CN110468104A - Inducing somatic transdifferentiation is sensory neuron and ganglionic composition and method - Google Patents
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Abstract
The invention discloses compositions and method that inducing somatic transdifferentiation is sensory neuron and neural ganglia structure, contain any combination transcription factor of Ascl1+Isl1, Ascl1+Brn3a, Ascl1+Brn3b, Ascl1+Brn3c, Ascl1+Isl1+Brn3a, Ascl1+Isl1+Brn3b, Ascl1+Isl1+Brn3c in composition, can be sensory neuron and neural ganglia structure somatic induction transdifferentiation.
Description
Technical field
The present invention relates to the field of biotechnology such as cell fate transdifferentiation, sensory neuron disease research, mainly sharp
It is maincenter/peripheral sensory neuron and class neuromere that the body cell of people or animal is induced to transdifferentiation in vitro with transcription factor
Structure provides the acquisition pattern of a new external evoked sensory neuron.
Background technique
Sensory neuron can experience outer signals and be divided into vision (vision), the sense of hearing (auditory), the sense of taste
(taste), the types receptor such as smell (smell), temperature (temperature), mechanical stimulus (mechanoreceptors),
It can also include blood receptor (blood receptors), nociceptor (nociceptors) etc. with signal in receptor.
Wherein nociceptor is divided into the subclass such as heat, cold receptor again.According to position distribution, sensory neuron can be divided into maincenter and outer
All sensory neurons.Maincenter sensory neuron includes perception vision, the sensory neuron of the sense of hearing, smell and the sense of taste, these nerves
The signal that member generates can directly be passed to brain.The input signal of peripheral sensory neuron is needed by spinal cord or peripheral nerve section
Then transfer could be passed to brain.The aggregation of peripheral sensory neuron cell body is agglomerating, forms dorsal ganglia and is connected to spinal cord,
Or it forms gasserian ganglion and is connected to brain, or form godling's warp knuckle (such as GI nerves section) of part.Due to gene
The reasons such as mutation, inflammation can cause sensory neuron abnormal, as to the diseases such as hyperpathia is sensitive or blunt.
, to many sensory nerve diseases, the pathogenesis of the especially diseases such as chronic ache is also not very clear for we, is led
Cause the drug development for these diseases slow.One of them important reason is caused by the shortage of disease model.Base
It is largely that nerve injury and chemicals are introduced by operation in the pain model of mouse and rat about more than 40 kinds [1]
What injury was realized, therefore the result obtained must be explained up in the background of model itself, cause many results in different models
Between it is widely different.More importantly since the mankind and rodent are widely different between species, much in mouse
Or the data obtained on rat and the mankind it is inconsistent.But repeating or verify these data directly on human body is clearly not
It is possible, it can only be realized by using the cell of the mankind, thus need different types of cell model, obtain sufficient amount
, different types of cell just become a kind of necessity.The sensory neuron for how reprogramming acquisition in vitro, as cell model
The pathogenesis and screening therapeutic agent for studying pains and other diseases, for understanding the pathogenesis of these diseases and providing newly
Treatment means and drug are extremely important.
It is a kind of obtain sensory neuron mode be by vitro directed differentiation embryonic stem cell (embryonic stem
Cell, ESC), multipotential stem cell (pluripotent stem cell, PSC) or induce multi-potent stem cell (induced
pluripotent stem cell,iPSC).Pain nerve member can be differentiated from people PSC, these neurons can be right
The stimulation of P2XR3, capsaicin are made a response [2], but without the sensory nerve element type for reporting other differentiation.
The mode that another kind obtains sensory neuron is to reprogram by cells transdifferentiate, such as by fibroblast as sense
Feel neuron.In the process, transcription factor Ascl1 (Mash1) is an important key-gene, independent [3] or combines it
Fibroblast transdifferentiation can be neuron by its transcription factor [4,5].Ascl1 and the combination of different specialization transcription factors,
Certain types of neuron, such as dopaminergic neuron [6-8], motor neuron [9] etc. can be converted into.
Two schemes are reported in two articles that Nature Neuroscience is delivered within 2015, it can will be at fiber
Cell reprogramming is sensory neuron.Woolf et al. discovery, using five kinds of transcription factors AsclI, Mytl1, Ngn1, Isl2 and
Klf7 can be that pain nerve is first [10] mouse embryonic fibroblasts or human fibroblasts reprogramming.Baldwin et al.
Another scheme is taken, is combined using transcription factor Ngn1 or Ngn2 with Brn3a, it can be by mouse or the fibroblast of people weight
It is programmed for the various sensory neurons [11] of expression TrkA/TrkB/TrkC.
As it can be seen that by body cell transdifferentiation be sensory neuron be an important R&D direction of medical development.
