CN110396500A - Composition and its application of the induced fibroblast directly to neuron transdifferentiation - Google Patents
Composition and its application of the induced fibroblast directly to neuron transdifferentiation Download PDFInfo
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Abstract
The present invention relates to neurodevelopment technical fields, and in particular to composition and its application of the induced fibroblast directly to neuron transdifferentiation.The present invention is provided to induced fibroblasts directly to the composition of neuron transdifferentiation, it includes composition A and/or composition B, wherein, composition A includes CHIR99021, LDN193189, RG108, Dorsomorphin, P7C3-A20, A83-01 and ISX9；Composition B includes Forskolin, Y27632, DAPT, PD0325901, Purmorphamine and P7C3-A20.Composition provided by the invention can significantly improve the efficiency of the neuronotropic direct transdifferentiation of fibroblast, to solve the problems, such as that the cell origin of neurodegenerative disease transplantation treatment provides effective way.
Description
Technical field
The present invention relates to neurodevelopment technical fields, and in particular to directly turns to divide to neuron for induced fibroblast
The composition of change and its application and induced fibroblast are directly to the method for neuron transdifferentiation.
Background technique
Neurodegenerative disease (Neurodegenerative disease) is that one kind is damaged by brain or spinal neuron
Mental disease caused by losing or lacking.Degenerative disease can deteriorate over time, will eventually lead to nerve
Sex dysfunction.Neurodegenerative disease mainly includes amyotrophic lateral sclerosis, Huntington disease, Parkinson, Alzheimer
Disease etc..Although the diseased region and the cause of disease of this kind of disease are different, nerve cell retrogression pathological changes are their common ground.
For many years, due to the complexity of brain function, the treatment of this kind of disease is always a problem.
The major way for slowing down neurodegenerative disease symptom is drug therapy, but is directed to the drug therapy of different syndromes
Targeting is poor.It and is current treatment mind by the cell therapy of grafted dopamine neurons or neural stem cell to lesion deleted areas
Through one of degenerative disease important method.Started from using the imagination that nerve cell carries out replacement therapy to neurodegenerative disease
Focal lesion such as Parkinson's disease, prosperous front yard disease etc., and ideal effect is achieved in an experiment.A large amount of zooperies are aobvious
Show, partially can repair or substitute the neuron of defect by nerve cells transplantation, partially restore its function and promote brain from
I repairs.It can survive after nerve cell implantation brain area, different parts can be moved to, it can partial reconstitution neural circuitry and function.
But nerve cells transplantation is there is also many problems, such as cell largely comes source problem, rejection and ethics etc.
Problem, and the appearance of cell reprogramming technology is to solve these problems to bring unlimited hope.
2006, Japanese Yamanaka study group first passage be overexpressed 4 transcription factors Oct3/4, Sox2, Klf4 and
C-Myc simultaneously uses retroviral infection technology, successfully reprograms l cell for embryonic stem cell-like cell,
And it is named as induced pluripotent stem cells (induced pluripotent stem cells, iPSCs).But multipotential stem cell
Complicated control measures are needed to the atomization of neuronal cell or other development cells of termination and the time is relatively very long.
With the appearance of cell reprogramming technology, more results of study show by the tissue-specific transcriptions of terminally differentiated cells because
The overexpression of son, initiator cell can be directly translated into mature neuronal cell directly across the state of pluripotency.
Direct transdifferentiation reduces the complexity of in vitro operation, has substantially evaded noble cells and has returned back to pluripotency
State bring risk, such as Tumor formation.However mature cell amplification ability is limited, it is difficult to obtain clinical required enough cells
Quantity affects the clinical value of this technology.Much studies have shown that small molecule compound can increase the effect of transdifferentiation
Rate and the process for promoting development, and small molecule compound is many kinds of, can replace the relevant regulatory factor of a variety of developments, and
And it is cheap, it can be the ability to easily control and operate on dosage and administration time, turn so small molecule compound becomes to improve
The primary candidate of differentiation efficiency.
