CN106635991A - Method for inducing human skin fibroblast into nerve cells and application - Google Patents
Method for inducing human skin fibroblast into nerve cells and application Download PDFInfo
- Publication number
- CN106635991A CN106635991A CN201611258215.9A CN201611258215A CN106635991A CN 106635991 A CN106635991 A CN 106635991A CN 201611258215 A CN201611258215 A CN 201611258215A CN 106635991 A CN106635991 A CN 106635991A
- Authority
- CN
- China
- Prior art keywords
- nerve cell
- concentration
- nerve
- cell
- hsf
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/71—Oxidoreductases (EC 1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
Abstract
The invention discloses a method for inducing human skin fibroblast into nerve cells. The method comprises steps as follows: a 24-pore plate containing a nerve cell culture solution is inoculated with human skin fibroblast with 5,000 cells per pore, and the nerve cell culture solution is changed once every 3 days. The nerve cell culture solution contains a DMEM culture medium, E61542, tranylcypromine, forskolin, brain-derived neurotrophic factors and a B-27 additive. By means of the method, the human skin fibroblast can be induced and trans-differentiated into cardiomyocytes, and the obtained nerve cells have specific molecule labels of normal nerve cells, have the normal nerve function and provide a new way for cell sources of regenerative medicine. Besides, the human skin fibroblast is widely sourced and low in cost.
Description
Technical field
The present invention relates to biomedicine technical field, more particularly to HSF's induction is the side of nerve cell
Method and application.
Background technology
Regenerative medicine based on stem cell transplantation technology defeats intractable DD to bring huge wishing for the mankind
Hope.Induced multi-potent stem cell, due to breaching the ethical hindrances of embryonic stem cell, was once considered as the new hope of regenerative medicine.So
And, its induced efficiency it is low, the unstability and high oncogenicity between batch makes it difficult to be directly used in disease treatment.
Since two thousand and ten, both at home and abroad some scientists by the specific transcription factor of expression realize by it is a type of into
Somatic conversion is another type of adult cell or adult stem cell.This method is referred to as " transdifferentiation ".By excess
The expression pedigree specific regulatory control factor, people can not only be such that adult cell dedifferentes as embryonic stem cell, moreover it is possible to realize not contemporaneity
The transdifferentiation process being tied to form between body cell.At present, by the specific reprogramming factor of forced expression in body cell, mediation body is thin
The direct transdifferentiation of born of the same parents is some preciousnesses or non-renewable cell, to solve the problems, such as that above-mentioned cell derived deficiency is provided
New approach.
However, the method and technology barrier of foreign gene overexpression is higher, expression vector may insert genome, introgressive line
There is hidden danger in system security, efficiency also has much room for improvement.Except the induction means of specific gene, some micromolecular compounds also by
Confirmation can promote cell to reprogram process.Several nearest researchs find that all transcription factors during reprogramming all may be used
To be substituted with small molecule.Micromolecular compound not only has cell membrane, easy manipulation and the low advantage of cost of infiltrating through, and little point
Son can realize the reprogramming of somatic cells process without the intervention of any foreign gene, and this undoubtedly obtains multipotent stem cells and carries for people
New method and thinking is supplied.
The content of the invention
Based on the technical problem that background technology is present, the purpose of the present invention is using wide material sources, fell with low cost
Skin fibroblast induces the method for nerve cell, and the application of gained nerve cell.
A kind of HSF's induction proposed by the present invention is the method for nerve cell, is comprised the steps：Will
HSF was inoculated in 24 orifice plates containing neuronal cell cultures liquid with 5000 cells/wells and is cultivated 28 days, per 3 days
Change liquid.
Preferably, neuronal cell cultures liquid includes：DMEM culture mediums, E61542, tranylcypromine, forskolin, Fu Sikelin,
BDNF, B-27 additives.
E61541 is the inhibitor in ALK5 sites in TGF-β signal path.
Tranylcypromine is the inhibitor of monoamine oxidase.
Forskolin is the activator of adenyl cyclase.
Fu Sikelin (Forskolin) is the natural diterpene that one kind is isolated from Indian plant (Coleus forskohlii)
Product, Forskolin is commonly used to improve cyclic adenosine monophosphate (cAMP) level in stechiology experimental study.
BDNF (BDNF) is a kind of protein with neurotrophic effect.Brain-derived neurotrophy
The factor and its acceptor are in nervous system wide expression.A kind of small molecule protein dimer BDNF structures, distribution and signal transduction
The secreting type mature polypeptide that BDNF molecule monomers are made up of 119 amino acid residues, albumen isoelectric point is 9.99, average molecular
Quality is 3.5 × 103, is mainly made up of β-pleated sheet and random coil secondary structure, is a kind of alkaline egg containing 3 disulfide bond
White matter.
B-27 additives are used for the growth of hippocampal neuron and other central nervous system (CNS) neurons and keep its short
Phase or long period of activity.