We prove that POU domain family member Brn3a/b/c [12-20] is for maincenter sensory nerve in previous work
The development of member and peripheral sensory neuron is essential.Other laboratories also turn out
LIM/homeodomain domain family member Isl1 is also required [21-24] to these neuronal developments.Body
One important evidence of outer cell differentiation or transdifferentiation is to simulate the natural differentiation process of internal cell in growth course.It manages according to this
By basis, we have invented achieve the purpose that cells transdifferentiate using the combination of the transcription factors such as Ascl1 and Brn3b or/Isl1.
Summary of the invention
The purpose of the present invention is to provide the combinations that inducing somatic transdifferentiation is sensory neuron and neural ganglia structure
Object, the preparation method of composition, using composition inducing somatic transdifferentiation be sensory neuron and neural ganglia structure side
Method.
In order to achieve the above object the present invention adopts the following technical scheme:
Inducing somatic transdifferentiation is the composition of sensory neuron and neural ganglia structure, and active constituent is selected from the following
A, b, c are any:
A. it carries the virion of combination transcription factor: carrying the virion of Ascl1+Isl1, carry Ascl1
The virion of+Brn3a carries the virion of Ascl1+Brn3b, carries the virion of Ascl1+Brn3c, carries
There is the virion of Ascl1+Isl1+Brn3a, carries the virion of Ascl1+Isl1+Brn3b, carry Ascl1+
It is one or more kinds of in the virion of Isl1+Brn3c；
B. the episome or plasmid of combination transcription factor are carried: carrying the episome or matter of Ascl1+Isl1
Grain, the episome for carrying Ascl1+Brn3a or plasmid, the episome for carrying Ascl1+Brn3b or plasmid carry
The episome or plasmid of Ascl1+Brn3c, the episome for carrying Ascl1+Isl1+Brn3a or plasmid carry
The episome or plasmid of Ascl1+Isl1+Brn3b carry one kind in the episome or plasmid of Ascl1+Isl1+Brn3c
Or it is two or more；
C. the mRNA/ protein mixture of transcription factor: the mRNA/ albumen of the mRNA/ protein of Ascl1, Isl1 is combined
Both matter mixture；Both the mRNA/ protein mixture of the mRNA/ protein of Ascl1, Brn3a；The mRNA/ albumen of Ascl1
Both the mRNA/ protein mixture of matter, Brn3b；Both the mRNA/ protein mixing of the mRNA/ protein of Ascl1, Brn3c
Object；The mRNA/ protein of Ascl1, the mRNA/ protein of Isl1, Brn3a mRNA/ protein three's mixture；Ascl1's
MRNA/ protein, the mRNA/ protein of Isl1, Brn3b mRNA/ protein three's mixture；The mRNA/ albumen of Ascl1
Matter, the mRNA/ protein of Isl1, Brn3c mRNA/ protein three's mixture in it is one or more kinds of.
Further, the virion for carrying Ascl1+Isl1, which refers to, carries the virion of Ascl1 and takes
The mixture of virion with Isl1, and/or the virion of Ascl1, Isl1 are carried simultaneously；
The virion for carrying Ascl1+Brn3a refers to the virion for carrying Ascl1 and carries Brn3a
Virion mixture, and/or carry the virion of Ascl1, Brn3a simultaneously；
The virion for carrying Ascl1+Brn3b refers to the virion for carrying Ascl1 and carries Brn3b
Virion mixture, and/or carry the virion of Ascl1, Brn3b simultaneously；
The virion for carrying Ascl1+Brn3c refers to the virion for carrying Ascl1 and carries Brn3c
Virion mixture, and/or carry the virion of Ascl1, Brn3c simultaneously；
The virion for carrying Ascl1+Isl1+Brn3a, refers to the virion for carrying Ascl1, carries
The virion of Isl1 carries virion three's mixture of Brn3a；And/or the disease of Ascl1, Isl1 are carried simultaneously
Malicious particle carries both virions of Brn3a mixture；And/or the virion of Ascl1, Brn3a are carried simultaneously,
Carry both virions of Isl1 mixture；And/or the virion of Brn3a, Isl1 are carried simultaneously, it carries
Both virions of Ascl1 mixture；And/or the virion of Ascl1, Isl1, Brn3a are carried simultaneously；
The virion for carrying Ascl1+Isl1+Brn3b, refers to the virion for carrying Ascl1, carries
The virion of Isl1 carries virion three's mixture of Brn3b；And/or the disease of Ascl1, Isl1 are carried simultaneously
Malicious particle carries both virions of Brn3b mixture；And/or the virion of Ascl1, Brn3b are carried simultaneously,
Carry both virions of Isl1 mixture；And/or the virion of Brn3b, Isl1 are carried simultaneously, it carries
Both virions of Ascl1 mixture；And/or the virion of Ascl1, Isl1, Brn3b are carried simultaneously；
The virion for carrying Ascl1+Isl1+Brn3c refers to the virion for carrying Ascl1, carries Isl1
Virion, carry virion three's mixture of Brn3c；And/or the virus of Ascl1, Isl1 are carried simultaneously
Grain, carries both virions of Brn3c mixture；And/or the virion of Ascl1, Brn3c are carried simultaneously, it carries
There is both virions of Isl1 mixture；And/or the virion of Brn3c, Isl1 are carried simultaneously, carry Ascl1's
Both virions mixture；And/or the virion of Ascl1, Isl1, Brn3c are carried simultaneously.
