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Method for inducing transdifferentiation of fibroblasts to nerve cells

A technology of fibroblasts and nerve cells, applied in the biological field, can solve the problems of efficiency to be improved, hidden dangers of system security, and high technical barriers.

Active Publication Date: 2017-02-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the method of overexpression of exogenous genes has high technical barriers, the expression vector may be inserted into the genome, there are hidden dangers in the safety of the introduction system, and the efficiency needs to be improved

Method used

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  • Method for inducing transdifferentiation of fibroblasts to nerve cells
  • Method for inducing transdifferentiation of fibroblasts to nerve cells
  • Method for inducing transdifferentiation of fibroblasts to nerve cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] (1) The preparation steps of mouse embryonic fibroblasts (MEFs) are as follows:

[0029] 1) Pretreatment of the culture vessel: Cover the bottom wall of the culture vessel with 0.2% gelatin, leave it at room temperature for 30 minutes, suck out the 0.2% gelatin, and set it aside at room temperature.

[0030] 2) Mice (wild-type mice or transgenic mice carrying GFAP-GFP reporter system) were injected with about 0.5 mL of avertin for anesthesia at 13.5 days of pregnancy, and the mice were killed by neck breaking, and immersed in 75% ethanol Disinfect for 5 minutes.

[0031] 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.

[0032] 4) Cut open the embryo sac with scissors, and move the embryos to a petri dish.

[0033] 5) Carefully remove the head and viscera of the embryo, transfer the torso of the em...

Embodiment 2

[0042] 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

[0043] HDAC inhibitors: NaB(N), VPA(V), TSA(A);

[0044] DNMT inhibitors: RG108(R), 5-AZA(5);

[0045] G9a inhibitor: BIX-01294(B);

[0046] Ezh2 inhibitors: DZNep (D), GSK126 (G);

[0047] LSD1 inhibitor: phencypromine (T);

[0048] AC inhibitor: Forskolin (F);

[0049] GSK3 inhibitor: CHIR99021(C);

[0050] MEK inhibitor: PD032590(P);

[0051] ALK5 inhibitors: A-83-01(8), E616452(6), SB431542(S).

[0052] The concentration of each small molecule compound is shown in Table 1.

[0053] Table 1

[0054] small molecule compound Concentration (μM) VPA(V) 500 NaB(N) 20 RG108(R), Forskolin(F), CHIR99021(C) 10 Phenylcypromine(T), E616452(6), SB431542(S) 5 5-AZA(5) 4 PD032590...

Embodiment 3

[0060] The TTFs cells prepared in Example 1 were inoculated on a 6-well plate for cell culture (20,000 cells per well), and different small molecule combinations were added to the medium from the second day, and then the medium was changed every four days. After 16 days of detection, the screening effect of different small molecule combinations was determined by counting the number of clones formed and qPCR analysis. Such as Figure 5 As shown, small molecules combined with 6TCF to treat TTFs can also obtain a-Actinin-positive cardiomyocyte-like cells and Tuj1-positive neuron-like cells.

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Abstract

The invention discloses a method for inducing transdifferentiation of fibroblasts to nerve cells. The method comprises the following steps: (1) culturing the fibroblasts for 20 to 30 hours; (2) transferring the fibroblasts into a culture medium containing an inducing micromolecular combination 6TCF, continuing to carry out culturing for 6 to 8 days, and replacing the culture medium once every 2 to 4 days during the culturing; (3) then, transferring the fibroblasts into a culture medium containing inducing micromolecular combinations 6TCF and 8CFV, continuing to carry out culturing for 7 to 16 days, and replacing the culture medium once every 2 to 4 days during the culturing, thereby obtaining the nerve cells, wherein 6 means E61541, T means tranylcypromine, C means CHIR99021, F means forskolin, 8 means A-83-01 and V means valproic acid. According to the method, through adding the inducing micromolecular combinations into the culture media, the fibroblasts can be trans-differentiated into the nerve cells through inducing, and the obtained nerve cells have specific molecular tags of normal nerve cells and have functions of the normal nerve cells, so that a new way is provided for solving the problem in cell sources of regenerative medicine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for inducing the transdifferentiation of fibroblasts into nerve cells. 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 transdifferentiation between adult cells of...

Claims

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Application Information

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IPC IPC(8): C12N5/079C12N5/071C12Q1/02
CPCC12N5/0618C12N2501/70C12N2501/71C12N2501/727C12N2503/02C12N2506/1307G01N33/5058G01N2500/10
Inventor 郭国骥韩晓平李侠张芬岑燕红
Owner ZHEJIANG UNIV
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