Preferably, in neuronal cell cultures liquid, the concentration of E61542 is 4～6 μm of ol/L, and the concentration of tranylcypromine is 4～6
μm ol/L, the concentration of forskolin is 8～12 μm of ol/L, and the concentration of Fu Sikelin is 4～6 μm of ol/L, brain-derived neurotrophy because
The concentration of son is 0.1～1 μ g/L, and the concentration of B-27 additives is 20～40mg/L.
Preferably, condition of culture is 37 DEG C, 5%CO2。
A kind of nerve cell that the present invention is also proposed, adopts above-mentioned HSF to induce the side for nerve cell
Method is obtained.
The above-mentioned nerve cell that the present invention is also proposed is used for the medicine for preparing prevention or treatment nerve cell dysfunction disease
Compositions.
Adult skin fibroblast induction transdifferentiation can be cardiac muscle cell by the present invention, and gained nerve cell has just
Normal Neuron-specific molecular label, and with normal nervous function, the cell derived problem for regenerative medicine is provided
A kind of new approach, while HSF's wide material sources, cheap.
Specific embodiment
Below, technical scheme is described in detail by specific embodiment.
Embodiment 1
A kind of HSF's induction is the method for nerve cell, is comprised the steps：By application on human skin into fiber
Cell is inoculated in 24 orifice plates containing neuronal cell cultures liquid with 5000 cells/wells and is cultivated 28 days, and liquid was changed per 3 days, cultivates bar
Part is 37 DEG C, 5%CO2。
Neuronal cell cultures liquid includes：DMEM culture mediums, E61542, tranylcypromine, forskolin, Fu Sikelin, brain source property
Neurotrophic factor, B-27 additives；The wherein concentration of E61542 is 4 μm of ol/L, and the concentration of tranylcypromine is 6 μm of ol/L, hair
The concentration of larynx element is 8 μm of ol/L, and the concentration of Fu Sikelin is 6 μm of ol/L, and the concentration of BDNF is 0.1 μ g/
The concentration of L, B-27 additive is 40mg/L.
Embodiment 2
A kind of HSF's induction is the method for nerve cell, is comprised the steps：By application on human skin into fiber
Cell is inoculated in 24 orifice plates containing neuronal cell cultures liquid with 5000 cells/wells and is cultivated 28 days, and liquid was changed per 3 days, cultivates bar
Part is 37 DEG C, 5%CO2。
Neuronal cell cultures liquid includes：DMEM culture mediums, E61542, tranylcypromine, forskolin, Fu Sikelin, brain source property
Neurotrophic factor, B-27 additives；The wherein concentration of E61542 is 6 μm of ol/L, and the concentration of tranylcypromine is 4 μm of ol/L, hair
The concentration of larynx element is 12 μm of ol/L, and the concentration of Fu Sikelin is 4 μm of ol/L, and the concentration of BDNF is 1 μ g/L,
The concentration of B-27 additives is 20mg/L.
Embodiment 3
A kind of HSF's induction is the method for nerve cell, is comprised the steps：By application on human skin into fiber
Cell is inoculated in 24 orifice plates containing neuronal cell cultures liquid with 5000 cells/wells and is cultivated 28 days, and liquid was changed per 3 days, cultivates bar
Part is 37 DEG C, 5%CO2。
Neuronal cell cultures liquid includes：DMEM culture mediums, E61542, tranylcypromine, forskolin, Fu Sikelin, brain source property
Neurotrophic factor, B-27 additives；The wherein concentration of E61542 is 5 μm of ol/L, and the concentration of tranylcypromine is 5 μm of ol/L, hair
The concentration of larynx element is 10 μm of ol/L, and the concentration of Fu Sikelin is 5 μm of ol/L, and the concentration of BDNF is 5 μ g/L,
The concentration of B-27 additives is 30mg/L.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, all should be included within the scope of the present invention.
Claims (6)
1. a kind of HSF's induction is the method for nerve cell, it is characterised in that comprised the steps：By fell
Skin fibroblast is inoculated in 24 orifice plates containing neuronal cell cultures liquid with 5000 cells/wells and is cultivated 28 days, is changed per 3 days
Liquid.
2. according to claim 1 HSF's induction is the method for nerve cell, it is characterised in that nerve is thin
Born of the same parents' nutrient solution includes：DMEM culture mediums, E61542, tranylcypromine, forskolin, Fu Sikelin, BDNF, B-
27 additives.
3. HSF's induction according to claim 1 or claim 2 is the method for nerve cell, it is characterised in that god
In Jing cell culture fluids, the concentration of E61542 is 4～6 μm of ol/L, and the concentration of tranylcypromine is 4～6 μm of ol/L, forskolin it is dense
Spend for 8～12 μm of ol/L, the concentration of Fu Sikelin is 4～6 μm of ol/L, and the concentration of BDNF is 0.1～1 μ
The concentration of g/L, B-27 additive is 20～40mg/L.
4. HSF's induction is the method for nerve cell according to any one of claim 1-3, and its feature exists
In condition of culture is 37 DEG C, 5%CO2。
5. a kind of nerve cell, it is characterised in that induced using the HSF as described in any one of claim 1-4
Method for nerve cell is obtained.