Further, the carrier of the virion is selected from slow virus, retrovirus, sendai virus, adenovirus, gland phase
Close virus and other pharmaceutically acceptable carriers.
Further, the episome for carrying Ascl1+Isl1 or plasmid refer to the episome for carrying Ascl1
Either plasmid and the episome of Isl1 or the mixture of plasmid are carried, and/or carries the free of Ascl1, Isl1 simultaneously
Body or plasmid；
The episome for carrying Ascl1+Brn3a or plasmid refer to the episome or plasmid for carrying Ascl1
With carry the episome of Brn3a perhaps the mixture of plasmid and/or simultaneously carry Ascl1, Brn3a episome or
Plasmid；
The episome for carrying Ascl1+Brn3b or plasmid refer to the episome or plasmid for carrying Ascl1
With carry the episome of Brn3b perhaps the mixture of plasmid and/or simultaneously carry Ascl1, Brn3b episome or
Plasmid；
The episome for carrying Ascl1+Brn3c or plasmid refer to the episome or plasmid for carrying Ascl1
With carry the episome of Brn3c perhaps the mixture of plasmid and/or simultaneously carry Ascl1, Brn3c episome or
Plasmid；
The episome or plasmid for carrying Ascl1+Isl1+Brn3a, refer to the episome for carrying Ascl1 or
Person's plasmid, carrying the episome of Isl1, perhaps plasmid carries the episome or plasmid three's mixture of Brn3a；And/or
Carrying the episome of Ascl1, Isl1 simultaneously, perhaps plasmid carries both episome or plasmids of Brn3a mixture；
And/or the episome of Ascl1, Brn3a is carried simultaneously perhaps plasmid carries the mixing of both episome or plasmid of Isl1
Object；And/or the episome of Brn3a, Isl1 is carried simultaneously perhaps plasmid carries both episome or plasmid of Ascl1
Mixture；And/or the episome or plasmid of Ascl1, Isl1, Brn3a are carried simultaneously；
The episome or plasmid for carrying Ascl1+Isl1+Brn3b, refer to the episome for carrying Ascl1 or
Person's plasmid, carrying the episome of Isl1, perhaps plasmid carries the episome or plasmid three's mixture of Brn3b；And/or
Carrying the episome of Ascl1, Isl1 simultaneously, perhaps plasmid carries both episome or plasmids of Brn3b mixture；
And/or the episome of Ascl1, Brn3b is carried simultaneously perhaps plasmid carries the mixing of both episome or plasmid of Isl1
Object；And/or the episome of Brn3b, Isl1 is carried simultaneously perhaps plasmid carries both episome or plasmid of Ascl1
Mixture；And/or the episome or plasmid of Ascl1, Isl1, Brn3b are carried simultaneously；
Carrying the episome of Ascl1+Isl1+Brn3c, perhaps plasmid refers to the episome or matter for carrying Ascl1
Grain, carrying the episome of Isl1, perhaps plasmid carries the episome or plasmid three's mixture of Brn3c；And/or simultaneously
Carrying the episome of Ascl1, Isl1, perhaps plasmid carries both episome or plasmids of Brn3c mixture；And/or
Carrying the episome of Ascl1, Brn3c simultaneously, perhaps plasmid carries both episome or plasmids of Isl1 mixture；
And/or the episome of Brn3c, Isl1 is carried simultaneously perhaps plasmid carries the mixing of both episome or plasmid of Ascl1
Object；And/or the episome or plasmid of Ascl1, Isl1, Brn3c are carried simultaneously.
The preparation step for carrying the virion of combination transcription factor is as follows:
1) it constructs viral vectors: combination transcription factor coded sequence clone being entered the carrier of virion, building transfection
Plasmid；
2) preparation virus: culture Viral packaging cell, plasmid prepared by step 1) are sharp together with viral packaging plasmid
With liposome or electricity turn or calcium chloride or calcium phosphate transfection Viral packaging cell, cultivated；
3) virion is collected from culture solution or/and Viral packaging cell, obtains the virus for carrying combination transcription factor
Particle.