2015, there are three research groups to find that the inducible mouse embryo fibroblast of small molecule compound is thin respectively both at home and abroad
Born of the same parents, human skin fibroblasts and National People's Congress's cerebro-cardiac apoplexy transdifferentiation are at neuron.Although source of people astroglia
Reach 80% or more to neuron transdifferentiation efficiency, but it is primarily present in brain and spinal cord, has on source obtains
Certain difficulty；And the relatively easily obtained human skin fibroblasts in source neuron under the induction of small molecule compound turns
Differentiation efficiency significantly limits its treatment potential in clinical application between 4%~10%.So how to pass through source
Relatively easy human skin fibroblasts, and by a kind of integration of non-genomic, rapidly and efficiently in a manner of by its transdifferentiation be
Neuron becomes problem in science urgently to be solved.The present invention is sent out by screening to a variety of small molecule compounds and combinations thereof
Having showed can be combined with inducing embryo fibroblast to the small molecule compound of neuron efficient transition, to pass through grafted dopamine neurons
The solution for treating the cell origin problem of neurodegenerative disease provides effective ways.
Summary of the invention
The technical issues of to solve in the prior art, the purpose of the present invention is to provide being capable of efficient induced fibroblast
Directly to the composition and induced fibroblast of neuron transdifferentiation directly to the method for neuron transdifferentiation.
To achieve the above object, technical scheme is as follows: by the neuronotropic direct transdifferentiation of fibroblast
It is related to the extremely complex regulatory mechanism of a variety of regulatory factors, the enzyme inhibitor of neuron differentiation associated signal paths, lures at activator
Agent is led to interact in neuron differentiation regulation process, it is common to participate in complicated differentiation regulation, and these small molecule chemical combination
Object also will affect the cell survival rate of neuron.The present invention is had found by the in-vitro screening combined to a variety of small molecule compounds
Can combination of the induced fibroblast directly to the small molecule compound of the efficient transdifferentiation of neuron, utilize these small molecules
The combined treatment fibroblast of compound can significantly improve fibroblast directly to the efficiency of neuron transdifferentiation, simultaneously
Guarantee higher cell survival rate.
Specifically, technical scheme is as follows:
In a first aspect, the present invention provide it is a kind of for induced fibroblast directly to the composition of neuron transdifferentiation,
It includes composition A and/or composition B；The composition A includes GSK-3 α/β inhibitor, BMP inhibitor, the transfer of DNA methyl
Enzyme inhibitor, AMPK inhibitor, ALK inhibitor, neuron differentiation inducer, neuroprotective activity agent；The composition B includes
Neuroprotective activity agent, adenyl cyclase activator, ROCK1 inhibitor, GSI-IX gamma-secretase inhibitors, mek inhibitor
With smooth receptor stimulating agent.
Preferably, the GSK-3 α/β inhibitor be selected from one of CHIR99021, TWS119, SB415286 or
It is a variety of；The BMP inhibitor is selected from one of LDN193189, LY364947, SB431542 or a variety of；The DNA methyl
Inhibitors are selected from one of RG108, Decitabine, SGI-1027 or a variety of；The AMPK inhibitor is choosing
From one of Dorsomorphin, HTH-01-015, WZ4003 or a variety of；The neuroprotective activity agent be selected from P7C3 or
One of its derivative is a variety of；The ALK inhibitor is selected from one of A83-01, SB525334, DMH1 or a variety of；
The neuron differentiation inducer is selected from the one or more of ISX9 or derivatives thereof；The adenyl cyclase activator is
Selected from one of Forskolin, SQ22536, ESI-09 or a variety of；The ROCK1 inhibitor be selected from Y27632,
Thiazovivin, Fasudil's is one or more；The GSI-IX gamma-secretase inhibitors be selected from DAPT, RO4929097,
One of LY411575 or a variety of；The mek inhibitor be selected from one of PD0325901, PD98059, BIX0218 or
It is a variety of；The smoothened receptors agonist is selected from one of Purmorphamine, Cyclopamine, GANT61 or a variety of.
It is further preferred that the GSK-3 α/β inhibitor is CHIR99021, the BMP inhibitor is LDN193189,
The dnmt rna inhibitor is RG108, and the AMPK inhibitor is Dorsomorphin, the neuroprotective activity
Agent is P7C3-A20, and the ALK inhibitor is A83-01, and the neuron differentiation inducer is ISX9, the adenylate cyclisation
Zymoexciter is Forskolin, and the ROCK1 inhibitor is Y27632, and the GSI-IX gamma-secretase inhibitors are DAPT, institute
Stating mek inhibitor is PD0325901, and the smoothened receptors agonist is Purmorphamine.Present invention discover that selecting above-mentioned spy
Fixed small molecule compound, a combination thereof can advantageously promote the neuronotropic direct transdifferentiation of fibroblast, more effectively
Improve transdifferentiation efficiency.
