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Preparation method and applications of young repair-type fibroblast

A fibroblast and inhibitor technology, applied in the field of cell biology, can solve problems such as off-target, genetic variation, and tumorigenesis

Pending Publication Date: 2019-11-08
YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the induction efficiency of iPS is low, and involves the introduction of exogenous genes, and genetic variation often occurs, which is not conducive to clinical application
Genetic modification has recently been proposed as a rejuvenation method, but this method still has the risk of off-target and tumorigenesis

Method used

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  • Preparation method and applications of young repair-type fibroblast
  • Preparation method and applications of young repair-type fibroblast
  • Preparation method and applications of young repair-type fibroblast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1. Obtainment and identification of repaired skin fibroblasts

[0101] 1. Human skin fibroblasts are inoculated into a 6-well plate and cultured in skin fibroblast culture medium for 24 hours;

[0102] 2. Replace the cell culture medium with rFib induction medium containing the small molecule cocktail mix V, and change the medium every 2 days;

[0103] 3. After culturing for 5 days with the rFib induction medium containing the small molecule cocktail mix Mix V, change the medium to the rFib induction medium containing the small molecule cocktail mix Mix P, and change the medium every 2 days;

[0104] 4. Use the rFib induction culture medium containing the small molecule cocktail mix Mix P for 7 days, then change the culture medium to HG-DMEM with 10% FBS, 10ng / mL bFGF, 100ng / mL PDGF-AB and 10ng / mL BMP4 ; Or HG-DMEM with 10% FBS added; Or use rFib medium for culture; Cells are treated for 3 days after long-term passage; Cells are treated for 3 days after the identificati...

Embodiment 2

[0147] Example 2 rFib cells have bone defect repair ability

[0148] Under the approval of the ethics committee, 8 to 10 weeks old NOD / SCID mice weighing 20 to 24 g were used to create a femoral defect model. 5 animals were used in each group. The modeling method was as follows: under the anesthesia of sodium pentobarbital, cut Cut the skin and subcutaneous tissue, and expose enough mid-femur by blunt separation between the rectus femoris and semitendon. The operation is performed at the center of the right femur near the center. A continuous bone defect of 4mm×1mm was constructed by surgery. Skin fibroblasts (Fib), bone marrow mesenchymal stem cells (bMSC) and rFib were stained with Hoechst 33342 (Thermo, NucBlue live cell), then mixed with Matrigel and transplanted into the defect site with 5×105 cells / mouse.

[0149] 28 days after transplantation, the mice were sacrificed by injection of a lethal dose of sodium pentobarbital. The mouse thighs were bluntly separated, fixed wit...

Embodiment 3

[0156] Example 3 rFib cells have cartilage defect repair ability

[0157] Articular cartilage defect model and cell transplantation

[0158] NOD / SCID mice weighing 20 to 24 g at 8 to 10 weeks of age were used. A modified articular cartilage model was used to evaluate the efficacy of rFib (Cheng et al., 2014). The articular cartilage defect (1.5mm×1mm) was constructed in the trochlear groove of the distal femur with a biopsy punch. Cell ((2.5×10 5 Each in 35μl Matrigel) was labeled with Hoechst 33342 and implanted into the defect site. Matrigel without cells was implanted as a control.

[0159] Figure 7 .In vivo cartilage repair experiment

[0160] A-B. Cartilage tissue samples and 10μm sections were stained with Safranin and Fast Green. In Safranin Fast Green staining, red is cartilage and green is bone tissue. Both young rFib (39 years old) and bMSC (31 years old) can repair cartilage defects. Old bMSC (62 years old) can generate new tissues, but there is no new cartilage. Howe...

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Abstract

The invention discloses a preparation method and applications of a young repair-type fibroblast, and belongs to the technical field of cell biology. The JAK-STAT signal path in the repair-type fibroblast provided by the invention is inhibited, and the cell has the characteristic of being young. The preparation method adopts a small molecular compound, a cytokine combination or a recombinant protein combination to inhibit a corresponding signal path in a common fibroblast, and can be used for the reprogramming of cells, tissues, organs and organisms or the rejuvenation of cells, tissues, organsand organisms. The repair-type fibroblast and a cell product thereof can be used for constructing tissue engineering materials, repairing the damage of mammalian tissues and organs and repairing aging and degenerative tissues and organs, delaying or reversing the aging process of cells, tissues, organs and organisms, and can be used for the immunoregulation of cells, tissues, organs and organisms. The repair-type fibroblast prepared by the technology has the characteristics of skin fibroblasts, and has the characteristics of mesenchymal stem cells, so the fibroblast is named as the repair type skin fibroblast.

Description

Technical field [0001] The present invention belongs to the technical field of cell biology, and relates to a method for preparing rejuvenated repair fibroblasts and applications thereof, especially regulating the JAK-STAT signal pathway to regulate cell differentiation, dedifferentiation, transdifferentiation, and youth Methods for chemical, aging, apoptosis and prolonging the life of the body and the preparation and application of cell products. Background technique [0002] Stem cells are considered the "Holy Grail" of regenerative medicine and anti-aging. When an individual begins to age, the stem cells in the body will also age, leading to malignant transformation or degeneration of multiple organs including bones, cartilage, heart, muscle, brain, skin, pancreas, liver, kidneys, and gastrointestinal tract. Abnormal immune system function is also related to aging. In fact, chronic inflammation of tissues and organs is the cause of their degeneration and aging. Older cells ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61K35/33A61P39/00A61L27/38
CPCC12N5/0656A61K35/33A61P39/00A61L27/3804C12N2501/03C12N2501/15C12N2501/415C12N2501/42C12N2501/065C12N2501/01C12N2501/385C12N2501/405C12N2501/135C12N2501/155C12N2501/115C12N2501/11C12N2501/165C12N2501/33C12N2501/21C12N2501/12C12N2501/24C12N2501/2302C12N2501/2306C12N2501/25C12N2501/145C12N2501/105C12N2500/38A61P43/00C12N2501/999C12N2501/72C12N2500/40C12N5/0663C12N2510/00C12N5/0603
Inventor 胡敏李燕皎
Owner YUNNAN JICI INSITUTE FOR REGENERATIVE MEDICINE CO LTD
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