A method of using above-mentioned composition inducing somatic transdifferentiation be sensory neuron and neural ganglia structure, packet
Include following steps:
(1) acquire and expand body cell；
(2) composition is infected body cell, makes to be overexpressed combination transcription factor: Ascl1+Isl1 or Ascl1 in body cell
+ Brn3a or Ascl1+Brn3b or Ascl1+Brn3c or Ascl1+Isl1+Brn3a or Ascl1+Isl1+Brn3b, or
Ascl1+Isl1+Brn3c；
(3) body cell after culture transfection, during which body cell transdifferentiation is sensory neuron and forms netted interconnection
Neural ganglia structure.
Body cell can be fibroblast, since above-mentioned transcription factor has the sequence conservation of height, humans and animals
The fibroblast in source has identical function.When acquiring fibroblast, according to conventional acquisition method, it can acquire small
Mouse embryo, childhood, any growth and development stage of adult fibroblast；Human fibroblasts source can be in surgical operation
The small strip obtained.
Preferably, the process of the step (2) is:
Body cell is added in the coated culture dish of poly-l-ornithine and laminin with culture medium and is cultivated；It removes
Culture medium is proportionally added into composition transfected somatic cell.
Preferably, the composition is the episome or plasmid for carrying combination transcription factor, or combination transcription
The mRNA/ protein mixture of the factor, the direct infection body cell in the way of liposome or electrotransfer or calcium phosphate, overexpression group
Close transcription factor.
Preferably, when the step (2) transfects, using CRISPR technology, by dCAS9-VP64 plasmid vector or have
The fusion protein of identical function, and the gRNA of targeting said combination transcription factor activate these transcription factors in body cell
Transcription, to be overexpressed these transcription factors；Or nuclease-VP64 the fusion protein by targeting said combination transcription factor
Or transcription of the fusion protein activation said combination transcription factor with the same function in body cell, to be overexpressed these turns
Record the factor；
Nuclease-VP64 the fusion protein includes ZFN, TALEN.
A kind of inducing somatic transdifferentiation is the sensory neuron that the method for sensory neuron and neural ganglia structure obtains
And the application of neural ganglia structure.Sensory neuron is separated using distinct methods, studies its various biochemical, physiology and other biological
Characteristic is learned, or directly carries out the applications such as external various experiments, drug screening using culture cell.
Contain Ascl1+Isl1 or Ascl1+Brn3a or Ascl1+Brn3b or Ascl1+Brn3c, these transcription factors
L cell can be reprogrammed the inhomogeneity for expression TrkA/TrkB/TrkC and various sensory neuron receptors
The sensory neuron and retinal ganglial cells of type.These sensory neurons and intracorporal sensory neuron are very close,
Gene expression profile having the same, identical electricity physiological signal stimulate identical reaction to sensory signal, and can be formed mutual
The class neural ganglia structure of connection；Especially Ascl1+Islet1+Brn3b or Ascl1+Islet1+Brn3a, or Ascl1+
Tri- kinds of transcription factor combinations of Islet1+Brn3c, reprogramming form the efficiency of sensory neuron and retinal ganglial cells
It can be higher.Reprogram obtained sensory neuron and retinal ganglial cells can be applied to sensory nerve disease model,
Drug screening and potential cellular transplantation therapy etc..
More preferably place is the present invention compared with prior art: it is various types of for can not only efficiently reprogramming fibroblast
The peripheral sensory neuron of type can also reprogram as maincenter sensory neuron such as retinal ganglial cells and the sense of hearing
Gangliocyte relevant with smell；Similar intracorporal different size of neural ganglia structure can be more formed, this is to have delivered
Method not available for.Therefore, the present invention can be obtained closer to intracorporal sensory neuron and class neuromere, in disease mould
Reliability and representativeness are had more in the application such as type and drug screening.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes part of this application, not
Inappropriate limitation of the present invention is constituted, in the accompanying drawings:
Fig. 1: Ascl1 slow virus and Isl1 slow-virus transfection fibroblast are induced as neural ganglia interconnected
The lab diagram of structure；Using after the fibroblast of Ascl1 and Isl1 slow-virus infection people or mouse, opened from the about the 12nd day
Begin, different size of neural chondritic is formed successively, wherein containing sensory neuron.