As the preferred embodiment of the present invention, the composition is made of composition A and composition B, the composition A
It is made of CHIR99021, LDN193189, RG108, Dorsomorphin, P7C3-A20, A83-01 and ISX9；The composition
B is made of Forskolin, Y27632, DAPT, PD0325901, Purmorphamine and P7C3-A20.
To better ensure that neuron while more effectively promotion fibroblast transdifferentiation direct to neuron
Screening is optimized to the consumption proportion of each small-molecule chemical object in cell survival rate, the present invention.
Preferably, the composition A includes following component:
The composition B includes following component:
Present invention discover that each small molecule compound can be effectively facilitated fibroblast with the combination of above-mentioned consumption proportion
While transdifferentiation direct to neuron, the cell survival rate of neuron is better ensured that.
It is further preferred that the composition A includes following component:
The composition B includes following component:
The present invention has found that the composition A and the composition B can be effective when being used alone by experimental verification
Improve the neuronotropic direct transdifferentiation efficiency of fibroblast；And when composition A and composition B are used in combination, improve at
The effect of the neuronotropic direct transdifferentiation efficiency of fibrocyte is more excellent；It uses when by composition A and composition B in cell culture
Different phase when adding respectively, can be while efficiently improving fibroblast neuronotropic direct transdifferentiation efficiency more
Guarantee higher cell survival rate well.
Second aspect, the present invention provide the induced fibroblast that is used for directly to the composition of neuron transdifferentiation
Following any application:
(1) induced development cell of termination Differentiating Into Neurons；
(2) induced fibroblast is to the direct transdifferentiation of neuron；
(3) neuron is prepared by fibroblast in vitro；
(4) product for preventing or treating neure damage or missing is prepared；
(5) product for preventing or treating neurodegenerative disease is prepared.
The present invention by composition described in experimental verification can induced fibroblast directly to neuron transdifferentiation, especially
Directly there is preferably induction facilitation to the transdifferentiation of glutamatergic neurons for fibroblast.
In the present invention, the fibroblast is preferably the fibroblast with lower differentiation degree, lower differentiation journey
The fibroblast of degree can better ensure that the composition plays the function of induction transdifferentiation.
As the preferred embodiment of the present invention, the fibroblast is embryo fibroblast.
The third aspect, the present invention is provided to prepare the product of neuron by fibroblast in vitro, it includes the use
In induced fibroblast directly to the composition of neuron transdifferentiation.
The product can be reagent or kit.
The kit also may include for preparing other reagents needed for neuron as fibroblast in vitro, including but
It is not limited to Fibroblast culture solution, neuron screening reagent etc..
Fourth aspect is used for the present invention is provided to prevent or treat the drug of neurodegenerative disease it includes described
Induced fibroblast is directly to the composition of neuron transdifferentiation.
The drug also may include the carrier and/or auxiliary material that pharmaceutical field allows.
5th aspect, the present invention provide a kind of induced fibroblast directly to the method for neuron transdifferentiation, pass through
It is added during Fibroblast cell-culture described directly real to the composition of neuron transdifferentiation for induced fibroblast
It is existing.
The present invention be further discovered that when by composition A and composition B respectively cell culture different phase add when, energy
It is enough to better ensure that higher cell survival while efficiently raising fibroblast neuronotropic direct transdifferentiation efficiency
Rate.
Preferably, described method includes following steps:
(1) using cell culture medium culture fibroblast 3-5 days of the addition composition A；
(2) cell culture fluid is changed to fresh cell culture medium, continues culture 3-5 days after adding the composition B.
In above-mentioned steps (2), 50% initial cell culture medium is preferably changed to Fresh cell culture medium.
In above-mentioned steps (2), preferably adds the composition B and continue after cultivating 3-5 days, then cell culture medium is all changed
At the cell culture medium for adding the composition A, step (1) and (2) is repeated once, then cell culture medium is changed into without described
The neural maturation medium of composition A and composition B continues to cultivate.
Cell culture medium described in above-mentioned steps (1) and (2) is preferably Neuronal induction media, the nerve-inducing training
The formula for supporting base is as follows: improvement Eagle culture medium (DMEM/F12) and Neurobasal medium (neural basal medium)
1:1 is prepared, while supplementing 1%N-2 wherein, 2%B-27,40ng/ml brain derived neurotrophic factor (BDNF), 20ng/ml glue
Cell plastid derived neurotrophic factor (GDNF), 20ng/ml insulin-like growth factor (IGF-1) and 1mM glutamine.
