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Small-molecular compound combination and method for inducing differentiated cells to prepare chondrocytes by using small-molecular compound combination

A small molecular compound and chondrocyte technology, applied in the field of cell biology, can solve the problems of cell induction and transdifferentiation, and the feasibility of transdifferentiation of similar cells is not high

Active Publication Date: 2018-05-22
深圳臻德济慈药品研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since there are about 25% genetic differences between humans and mice, it is not feasible to apply the above-mentioned successful patent application technology scheme in mouse cell experiments to human cells of the same kind; on the other hand, due to the induction of similar cells The specific theoretical basis and technical means of transdifferentiation to obtain different target cells are not the same. The applicant used the technical solutions reported above to repeat the experiments, but failed to successfully apply the transdifferentiation technical solutions applied to mouse cells to similar human cells. transdifferentiation, and failed to induce transdifferentiation of human differentiated cells into chondrocytes

Method used

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  • Small-molecular compound combination and method for inducing differentiated cells to prepare chondrocytes by using small-molecular compound combination
  • Small-molecular compound combination and method for inducing differentiated cells to prepare chondrocytes by using small-molecular compound combination
  • Small-molecular compound combination and method for inducing differentiated cells to prepare chondrocytes by using small-molecular compound combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] 1. Isolation of skin fibroblasts

[0157] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from the donor, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0158] 1.2 Subsequent passage of cells to a large number of expansion, the number of cell generations between the 6th and 12th passages is used for the induction of transdifferentiation into chondrocytes. The day before initiation of differentiation (Day-1), inoculate cell density at 1-2.5×10 4 / cm 2 Cultured at 37°C, 5% CO 2 in the incubator.

[0159] 2. The first stage induction of skin fibroblasts

[0160] After the treatment in the second step above, completely replace it with the second-stage culture medium for cell culture. The culture time is 6-12 days, at 37°C, 5% CO 2 environment to grow cells. The second st...

Embodiment 2

[0168] 1. Isolation of skin fibroblasts

[0169] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from the donor, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0170] 1.2 Subsequent passage of cells to a large number of expansion, the number of cell generations between the 6th and 12th passages is used for the induction of transdifferentiation into chondrocytes. The day before initiation of differentiation (Day-1), inoculate cell density at 1-2.5×10 4 / cm 2 Cultured at 37°C, 5% CO 2 in the incubator.

[0171] 2. Pretreatment of skin fibroblasts

[0172] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the first-stage culture medium, and culture the cells for 4 to 6 days. The first-stage culture medium refers to: 10% fetal bovine ...

Embodiment 3

[0182] 1. Isolation of skin fibroblasts

[0183] 1.1 Obtain a skin tissue block with a diameter of about 1 cm from the donor, separate the skin fibroblasts by the adherent method, and culture the separated cells in the basal culture medium: 10% fetal bovine serum (Hyclone) + 100U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0184] 1.2 Subsequent passage of cells to a large number of expansion, the number of cell generations between the 6th and 12th passages is used for the induction of transdifferentiation into chondrocytes. The day before initiation of differentiation (Day-1), inoculate cell density at 1-2.5×10 4 / cm 2 Cultured at 37°C, 5% CO 2 in the incubator.

[0185] 2. Pretreatment of skin fibroblasts

[0186] 2.1 When starting transdifferentiation (Day0), completely replace the basal culture medium with the first-stage culture medium, and culture the cells for 4 to 6 days. The first-stage culture medium refers to: 10% fetal bovine ...

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Abstract

The invention discloses a small-molecular compound combination and a method for inducing differentiated cells to prepare chondrocytes by using the small-molecular compound combination. The method is used for preparing the chondrocytes by directional induction of the differentiated cells. The directed induction includes inhibition of the activity of lysine deacetylase, inhibition of a signal pathway of TGF-beta, inhibition of the activity of PKC, inhibition of the activity of DNA methyltransferase, inhibition of the activity of histone methyltransferase, activation of a WNT / beta-catenin signalpathway, activation of a cAMP signal pathway and activation of an RA signal pathway; the small-molecular compound combination includes compounds for regulating and controlling the signal pathways and / or enzyme activities. Through phased induction of the small-molecular compounds, the differentiated cells can be differentiated into the chondrocytes, precision quality control can be achieved in eachstep, and standardization operation and large-scale production are facilitated. The method provided by the invention has wide donor sources, a patient himself can be used as a donor, and the chondrocytes needed for basic research, clinical treatment, or tissue engineering production can be obtained in a relatively short period of time.

Description

technical field [0001] The invention relates to the fields of cell biology, tissue engineering and regenerative medicine, in particular to a combination of small molecular compounds and a method for preparing chondrocytes from cells induced by the combination of small molecular compounds. Background technique [0002] Articular cartilage covers the ends of the bones and provides shock absorption and lubrication for the glenoid socket. As an avascular, alymphatic, and anervous tissue, cartilage has poor intrinsic healing capabilities. Cartilage damage has caused major economic and social problems worldwide and is now the fourth leading cause of disability, as trauma or age-related cartilage defects can lead to a variety of diseases such as osteoarthritis (OA). According to reports, about 9% of the people aged 30 and over in the United States have hip or knee OA patients, and the annual diagnosis and treatment costs are as high as 28.6 billion US dollars. Therefore, safe and...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2501/01C12N2501/115C12N2501/135C12N2501/15C12N2501/155C12N2501/405C12N2501/415C12N2501/72C12N2501/73
Inventor 胡敏李燕皎王兆杰
Owner 深圳臻德济慈药品研发有限公司
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