The cell for the sensory neuron expression nerve cell molecular labeling Map2 that Fig. 2: Ascl1 and Isl1 is reprogrammed is glimmering
Photoactivated antibody Coloration experiment figure；
The cell for the sensory neuron expression nerve cell molecular labeling Tuj1 that Fig. 3: Ascl1 and Isl1 is reprogrammed is glimmering
Photoactivated antibody Coloration experiment figure；
The cell for the sensory neuron expression nerve cell molecular labeling Dcx that Fig. 4: Ascl1 and Isl1 is reprogrammed is glimmering
Photoactivated antibody Coloration experiment figure；
The cell fluorescence for the sensory neuron expression specificity molecular labeling TrkA that Fig. 5: Ascl1 and Isl1 is reprogrammed
Antibody Coloration experiment figure；
The cell for the sensory neuron expression specificity molecular labeling Brn3a that Fig. 6: Ascl1 and Isl1 is reprogrammed is glimmering
Photoactivated antibody Coloration experiment figure；
The cell fluorescence for the sensory neuron expression specificity molecular labeling RPF that Fig. 7: Ascl1 and Isl1 is reprogrammed
Antibody Coloration experiment figure, figure Green fluorescence are Tuj1 antibody signal, and red fluorescence is RPF antibody signal.
The cell fluorescence for the sensory neuron expression inhibiting molecular labeling TH that Fig. 8: Ascl1 and Isl1 is reprogrammed is anti-
Body Coloration experiment figure, figure Green fluorescence are Tuj1 antibody signal, and red fluorescence is TH signal.
Specific embodiment
It is below that embodiment is detailed using " Ascl1 and Isl1 slow virus is by l cell transdifferentiation as sensory neuron "
Detailed process and details of the invention are carefully described.
(1) building of slow virus carrier (plasmid)
Mouse Ascl1 (NM_008553.4,696bp) and Isl1 (NM_021459.4,1050bp) coded sequence are distinguished
Clone enters the slow virus carrier (Tet-O-FUW) of the different regulating and controlling sequences containing tet-on, obtains plasmid Tet-O-FUW-
Ascl1 and plasmid Tet-O-FUW-Isl1.Purpose is future and RTTA slow virus cotransfection, and drug Dox can be added
(Doxycycline) expression of the control gene in body cell.After DNA sequencing verifies correct clone, the matter of transfection is prepared
Grain.
(2) preparation of slow virus
1) 24 hours before plasmid transfection, in 10cm Tissue Culture Dish middle berth 4.0x106HEK293T cell, be added 6ml
MEF culture medium is cultivated.
2) before transfecting, former culture medium is removed, changes the fresh MEF culture medium preheated in advance of 9ml into.
3) it prepares Ascl1 slow virus: in the 1.5ml centrifuge tube equipped with 500 μ l Opti-MEM culture mediums, plasmid is added
Tet-O-FUW-Ascl1 and slow virus packaging plasmid pMDLg/pRRE (5ug), pRSV-rev (2.5ug), pMD2.G
(2.5ug), mixes well, and obtains Opti-MEM/DNA mixed liquor；Prepare another again equipped with 460 μ l Opti-MEM culture mediums
1.5ml centrifuge tube, the lipofectamin2000 of 40ul is added, mixes gently, room temperature condition be incubated for 5min, obtain Opti-
2000 mixed liquor of MEM/lipofectamine；Opti-MEM/ is added in 2000 mixed liquor of Opti-MEM/lipofectamine
In DNA mixed liquor, be added among the liquid, avoid it is adherent plus, mix gently, be incubated for 20-30 minutes at room temperature.
4) it by the mixed liquor (about 1ml) of Opti-MEM/lipofectamine 2000/DNA, drops evenly in culture
In the 10cm culture dish of HEK293T, 37 DEG C of CO2 incubator cultures.
5) supernatant, the filtering of 0.45um sterilizing filter are collected after 48 hours, packing saves, and 4 DEG C are two weeks storable, and -80
It DEG C can long-term preservation.Once thawing, preferably it is finished or abandons, avoid multigelation.
6) it prepares Isl1 slow virus: changing plasmid Tet-O-FUW-Ascl1 into plasmid Tet-O-FUW-Isl1, remaining step
Ibid；
Preparation RTTA slow virus: plasmid Tet-O-FUW-Ascl1 is changed into plasmid TUW-RTTA, remaining step is same as above；
Detect virus titer.