It is further preferred that in above-mentioned steps (1), the composition A be added to CHIR99021, LDN193189,
The concentration of RG108, Dorsomorphin, P7C3-A20, A83-01 and ISX9 is respectively 3-10 μM, 0.25-3 μM, 10-30 μM,
1-3μM、1-3μM、0.25-1μM、10-30μM。
It is further preferred that in above-mentioned steps (2), the composition B be added to Forskolin, Y27632,
The concentration of DAPT, PD0325901, Purmorphamine and P7C3-A20 are respectively 10-30 μM, 2-6 μM, 2-6 μM, 0.25-1 μ
M、1-10μM、1-3μM。
As the preferred embodiments of the invention, in above-mentioned steps (1), the composition A be added to
The concentration of CHIR99021, LDN193189, RG108, Dorsomorphin, P7C3-A20, A83-01 and ISX9 is respectively 3 μM,
0.25μM、10μM、1μM、3μM、1μM、10μM。
As the preferred embodiments of the invention, in above-mentioned steps (2), the composition B be added to
The concentration of Forskolin, Y27632, DAPT, PD0325901, Purmorphamine and P7C3-A20 are respectively 10 μM, 5 μM, 2
μM、1μM、1μM、3μM。
As the preferred embodiments of the invention, the induced fibroblast is directly to the method packet of neuron transdifferentiation
Include following steps:
(1) after the fibroblast of culture being digested to individual cells, it is layered on the coated sheet glass of poly-ornithine
Upper culture 24 hours；
(2) fibroblast culture medium full dose is changed into liquid into the Neuronal induction media for adding the composition A；
(3) after cultivating 3 days, cell culture medium half is measured and changes liquid into the Neuronal induction media for adding the composition B；
(4) after cultivating 3 days, then cell culture medium full dose changed into liquid into the Neuronal induction media for adding the composition A,
After culture 3 days, half amount of cell culture medium is changed into liquid and (repeats step (2) at the Neuronal induction media for adding the composition B
(3) primary), after culture 3 days, then changes culture medium into neural maturation medium and continue to cultivate.
The formula of Neuronal induction media of the present invention is as follows: improvement Eagle culture medium (DMEM/F12) and nerve
Basal medium (neural basal medium) 1:1 is prepared, while supplementing 1%N-2 wherein, 2%B-27,40ng/ml brain
Derived neurotrophic factor (BDNF), 20ng/ml glial cell line-derived neurotrophic factor (GDNF), 20ng/ml Insulin-Like are raw
The long factor (IGF-1) and 1mM glutamine.
It is of the present invention nerve maturation medium formula it is as follows: in NIM culture medium supplement 20ng/ml neurotrophic because
Son -3 (NT-3) and 2 μM of Y27632
6th aspect, the present invention provides a kind of glutamatergic neurons, for from the induced fibroblast directly to
The method of neuron transdifferentiation is prepared.
7th aspect, the present invention is provided to treat the product of neurodegenerative disease, it includes induced by described into fibre
The glutamatergic neurons that dimension cell is directly prepared to the method for neuron transdifferentiation.
The beneficial effects of the present invention are: the combination of small molecule compound provided by the invention can efficiently induce into fiber
The neuronotropic direct transdifferentiation of cell, significantly improves the ratio and efficiency of the neuronotropic direct transdifferentiation of fibroblast
(transdifferentiation efficiency reaches as high as 80% or more), while guaranteeing higher cell survival rate, realize the side integrated with non-genomic
The direct transdifferentiation of the relatively easy fibroblast in source is neuron by formula, significantly shortens the week for preparing a large amount of neurons
Phase, to solve the problems, such as that the cell origin of neurodegenerative disease transplantation treatment provides effective way.
The present invention carries out bioelectrical activity monitoring, cellular genome by the neuron obtained to fibroblast transdifferentiation
The expression trend analysis of RNA and the verifying of internal cells viability, it was demonstrated that utilize small molecule compound provided by the invention
The neuron that combination induction obtains can be used as the cell origin of the transplantation treatment of neurodegenerative disease.