6. a kind of nerve cell as claimed in claim 5 is used for the medicine for preparing prevention or treatment nerve cell dysfunction disease
Composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611258215.9A CN106635991A (en) | 2016-12-30 | 2016-12-30 | Method for inducing human skin fibroblast into nerve cells and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611258215.9A CN106635991A (en) | 2016-12-30 | 2016-12-30 | Method for inducing human skin fibroblast into nerve cells and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106635991A true CN106635991A (en) | 2017-05-10 |
Family
ID=58837478
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611258215.9A Pending CN106635991A (en) | 2016-12-30 | 2016-12-30 | Method for inducing human skin fibroblast into nerve cells and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106635991A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804593A (en) * | 2019-10-17 | 2020-02-18 | 苏州大学 | Small molecular compound combination for inducing skin fibroblast to directly transdifferentiate towards neuron and application |
| CN112513257A (en) * | 2018-06-14 | 2021-03-16 | 中央研究院 | Methods of generating induced oligodendrocyte lineage cells and treatments using same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894060A (en) * | 2014-03-03 | 2015-09-09 | 中国科学院上海生命科学研究院 | Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof |
| CN105861428A (en) * | 2016-04-07 | 2016-08-17 | 浙江大学 | Inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells and application of inducing culture medium |
-
2016
- 2016-12-30 CN CN201611258215.9A patent/CN106635991A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894060A (en) * | 2014-03-03 | 2015-09-09 | 中国科学院上海生命科学研究院 | Method for inducing transdifferentiation of somatic cells into neural stem cells and application thereof |
| CN105861428A (en) * | 2016-04-07 | 2016-08-17 | 浙江大学 | Inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells and application of inducing culture medium |
Non-Patent Citations (2)
| Title |
|---|
| E TIAN,ET AL.: "Small-Molecule-Based Lineage Reprogramming Creates Functional Astrocytes", 《CELL REPORTS 》 * |
| WENXIANG HU,ET AL.: "Direct Conversion of Normal and Alzheimer’s Disease Human Fibroblasts into Neuronal Cells by Small Molecules", 《CELL STEM CELL》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112513257A (en) * | 2018-06-14 | 2021-03-16 | 中央研究院 | Methods of generating induced oligodendrocyte lineage cells and treatments using same |
| CN110804593A (en) * | 2019-10-17 | 2020-02-18 | 苏州大学 | Small molecular compound combination for inducing skin fibroblast to directly transdifferentiate towards neuron and application |
| CN110804593B (en) * | 2019-10-17 | 2021-09-03 | 苏州大学 | Small molecular compound combination for inducing skin fibroblast to directly transdifferentiate towards neuron and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9447383B2 (en) | MDCK-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells | |
| Ge et al. | Local production of astrocytes in the cerebral cortex | |
| CN108384755A (en) | A method of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating | |
| Kikuchi et al. | Midkine, a novel neurotrophic factor, promotes survival of mesencephalic neurons in culture | |
| CN105331634B (en) | A kind of induced fibroblast transdifferentiation is the method and its application of neuronal cell | |
| CN107418928A (en) | A kind of human mesenchymal stem cell Osteoinductive differentiation culture medium and preparation method | |
| CN106635991A (en) | Method for inducing human skin fibroblast into nerve cells and application | |
| CN109234223A (en) | Low albumen serum-free cell culture medium | |
| CN106754718A (en) | A kind of human embryo stem cell slewing is broken up to the method for retinal pigment epithelium | |
| CN106479977B (en) | People source iPS stem cell in vitro directed differentiation is the kit and method of nerve cell | |
| CN103215225A (en) | Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell | |
| CN104789531B (en) | A kind of method that umbilical cord mesenchymal stem cells are induced differentiation into dopaminergic neuron | |
| CN108220369A (en) | A kind of method for producing lepirudin 023 ludon | |
| CN102747029B (en) | Culture method for retina progenitor cells and culture medium thereof | |
| CN104974982B (en) | A kind of method of in vitro culture testis tissue inductive formation spermatoblast | |
| CN104726400B (en) | Non-animal derived component cultural method of the human pluripotent stem cells to germline | |
| CN105087475A (en) | Cell culture fluid, application of cell culture fluid and method of inducting DPSCs to differentiate into neuron-like cells | |
| CN109294920A (en) | A method of addition nano material induces haematococcus pluvialis efficient accumulation astaxanthin | |
| CN107574147A (en) | A kind of mescenchymal stem cell Proliferation, Differentiation nutrient solution using atractylenolide as trophic factors | |
| CN104830686B (en) | A kind of method preparing cell climbing sheet by electrostatic spinning | |
| CN102406933A (en) | Preparation method for measles attenuated live vaccine | |
| CN107287150A (en) | A kind of construction method of 3D epidermises model | |
| Ziegler et al. | Rat adrenal non-chromaffin cells contain a neurite outgrowth-promoting factor immunologically different from nerve growth factor | |
| CN103536913A (en) | Method for producing avian influenza virus bivalent inactivated vaccine by use of MDCK cell line | |
| CN101705205A (en) | Method for inducing differentiation of neural stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170510 |
|
| RJ01 | Rejection of invention patent application after publication |