(3) preparation of mouse embryonic fibroblasts (MEF)
1) the C57BL/6j mice embryonic for taking E13.5-E16.5, is put into the 10cm culture dish for filling HBSS, simple to rinse
It is transferred to another new 10cm culture dish for filling HBSS afterwards；
2) blank is put into microscissors removal head, backbone, four limbs and internal organ, remaining tissue under microscope
10cm culture dish；
3) it is put into no more than 8 embryos in each culture dish, 0.25% trypsase (0.25%trypsin- of 1ml is added
EDTA), with scissors by tissue shear to homogeneous state.It is incubated for 15 minutes in 37 DEG C of incubators, during which rocks 1-2 times；
4) appropriate MEF culture medium is added to be dispelled tissue with 10ml pipette.Every 2-3ml culture medium is put into 1 10cm training
It supports in ware；
5) MEF culture medium is added, in 37 DEG C of CO2It is cultivated in cell incubator；
6) culture medium after cell covers with (general 1-2 days), is removed, PBS is rinsed one time, and 0.25% tryptose of 1ml is added
Enzyme, 37 DEG C digest 3 minutes.Add 5ml MEF culture medium, is blown and beaten up and down with pipette after breaing up cell for several times, be transferred to 15ml centrifugation
Pipe；
7) it is centrifuged, 1000rpm/5min.Supernatant is removed, after adding MEF culture medium to be resuspended, divides disk to expand with the ratio of 1:4, i.e.,
Cell re-suspension liquid is divided into 4 parts to expand respectively；
8) after cell covers with, culture medium is removed, PBS is rinsed one time, and 0.25% trypsase of 1ml, 37 DEG C of digestion are added
3 minutes.Add 5ml MEF culture medium, blows and beats 1000rpm/5min centrifugation for several times up and down with pipette.Supernatant is removed, 5ml is added
Freezing media；
9) cell suspension is transferred to cryopreservation tube, 1ml/ pipe rapidly；Cryopreservation tube is placed on ice immediately, then is transferred to -80 DEG C of guarantors
It deposits case to stay overnight, next day moves into liquid nitrogen and saves for a long time.
(4) Tissue Culture Dish/plate coating
1) the preservation liquid of poly-l-ornithine and laminin is slowly dissolved at 4 DEG C；
2) by 100 × poly-l-ornithine save liquid be diluted to 1 in cold sterile ddH2O ×, culture is added
Ware/plate makes it that culture dish/board bottom portion be completely covered, and 37 DEG C of incubators are incubated at least 1 hour；
3) poly-l-ornithine solution is removed, and is rinsed once with sterile ddH2O；By laminin cold sterile
ddH2It is diluted to the concentration of 5ug/ml in O, culture dish is added, equally makes it that culture dish bottom be completely covered, 37 DEG C of incubators are incubated
It educates at least 1 hour；
4) laminin is removed, PBS rinsing is added, it is spare.
(5) induction MEF transdifferentiation is sensory neuron
1) by MEFs with 3-4 × 104The density in/hole is laid on 12 orifice plates being coated with, and 1ml MEF culture medium/hole, CO is added2
37 DEG C of cultures in incubator；
2) after 24 hours, MEF culture medium is removed, every hole is added the mixed liquor of 1ml slow virus and MEF culture medium and promotees to turn
Stain polybrene (10ug/ml).Ascl1 slow virus, Isl1 slow virus and RTTA slow virus are transfected with the ratio of 1:1:1
MEFs；In order to guarantee higher reprogramming efficiency, the amount of slow virus needs to do concentration gradient test in advance, in order to avoid excessive disease is added
Poison leads to MEF cell mortality；
3) after transfecting 16 hours, the mixed liquor of virus with culture medium is removed, changes DMEM/F12 culture medium (additional 1x into
B27,10ng/ml bFGF, 10ng/ml BDNF, 10ng/ml GDNF, 100ng/ml IGF-1 and 2ng/ml
doxycycline)；
4) during reprogramming, a fresh culture is changed within every two days；
5) after transfecting 12 days, so that it may neuron is gradually observed in culture dish, because forming different size of mind successively
Through chondritic, it can also be observed that different size of neuron agglomerate interconnected.
As shown in Figure 1, after above-mentioned steps, the fibroblast shape of Ascl1 slow virus and Isl1 slow-virus transfection
State structure changes, and forms many different size of cell masses interconnected, wherein including sensory neuron.
(6) immunofluorescence dyeing of cell is cultivated
1) culture medium is carefully removed, PBS is added, is rinsed 3 minutes；
2) PBS is removed, 4%PFA is added, fixes 10-15mins at room temperature；
3) PFA is removed, twice with PBS rinsing；Confining liquid is added, closes at room temperature at least 1 hour；
4) confining liquid is removed, primary antibody is added and (is diluted in 0.1%Triton X-100 and 2%normal donkey
In the PBS of serum), it is incubated at room temperature at least 1 hour；
5) primary antibody is removed, PBS is rinsed twice, and secondary antibody is added, and (1:1000 is diluted in 2%normal donkey serum's
In PBS), it is protected from light incubation 1 hour；
6) secondary antibody is removed, PBS is rinsed twice, is added in the darkroom DAPI (0.5 μ g/ml, be dissolved in PBS) and is incubated for 3 minutes；
7) DAPI is removed, PBS is added；Mountant is dripped on glass slide, is carefully taken out coverslip with tweezers, carefully
It tips upside down in mountant, stands, take pictures in case being observed at Laser Scanning Confocal Microscope (Carl Zeiss, LSM700).
Such as Fig. 2-8, can show as an example immunofluorescent staining as a result, different cell markings using different
Primary antibody, secondary antibody, can clearly show coloration result.