Detailed description of the invention
After compositions-treated fibroblast of the Fig. 1 to use small molecule compound in experimental group 1 in the embodiment of the present invention 5
The metamorphosis of different phase cell and the detection of expression result of neuron specific markers' object, wherein A is microscope light field view
Yezhong, fibroblast is when adding the 7th, 14,21 day of compositions-treated of small molecule compound, neuronotropic form
Variation, wherein column diagram is the neuronotropic transdifferentiation efficiency of fibroblast counted according to metamorphosis；B is immunofluorescence
The detection of expression to specific marker object DCX, MAP2, TAU of neuron is dyed, wherein column diagram is the various minds of expression of statistics
Induction of neuronal ratio through first specific marker object.
Fig. 2 is to the expression position of Induction of neuronal and to discharge the immune of mediator specific type in the embodiment of the present invention 5
Fluorescence detection statistical result.
Fig. 3 is the external bioelectrical activity detection of Induction of neuronal in experimental example 1 of the present invention, wherein A is prominent memebrane protein
The immunofluorescence dyeing testing result of synapsin I；B is the induction action potential in different time sections to Induction of neuronal
Testing result；C is the movable testing result of sodium and pot assium current；D is the testing result of spontaneous action potentials.
Fig. 4 is that the express spectra of high throughput RNA sequence verification fibroblast to neuron in experimental example 2 of the present invention changes；
Wherein, A is that fibroblast, Induction of neuronal and Human embryo neuron differential expression compose thermal map；B is that q-PCR detection nerve is special
The expression of different transcription factor changes；C is that q-PCR detection changes at the expression of fiber specific transcription factor.
Fig. 5 is the internal transplantation effect detection of Induction of neuronal in experimental example 3 of the present invention, wherein A is transplanting inducing neural
The flow diagram of member；B is the mature expression label analyte detection of nerves within the body member after transplanted cells.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The screening that embodiment 1 is directly combined to the small molecule compound of neuron transdifferentiation for induced fibroblast
The present invention is first in the base of CHIR99021, LDN193189, SB431542, RG108 and Dorsomorphin combination
It is screened on plinth and new small molecule is added one by one.By a series of experiment screening, tentatively obtain containing CHIR99021,
LDN193189,SB431542,RG108,Dorsomorphin,DMH1,Parnate,SU5402,Forskolin,Y27632,
The combination of 16 small molecule compounds of DAPT, Purmorphamine, ISX9, IBET151, SU16F, and P7C3-A20,
The neuronotropic transformation efficiency of inducing embryo fibroblast is 40% or so.In order to identify in the combination inducing embryo into fibre
Cell is tieed up directly to small molecule compound necessary to neuron transdifferentiation, using one small molecule of each removal, and uses remaining
The fibroblastic mode of small molecule processing carries out subsequent screening.By one wheel screening, Parnate, IBET151, SU5402,
SU16F and DMH1 is removed from the combination of above-mentioned 16 small molecule compounds, the mind of remaining 11 small molecule compounds combination
It is 61% through first induced efficiency.In order to further increase transformation efficiency, on the basis of 11 small molecules, further add other small
Molecular compound is screened, and the selection result shows to remove SB431542 while A83-01, PD0325901 is added, and can make to lure
It leads efficiency and reaches 75% or more, but be added at one time the death that more small molecule be easy to cause part cell.In order to guarantee
12 small molecule combinatorials are divided into two groups by the survival rate of inducing cell, the present invention, and first group includes small molecule compound
CHIR99021, LDN193189, RG108, Dorsomorphin, P7C3-A20, A83-01 and ISX9, second group includes small molecule
Compound Forskolin, Y27632, DAPT, PD0325901, Purmorphamine and P7C3-A20, using this two groups of chemical combination
Object successively handles fibroblast, as a result, it has been found that inducing cell can be with long-term surviving, and the induced efficiency of neuron is further
Increase, can reach 82%.
