Method of inducing fibroblasts into chondrocyte like cells

A fibroblast and chondrocyte technology, applied in the field of cell biology technology and regenerative medicine, can solve problems such as clinical application limitations and safety hazards

Inactive Publication Date: 2017-10-20
中国人民解放军联勤保障部队第九二〇医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, scientists have used induced pluripotent stem cells to differentiate bone and cartilage tissues (Hong SG, et al., CellReports, 2014, 7 (4): 1298-1309), however, reprogramming and lineage reprogramming technologies are currently the most effective The method is a vector-based transcription factor-mediated method. Since this method involves gene introduction, there are potential safety hazards, so it needs to be strictly limited in clinical application.

Method used

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  • Method of inducing fibroblasts into chondrocyte like cells
  • Method of inducing fibroblasts into chondrocyte like cells
  • Method of inducing fibroblasts into chondrocyte like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of inducers.

[0029] In this example, the inducer is used for the reprogramming of fibroblasts. The inducer is an extract of crucian carp oocytes, and crucian carp eggs are preferably in the yolk-filled phase.

[0030](1) Completely immerse crucian carp in 75% alcohol for 15-20 minutes to fully disinfect and prevent bacterial contamination to the greatest extent.

[0031] (2) Take out the fish eggs under aseptic conditions in a fume hood, then put them into a pre-cooled mortar, and place the mortar on ice for thorough grinding.

[0032] (3) After grinding, add pre-cooled physiological saline, centrifuge at 1000rpm at 4°C for 10min, then centrifuge at 3000rpm at 4°C for 10min, and take the supernatant.

[0033] (4) Use a 0.22 micron pore filter to filter the supernatant obtained in step (3), the filtrate is the prepared fish oocyte extract, and use the Coomassie brilliant blue method to determine the concentration of the prepared fish oocyte extract. prote...

Embodiment 2

[0036] Preparation of human skin fibroblasts.

[0037] The fibroblasts in this example are derived from the foreskin tissue of healthy males aged 20-25, and the preparation method thereof includes the following steps.

[0038] (1) Collection of human skin tissue: select the foreskin tissue of healthy men aged 20-25, and cut about 1×1cm under sterile conditions 2 spare. The foreskin tissue was provided by the Urology Department of the Kunming General Hospital of the PLA. The use of the foreskin tissue was approved by the Medical Ethics Committee of the Kunming General Hospital of the PLA. The collection and use of the foreskin tissue were obtained with the informed consent of the patients.

[0039] (2) Preparation of human skin fibroblasts: the skin tissue collected in the step (1) was repeatedly washed with saline and then placed in a 1×PBS solution containing 100 U / ml ampicillin and 100 mg / ml streptomycin sulfate Rinse three times, discard fat and connective tissue, and the...

Embodiment 3

[0041] Induction of fish oocyte extracts from human skin fibroblasts.

[0042] Inducing the human skin fibroblasts prepared in Example 2 with the inducer prepared in Example 1, so that the human skin fibroblasts are apparently reprogrammed into induced pluripotent cells under the induction of the fish oocyte extract Stem cells, the method comprising the following steps.

[0043] (1) Induction of fibroblasts: when the human skin fibroblasts prepared in Example 2 were subcultured to the fourth generation and the fibroblasts grew to 80% cell confluence, fish with a protein concentration of 10 mg / ml was added The oocyte extract was induced and cultured for 72 hours, so that the fibroblasts were apparently reprogrammed into induced pluripotent stem cells, and the final concentration of the fish oocyte extract used for induction was 10-20ug / ml.

[0044] (2) Identification of induced pluripotent stem cells: When the step (1) is completed, the apparent reprogramming of human skin fib...

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Abstract

The invention relates to a method for inducing fibroblasts into chondrocyte like cells, and belongs to the field of cell biology and regenerative medicine. The method aims at the general problems that carrier mediation and transgenic operation are needed, requirements on experimental facilities and technical level are relatively high, the security of produced chondrocytes is relatively poor and the like in methods for transdifferentiation of the fibroblasts into the chondrocytes in the prior art, and uses fish oocyte extract for inducing reprogramming of the fibroblasts into the chondrocyte like cells. The chondrocyte like cells are obtained only by use of the fish oocyte extract for inducing and use of a chondroblast induction medium for directional inducing culture without introduction of exogenous genes, so that experimental operation is relatively simple, and the obtained chondrocyte like cells are better in security, and have a broad application prospect in the field of cartilage tissue engineering and regenerative medicine.

Description

technical field [0001] The invention relates to a method for inducing fibroblasts to be chondrocyte-like cells, belonging to the fields of cell biology technology and regenerative medicine. Background technique [0002] Cartilage tissue is a tissue rich in proteoglycans, collagen, and hyaluronic acid. It is composed of chondrocytes and cartilage matrix. It mainly supports and reduces friction in the body. However, cartilage tissue lacks blood vessels, so it is difficult to repair cartilage tissue once it is damaged, even if it is a small cartilage tissue injury, it is difficult to repair naturally. [0003] At present, the clinical methods for repairing cartilage tissue damage mainly include cartilage and subchondral bone removal, microfracture and subchondral bone drilling, autologous osteochondral transplantation and allogeneic osteochondral transplantation, cartilage or periosteum transplantation, etc. , et al. Chinese Journal of Traumatic Orthopedics, 2014,16(6):537-539...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2500/82C12N2506/1307
Inventor 朱向情潘兴华陈强
Owner 中国人民解放军联勤保障部队第九二〇医院
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