The cell fluorescence antibody coloration result of Fig. 2 shows that the sensory neuron expression that reprogramming fibroblast obtains is refreshing
Through cell marker molecules Map2；
The cell fluorescence antibody coloration result of Fig. 3 shows that the sensory neuron expression that reprogramming fibroblast obtains is refreshing
Through cell marker molecules Tuj1；
The cell fluorescence antibody coloration result of Fig. 4 shows that the sensory neuron expression that reprogramming fibroblast obtains is refreshing
Through cell marker molecules Dcx；
The cell fluorescence antibody coloration result of Fig. 5 shows that the sensory neuron that Ascl1 and Isl1 are reprogrammed is expressed special
Different molecular labeling TrkA；
The cell fluorescence antibody coloration result of Fig. 6 shows that the sensory neuron that Ascl1 and Isl1 are reprogrammed is expressed special
Different molecular labeling Brn3a；
The cell fluorescence antibody coloration result of Fig. 7 shows that the sensory neuron that Ascl1 and Isl1 are reprogrammed is expressed special
Different molecular labeling RPF；Fig. 7 Green fluorescence is Tuj1 antibody signal, and red fluorescence is RPF signal.
The cell fluorescence antibody coloration result of Fig. 8 shows that the sensory neuron that Ascl1 and Isl1 are reprogrammed expresses suppression
Property molecular labeling TH processed, it is shown in red in figure；TH is one of the required enzyme for synthesizing GABA precursor；Display green is neural thin
Born of the same parents' mark molecule Tuj1.
(7) formula of main agents
1) MEF culture medium
2) freezing media
MEF media 70ml；
FBS 20ml；
DMSO 10ml。
Certainly, other than infestation with virus particles MEFs, episome/plasmid sense of combination transcription factor can also be carried
MEFs is contaminated, combines the mRNA/ protein mixture infection MEFs of transcription factor to realize a fibroblast transdifferentiation for feeling
Neuron and neuromere.
The general mind of Induction of neuronal (induced sensoryNeurons, iSN) expression that technical scheme obtains
Various cell marker molecules through member, including Tuj1, Map2, Synapsin, Tau etc..
The various specific molecular markers of sensory neuron and retinal ganglial cells in the iSN expression body of acquisition, packet
Include Thy1, RPF, TrkA/B/C, Isl1 etc..
ISN expression excitatory neurotransmitter vGlut1/2/3, inhibitory neurotransmitter GABA and the correlation of acquisition
The molecular labelings such as synzyme TH, Gad65/67.
The iSN expression of acquisition can perceive the relevant receptor of the signals such as hot and cold, damage, mechanical stimulus and molecule.
The iSN of acquisition expresses the channels such as sodium, potassium, calcium, and electro physiology detection shows reaction identical with internal sensory neuron.
The iSN of acquisition is gathered into different size of small cell cluster interconnected, the structure of similar nerves within the body section.
To sum up, the present invention induces sensory neuron and intracorporal sensory neuron closer, and practicability and reliability are higher.
It is provided for the embodiments of the invention technical solution above to be described in detail, specific case used herein
The principle and embodiment of the embodiment of the present invention are expounded, the explanation of above embodiments is only applicable to help to understand this
The principle of inventive embodiments；At the same time, for those skilled in the art, according to an embodiment of the present invention, in specific embodiment party
There will be changes in formula and application range, in conclusion the contents of this specification are not to be construed as limiting the invention.
Claims (10)
1. the composition that inducing somatic transdifferentiation is sensory neuron and neural ganglia structure, it is characterised in that:
Active constituent a, b, c selected from the following is any:
A. it carries the virion of combination transcription factor: carrying the virion of Ascl1+Isl1, carry Ascl1+
The virion of Brn3a carries the virion of Ascl1+Brn3b, carries the virion of Ascl1+Brn3c, carries
There is the virion of Ascl1+Isl1+Brn3a, carries the virion of Ascl1+Isl1+Brn3b, carry Ascl1+
It is one or more kinds of in the virion of Isl1+Brn3c；
B. the episome or plasmid of combination transcription factor are carried: carrying the episome of Ascl1+Isl1 or plasmid, takes
Episome or plasmid with Ascl1+Brn3a, the episome for carrying Ascl1+Brn3b or plasmid carry Ascl1
The episome or plasmid of+Brn3c, the episome for carrying Ascl1+Isl1+Brn3a or plasmid carry Ascl1+
The episome or plasmid of Isl1+Brn3b carry one or two in the episome or plasmid of Ascl1+Isl1+Brn3c
More than；
C. the mRNA/ protein mixture of transcription factor: the mRNA/ protein two of the mRNA/ protein of Ascl1, Isl1 is combined
Person's mixture；Both the mRNA/ protein mixture of the mRNA/ protein of Ascl1, Brn3a；The mRNA/ protein of Ascl1,
Both the mRNA/ protein of Brn3b mixture；Both the mRNA/ protein mixture of the mRNA/ protein of Ascl1, Brn3c；
The mRNA/ protein of Ascl1, the mRNA/ protein of Isl1, Brn3a mRNA/ protein three's mixture；Ascl1's
MRNA/ protein, the mRNA/ protein of Isl1, Brn3b mRNA/ protein three's mixture；The mRNA/ albumen of Ascl1
Matter, the mRNA/ protein of Isl1, Brn3c mRNA/ protein three's mixture in it is one or more kinds of.