Embodiment 2 is for induced fibroblast directly to the composition (1) of neuron transdifferentiation
The present embodiment provides a kind of for induced fibroblast directly to the composition of neuron transdifferentiation, by combining
Object A and composition B composition；
Wherein, composition A is made of following small molecule compound:
Composition B is made of following small molecule compound:
Embodiment 3 is for induced fibroblast directly to the composition (2) of neuron transdifferentiation
The present embodiment provides a kind of for induced fibroblast directly to the composition of neuron transdifferentiation, by combining
Object A and composition B composition；
Wherein, composition A is made of following small molecule compound:
Composition B is made of following small molecule compound:
Embodiment 4 is for induced fibroblast directly to the composition (3) of neuron transdifferentiation
The present embodiment provides a kind of for induced fibroblast directly to the composition of neuron transdifferentiation, by combining
Object A and composition B composition；
Wherein, composition A is made of following small molecule compound:
Composition B is made of following small molecule compound:
The composition induced fibroblast of 5 small molecule compound of embodiment is directly to neuron transdifferentiation
The present embodiment is with the composition of the small molecule compound in the embryo fibroblast of people and above-described embodiment 2-4
Example carries out fibroblast and carries out neuronotropic direct transdifferentiation, and the specific method is as follows:
(1) after the fibroblast of culture being digested to individual cells first, it is layered on the coated glass of poly-ornithine
Glass on piece (10000-15000 cell/24 orifice plate single holes) is cultivated 24 hours；
(2) fibroblast culture medium full dose is changed into liquid into the Neuronal induction media (neural of addition composition A
Induction medium, NIM)；
(3) after cultivating 3 days, cell culture medium half is measured and changes liquid into the NIM culture medium containing composition B；
(4) after cultivating 3 days, then cell culture medium full dose changed into liquid into the Neuronal induction media for adding the composition A,
After culture 3 days, half amount of cell culture medium is changed into liquid and (repeats step (2) at the Neuronal induction media for adding the composition B
(3) once, the additive amount of composition A and B is identical as step (2) and (3)), culture 3 days after, by culture medium change into nerve at
Ripe culture medium (neural maturation medium, NMM) continues to cultivate.
The formula of above-mentioned culture medium is as follows:
NIM formula: improvement Eagle culture medium (DMEM/F12, Gibco:11320082) and Neurobasal medium
(neural basal medium, Gibco:21103049) 1:1 is prepared, while supplementing 1%N-2,2%B-27 wherein,
40ng/ml brain derived neurotrophic factor (BDNF), 20ng/ml glial cell line-derived neurotrophic factor (GDNF), 20ng/ml pancreas
Island element like growth factor (IGF-1) and 1mM glutamine.
NMM formula: 20ng/ml neurotrophic factor -3 (NT-3) and 2 μM of Y27632 are supplemented in NIM culture medium.
Experimental group 1, experimental group 2 and experimental group 3 is respectively set as experimental group in group to handle through the above method,
In, experimental group 1 is composition A to CHIR99021, LDN193189 in the step (2) in addition embodiment 2, RG108,
The concentration of Dorsomorphin, P7C3-A20, A83-01 and ISX9 are respectively 3 μM, 0.25 μM, 10 μM, 1 μM, 3 μM, 1 μM, 10 μ
M；Composition B to Forskolin, Y27632, DAPT, PD0325901 in the step (3) in addition embodiment 2,
The concentration of Purmorphamine and P7C3-A20 is respectively 10 μM, 5 μM, 2 μM, 1 μM, 1 μM, 3 μM；Experimental group 2 is in step
(2) in addition embodiment 3 in composition A to CHIR99021, LDN193189, RG108, Dorsomorphin, P7C3-A20,
The concentration of A83-01 and ISX9 is respectively 10 μM, 3 μM, 30 μM, 3 μM, 1 μM, 0.25 μM, 30 μM；Addition is implemented in step (3)
The concentration of composition B to Forskolin, Y27632, DAPT, PD0325901, Purmorphamine and P7C3-A20 in example 3
Respectively 30 μM, 2 μM, 6 μM, 0.3 μM, 3 μM, 1 μM；Experimental group 3 be composition A in the step (2) in addition embodiment 4 extremely
The concentration of CHIR99021, LDN193189, RG108, Dorsomorphin, P7C3-A20, A83-01 and ISX9 is respectively 30 μM,
0.25μM,10μM,0.25μM,0.25μM,1μM,3μM；Composition B in step (3) in addition embodiment 4 is extremely
The concentration of Forskolin, Y27632, DAPT, PD0325901, Purmorphamine and P7C3-A20 are respectively 30 μM, 0.6 μ
M,0.6μM,0.1μM,10μM,3μM.To be not added with the processing group of small molecule compound as a control group, control group is by experimental group
The composition of the small molecule compound of middle addition replaces with the 0.5%DMSO of equivalent.
