a method for restraining mesenchymal stem cells from differentiating into fat cells. A purpose of restraining the mesenchymal stem cells from differentiating into the fat cells is realized by the following steps of: constructing recombinant retrovirus plasmids containing ICAM-1 (intercellular cell adhesion molecule) genes, carrying out co-transfection on the packaging cells by the recombinant retrovirus plasmids together with packaging plasmids, collecting relevant viral supernatants, and infecting the mesenchymal stem cells. According to the method for restraining the mesenchymal stem cells from differentiating into the fat cells, the separation method is simple in operation, convenient and practical, the efficiency (more than 90% of ICAM-1 can be highly expressed) for transfecting the mesenchymal stem cells is high, and the obtained mesenchymal stem cells are exuberant in in-vitro multiplication and can be passed down for multiple times (more than 50 generations). Thus, as the method and a culture technique system for restraining mesenchymal stem cells from differentiating into fat cells are established, a foundation for research and application of the mesenchymal stem cells is laid.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for inhibiting the differentiation of mesenchymal stem cells into adipocytes. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) are adult stem cells with multi-directional differentiation potential, which can differentiate into osteoblasts, chondrocytes, adipocytes and other tissue types under suitable conditions. Under physiological conditions, mesenchymal stem cells participate in the growth, development and metabolism of the body by differentiating into multiple progeny cells, and participate in tissue repair under pathological conditions. However, it is worth noting that when strong damage factors persist and exceed the body's regulatory range, mesenchymal stem cells will also participate in the pathological process of the disease and aggravate tissue damage. For example, in liver disease, mesenchymal stem ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 张毅朱恒徐芬芬陈继德刘元林
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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