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Induction medium for inducing transdifferentiation of fibroblast into adipocyte and application thereof

A technology for fibroblasts and induction medium, which can be used in cell culture active agents, embryonic cells, animal cells, etc., and can solve the problem of not seeing fat cells.

Active Publication Date: 2016-07-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there has been no report on the direct acquisition of adipocytes through transdifferentiation

Method used

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  • Induction medium for inducing transdifferentiation of fibroblast into adipocyte and application thereof
  • Induction medium for inducing transdifferentiation of fibroblast into adipocyte and application thereof
  • Induction medium for inducing transdifferentiation of fibroblast into adipocyte and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) The preparation steps of mouse embryonic fibroblasts (MEFs) are as follows:

[0034] 1) Pretreatment of the culture vessel: Cover the bottom wall of the culture vessel with 0.2% gelatin, place it at room temperature for 30 minutes, suck out the 0.2% gelatin, dry it at room temperature and set aside.

[0035] 2) After injecting about 0.5 mL of avertin to a mouse on the 13.5th day of pregnancy for anesthesia, execute the mouse by neck dislocation, and immerse it in 75% alcohol for disinfection for 5 minutes.

[0036] 3) Wipe the abdomen with 75% ethanol, cut the skin and pull the skin back to expose the abdominal wall. Cut the abdominal wall to expose the uterus. Move the uterus to a 100mm dish and wash it three times with 10mL PBS.

[0037] 4) Cut open the embryo sac with scissors, and move the embryos to a petri dish.

[0038] 5) Carefully remove the head and viscera of the embryo, transfer the torso of the embryo to a penicillin vial, and wash three times with 2...

Embodiment 2

[0047] In order to explore the potential role of small molecules in changing cell fate, more than 10 small molecule compounds and their combinations that have been proven to have an impact on reprogramming were systematically analyzed and screened, including

[0048] HDAC inhibitors: NaB(N), VPA(V), TSA(A);

[0049] DNMT inhibitors: RG108(R), 5-AZA(5);

[0050] G9a inhibitor: BIX-01294(B);

[0051] Ezh2 inhibitors: DZNep (D), GSK126 (G);

[0052] LSD1 inhibitor: phencypromine (T);

[0053] AC inhibitor: Forskolin (F);

[0054] GSK3 inhibitor: CHIR99021(C);

[0055] MEK inhibitor: PD032590(P);

[0056] ALK5 inhibitors: A-83-01(8), E616452(6), SB431542(S).

[0057] The experimental process is as follows figure 1 As shown, 1000 MEFs cells prepared in Example 1 were inoculated on a 96-well plate, and different small molecule combinations were added to the medium from the second day, and then the medium was changed every four days, and detected on the 16th day, by statistica...

Embodiment 3

[0063] The TTFs cells prepared in Example 1 were inoculated on a cell culture dish, and different small molecule combinations were added to the medium from the next day, and then the medium was changed every four days, and detected on the 16th day, by counting the number of clones formed and qPCR analysis to determine the screening effect of different small molecule combinations. like Figure 5 As shown, small molecules combined with 6TCF to treat TTFs can also obtain a-Actinin-positive adipocytes and Tuj1-positive neurons.

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Abstract

The invention discloses an induction medium inducing transdifferentiation of fibroblast into adipocyte, an induction method and application thereof. The induction medium contains a basal culture medium and an induction micromolecule combination, wherein the induction micromolecule combination is SG or 6TF, S is SB431542, G is GSK126, 6 is E61541, T is tranylcypromine, and F is forskolin. The induction medium can induce and trans-differentiate the fibrolast into the adipocyte, the obtained adipocyte contains a normal adipocyte specificity molecular tag and has a normal fat adipogenesis function, and a new approach is provided for solving the cellular source problem of regenerative medicine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an induction medium for inducing the transdifferentiation of fibroblasts into adipocytes and an application thereof. Background technique [0002] Most multicellular organisms develop from totipotent fertilized eggs. The fertilized egg formed by the combination of sperm and egg undergoes step-by-step lineage differentiation and finally develops into a mature individual. Among them, the determination of cell fate is the result of the synergy between thousands of exogenous signals and endogenous factors. This process is complex and difficult to control. In the rapid development of life science in recent years, one of the most striking achievements is that people can change the fate of cells through exogenous pathways. By overexpressing lineage-specific regulatory factors, people can not only dedifferentiate adult cells into embryonic stem cells, but also realize the process of transd...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/073C12Q1/02
CPCC12N5/0653C12N2501/70C12N2501/71C12N2501/72C12N2501/727C12N2503/02C12N2506/02G01N33/5008
Inventor 郭国骥李侠韩晓平张芬岑燕红
Owner ZHEJIANG UNIV
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