Morphological observation is carried out to the above-mentioned Induction of neuronal being prepared.Morphological observations show embodiment 2-4
The composition of the small molecule compound of offer can efficiently neuronotropic Morphological Transitions of inducing embryo fibroblast, and
And with the increase of small molecule processing time, the efficiency of transdifferentiation is gradually increased, at the composition of addition small molecule compound
The 14th day value that peaks after reason, wherein the transdifferentiation efficiency of experimental group 1 reaches 82% (A of Fig. 1), and experimental group 2 turns to divide
Change efficiency and reach 79%, the transdifferentiation efficiency of experimental group 3 reaches 75%.And there is no obvious neuron cells for control group at this time
Transformation.
Neuron marker detection further is carried out to the Induction of neuronal of acquisition, is tested and is detected using immunofluorescence dyeing
Neuron marker the result shows that, form can express classical neuron marker DCX, MAP2 similar to the cell of neuron
With TAU (B of Fig. 1).According to neuron expression position and neural markers analyte detection result it is found that the composition of small molecule compound
Main induced fibroblast is changed to the glutamatergic neurons at akrencephalon position, and (its ratio for accounting for Induction of neuronal is up to
89%) ratio, and to GABAergic neuron changed is less than 10%, cholinergic neuron, dopaminergic neuron
Expression marker does not detect (Fig. 2) then.
The external electrophysiological function of 1 Induction of neuronal of experimental example detects
To promote the neuron of induction further mature, the Induction of neuronal that experimental group 1 is prepared in embodiment 5 is passed through
It is layered on spongiocyte and is cultivated again after digestion.On spongiocyte after 30 days, detected by immunofluorescence dyeing
Specifically expressed synaptic membrane protein synapsin I (A of Fig. 3) in mature neuron.
The electric current and action potential of detection Induction of neuronal are tested by whole-cell patch-clamp, the results showed that, inducing neural
Member can detecte sodium and pot assium current and induction and spontaneous action potentials (B, C and D of Fig. 3).
The express spectra of 2 high throughput RNA sequence verification fibroblast of experimental example to neuron changes
The Induction of neuronal and fibroblastic express spectra obtained for analysis experimental example 1 changes, and is surveyed by RNA chip
Sequence compares fibroblast, Induction of neuronal and Human embryo primary neuron full-length genome express spectra.
Sequencing analysis the result shows that, after the compositions-treated fibroblast with the small molecule compound of embodiment 1, initially
Initiator cell with the variation of time start increase expression neuronal specificity transcription factor, gradually show Human embryo mind
Expression trend (A of Fig. 4) through member.
By the method for quantitative fluorescent PCR, further demonstrate the high transcription factor Ascl of neuron expression content,
The expression of NeuroD1, Brn2, Myt1l, NGN2, NeuroD4 in Induction of neuronal with small molecule compound composition
The processing time extends and gradually increases (B of Fig. 4), while the high idiosyncratic transcription factor Thy1 of fibroblast expression contents,
Col1a1, ELN, DKK3 then gradually decrease (C of Fig. 4) as the processing time of the composition of small molecule compound extends.
The internal transplantation effect of 3 Induction of neuronal of experimental example detects
In order to verify whether the external evoked neuron of the preparation of embodiment 5 can survive in vivo, and it is integrated into mouse
Intracorporal neural circuitry is digested and is transplanted to small after 1 small molecular compound of experimental group is handled fibroblast 9 days
The cerebral cortical sites of mouse, and section perfusion, slice in different times, carry out neural meta-tag by histogenic immunity fluorescent staining
The detection of albumen.
The result shows that embodiment 5 prepare Induction of neuronal can survive in vivo and gradually express neuron at
Ripe labelled protein MAP2, NEUN (Fig. 5).
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. it is a kind of for induced fibroblast directly to the composition of neuron transdifferentiation, which is characterized in that including composition A
And/or composition B；The composition A includes GSK-3 α/β inhibitor, BMP inhibitor, dnmt rna inhibitor, AMPK
Inhibitor, ALK inhibitor, neuron differentiation inducer, neuroprotective activity agent；
The composition B includes neuroprotective activity agent, adenyl cyclase activator, ROCK1 inhibitor, GSI-IX γ secretion
Enzyme inhibitor, mek inhibitor and smooth receptor stimulating agent.
