Method for inducing human skin fibroblasts to differentiate into adipocytes in vitro

A fibroblast, differentiation-inducing technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem that skin wound repair and scar repair cannot be applied, tissue sources are limited, and human skin fibroblasts cannot be used. Induced differentiation, etc.

Active Publication Date: 2021-06-18
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The source of cells in this protocol is primary mouse embryonic fibroblasts (MEF), which is relatively cumbersome and limited in tissue sources; in addition, this protocol cannot induce the differentiation of human skin fibroblasts into adipocytes, so it cannot be applied clinically Skin wound repair and scar repair etc.

Method used

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  • Method for inducing human skin fibroblasts to differentiate into adipocytes in vitro
  • Method for inducing human skin fibroblasts to differentiate into adipocytes in vitro
  • Method for inducing human skin fibroblasts to differentiate into adipocytes in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Method for inducing human skin fibroblasts to differentiate into fat cells in vitro

[0045] In this example, the isolated primary human skin fibroblasts (passage 6) were taken as an example, and the differentiation mode of "6+2+8=16 days" was adopted to induce the differentiation of primary human skin fibroblasts into adipocytes in vitro . The specific method is as follows:

[0046] 1. Make about 10 5 HDF cells were subcultured into a 35mm culture dish, added with 4ml of basal medium, and cultured in an incubator for 2 days. Cell culture conditions are 37 °C, 5% CO 2 , the same below.

[0047] 2. When the confluence of the cells reaches 95%, replace the medium with 3ml induction differentiation medium ①, replace it every two days, and induce culture for 6 days.

[0048] 3. Replace the induction differentiation medium ① with 3ml induction differentiation medium ②, and culture for 2 days.

[0049] 4. Replace the differentiation-inducing medium ② with 4ml ba...

Embodiment 2

[0086] Example 2 Induced Differentiation Medium ① Composition Optimization Experiment

[0087] The specific experimental steps of this example are the same as in Example 1, and the differentiation mode of "6+2+8=16 days" is also adopted to induce differentiation of primary human skin fibroblasts (6th passage). The difference is that the following adjustments were made to the composition of the induction differentiation medium ①:

[0088] Control group: differentiation induction medium ① containing 1 μM dexamethasone, 20 μg / mL insulin, 2 μM rosiglitazone, 0.5 mM 1-methyl-3-isobutylxanthine;

[0089] Dexamethasone-free group: differentiation induction medium ① containing 20 μg / mL insulin, 2 μM rosiglitazone, 0.5 mM 1-methyl-3-isobutylxanthine;

[0090] Insulin-free group: induction differentiation medium ① containing 1 μM dexamethasone, 2 μM rosiglitazone, 0.5 mM 1-methyl-3-isobutylxanthine;

[0091] No rosiglitazone group: differentiation induction medium ① containing 1 μM de...

Embodiment 3

[0097] Embodiment 3 The generation number of human skin fibroblasts is on the effect of inducing differentiation

[0098] The specific experimental steps of this example are the same as in Example 1, and the differentiation mode of "6+2+8=16 days" is also adopted to induce differentiation of primary human skin fibroblasts of different passages. The 3rd, 7th, 10th and 12th passage cells were selected for experiments.

[0099] The results of oil red staining are shown in Figure 7 , as shown in the figure, with the increase of the number of cell generations, the oil red staining gradually decreased, and almost no oil red staining was detected in the 12th passage cells.

[0100] For statistical results of oil red staining, see Figure 8 , as shown in the figure, with the increase of cell generations, the number of oil red stained cells gradually decreased, and the number of oil red stained cells in the 12th generation cells was almost zero.

[0101] For the test results of mat...

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Abstract

The present invention provides a method for inducing human skin fibroblasts to differentiate into adipocytes in vitro, the method comprising: A. In vitro culture and passage of human skin fibroblasts in a basal medium, and when the cell confluence reaches 80- 100%, carry out induced differentiation; B. Replace the basal medium of step A with induced differentiation medium ①, cultivate for 6-10 days, and change the medium every two days; then, replace induced differentiation medium ① with induced differentiation Medium ②, cultured for 2-4 days; finally changed to basal medium, cultured for 8-14 days, and changed the medium every two days. The invention successfully solves the problem of inducing and differentiating human skin fibroblasts into adipocytes; by inducing the skin fibroblasts to differentiate into adipocytes, the scars left by the skin during wound healing are reduced, and the scars left by the skin during wound healing are reduced, which is a good solution for clinical wound repair and treatment. Scar repair, etc. provide new ideas and new treatments.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for inducing human skin fibroblasts to differentiate into fat cells in vitro. Background technique [0002] At present, the in vitro induction and differentiation methods of adipocytes are basically the same, and they are all schemes to induce the differentiation of primary mouse embryonic fibroblasts into adipocytes. This protocol is currently a relatively mature adipocyte induction and differentiation protocol, which is widely used in the research of fat metabolism and adipocyte induction and differentiation. The source of cells in this protocol is primary mouse embryonic fibroblasts (MEF), which is relatively cumbersome and limited in tissue sources; in addition, this protocol cannot induce the differentiation of human skin fibroblasts into adipocytes, so it cannot be applied clinically Skin wound repair and scar repair etc. Contents of the invention [0003] The purp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077A61L27/38
CPCA61L27/3804A61L27/3895C12N5/0653C12N2500/30C12N2500/40C12N2500/84C12N2501/33C12N2506/1307
Inventor 张传茂王向阳
Owner PEKING UNIV
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