2. composition according to claim 1, which is characterized in that the GSK-3 α/β inhibitor be selected from CHIR99021,
One of TWS119, SB415286 or a variety of；The BMP inhibitor is selected from LDN193189, LY364947, SB431542
One of or it is a variety of；The dnmt rna inhibitor is selected from one of RG108, Decitabine, SGI-1027
Or it is a variety of；The AMPK inhibitor is selected from one of Dorsomorphin, HTH-01-015, WZ4003 or a variety of；It is described
Neuroprotective activity agent is selected from one of P7C3 or derivatives thereof or a variety of；The ALK inhibitor be selected from A83-01,
One of SB525334, DMH1 or a variety of；The neuron differentiation inducer be one kind selected from ISX9 or derivatives thereof or
It is a variety of；The adenyl cyclase activator is selected from one of Forskolin, SQ22536, ESI-09 or a variety of；It is described
ROCK1 inhibitor is selected from the one or more of Y27632, Thiazovivin, Fasudil；The GSI-IX gamma secretase suppression
Preparation is selected from one of DAPT, RO4929097, LY411575 or a variety of；The mek inhibitor be selected from PD0325901,
One of PD98059, BIX0218 or a variety of；The smoothened receptors agonist be selected from Purmorphamine,
One of Cyclopamine, GANT61 or a variety of；
Preferably, the GSK-3 α/β inhibitor is CHIR99021, and the BMP inhibitor is LDN193189, the DNA methyl
Inhibitors are RG108, and the AMPK inhibitor is Dorsomorphin, and the neuroprotective activity agent is P7C3-
A20, the ALK inhibitor are A83-01, and the neuron differentiation inducer is ISX9, and the adenyl cyclase activator is
Forskolin, the ROCK1 inhibitor are Y27632, and the GSI-IX gamma-secretase inhibitors are DAPT, and the MEK inhibits
Agent is PD0325901, and the smoothened receptors agonist is Purmorphamine.
3. composition according to claim 1 or 2, which is characterized in that the composition A includes following component:
The composition B includes following component:
Preferably, the composition A includes following component:
The composition B includes following component:
4. following any application of the described in any item compositions of claims 1 to 3:
(1) induced development cell of termination Differentiating Into Neurons；
(2) induced fibroblast is to the direct transdifferentiation of neuron；
(3) neuron is prepared by fibroblast in vitro；
(4) product for preventing or treating neure damage or missing is prepared；
(5) product for preventing or treating neurodegenerative disease is prepared；
Preferably, the neuron is glutamatergic neurons.
5. the product for preparing neuron by fibroblast in vitro, which is characterized in that include any one of claims 1 to 3
The composition.
6. the drug for preventing or treating neurodegenerative disease, which is characterized in that include any one of claims 1 to 3 institute
The composition stated.
7. a kind of induced fibroblast is directly to the method for neuron transdifferentiation, which is characterized in that in Fibroblast cell-culture
The described in any item compositions of claims 1 to 3 are added in the process；
Preferably, described method includes following steps:
(1) using cell culture medium culture fibroblast 3-5 days of the addition composition A；
(2) cell culture medium is changed to fresh cell culture medium, continues culture 3-5 days after adding the composition B；
It is highly preferred that being changed to Fresh cell culture medium for the 50% of cell culture medium in step (2)；
It adds the composition B to continue after cultivating 3-5 days, then cell culture medium is all changed into the thin of the addition composition A
Born of the same parents' culture medium repeats step (1) and (2) and once, then by culture medium is changed to the nerve without the composition A and composition B
Maturation medium continues to cultivate.
8. the method according to the description of claim 7 is characterized in that the composition A be added to CHIR99021,
The concentration of LDN193189, RG108, Dorsomorphin, P7C3-A20, A83-01 and ISX9 are respectively 3-10 μM, 0.25-3 μ
M,10-30μM,1-3μM,1-3μM,0.25-1μM,10-30μM；The composition B be added to Forskolin, Y27632,
The concentration of DAPT, PD0325901, Purmorphamine and P7C3-A20 are respectively 10-30 μM, 2-6 μM, 2-6 μM, 0.25-1 μ
M、1-10μM、1-3μM。
9. a kind of glutamatergic neurons, which is characterized in that be prepared by claim 7 or 8 the methods.
10. the product for treating neurodegenerative disease, which is characterized in that include Glutamatergic mind as claimed in claim 9
Through member.
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