2. inducing somatic transdifferentiation according to claim 1 is the composition of sensory neuron and neural ganglia structure,
It is characterized by:
The virus that the virion for carrying Ascl1+Isl1 refers to the virion for carrying Ascl1 and carries Isl1
The mixture of particle, and/or the virion of Ascl1, Isl1 are carried simultaneously；
The disease that the virion for carrying Ascl1+Brn3a refers to the virion for carrying Ascl1 and carries Brn3a
The mixture of malicious particle, and/or the virion of Ascl1, Brn3a are carried simultaneously；
The disease that the virion for carrying Ascl1+Brn3b refers to the virion for carrying Ascl1 and carries Brn3b
The mixture of malicious particle, and/or the virion of Ascl1, Brn3b are carried simultaneously；
The disease that the virion for carrying Ascl1+Brn3c refers to the virion for carrying Ascl1 and carries Brn3c
The mixture of malicious particle, and/or the virion of Ascl1, Brn3c are carried simultaneously；
The virion for carrying Ascl1+Isl1+Brn3a, refers to the virion for carrying Ascl1, carries Isl1
Virion, carry virion three's mixture of Brn3a；And/or the virus of Ascl1, Isl1 are carried simultaneously
Grain, carries both virions of Brn3a mixture；And/or the virion of Ascl1, Brn3a are carried simultaneously, it carries
There is both virions of Isl1 mixture；And/or the virion of Brn3a, Isl1 are carried simultaneously, carry Ascl1's
Both virions mixture；And/or the virion of Ascl1, Isl1, Brn3a are carried simultaneously；
The virion for carrying Ascl1+Isl1+Brn3b, refers to the virion for carrying Ascl1, carries Isl1
Virion, carry virion three's mixture of Brn3b；And/or the virus of Ascl1, Isl1 are carried simultaneously
Grain, carries both virions of Brn3b mixture；And/or the virion of Ascl1, Brn3b are carried simultaneously, it carries
There is both virions of Isl1 mixture；And/or the virion of Brn3b, Isl1 are carried simultaneously, carry Ascl1's
Both virions mixture；And/or the virion of Ascl1, Isl1, Brn3b are carried simultaneously；
The virion for carrying Ascl1+Isl1+Brn3c refers to the virion for carrying Ascl1, carries the disease of Isl1
Malicious particle carries virion three's mixture of Brn3c；And/or the virion of Ascl1, Isl1 are carried simultaneously, it takes
Both virions with Brn3c mixture；And/or the virion of Ascl1, Brn3c are carried simultaneously, carry Isl1
Both virions mixture；And/or the virion of Brn3c, Isl1 are carried simultaneously, carry the virus of Ascl1
Both grains mixture；And/or the virion of Ascl1, Isl1, Brn3c are carried simultaneously.
3. the combination that inducing somatic transdifferentiation according to claim 1 or 2 is sensory neuron and neural ganglia structure
Object, it is characterised in that:
The carrier of the virion is selected from slow virus, retrovirus, sendai virus, adenovirus, adeno-associated virus.
4. inducing somatic transdifferentiation according to claim 1 is the composition of sensory neuron and neural ganglia structure,
It is characterized by:
The episome for carrying Ascl1+Isl1 or plasmid refer to the episome for carrying Ascl1 or plasmid and carrying
There is the episome of Isl1 perhaps the mixture of plasmid and/or to carry the episome or plasmid of Ascl1, Isl1 simultaneously；
The episome for carrying Ascl1+Brn3a or plasmid refer to the episome for carrying Ascl1 or plasmid and take
Episome with Brn3a perhaps the mixture of plasmid and/or carries the episome or plasmid of Ascl1, Brn3a simultaneously；
The episome for carrying Ascl1+Brn3b or plasmid refer to the episome for carrying Ascl1 or plasmid and take
Episome with Brn3b perhaps the mixture of plasmid and/or carries the episome or plasmid of Ascl1, Brn3b simultaneously；
The episome for carrying Ascl1+Brn3c or plasmid refer to the episome for carrying Ascl1 or plasmid and take
Episome with Brn3c perhaps the mixture of plasmid and/or carries the episome or plasmid of Ascl1, Brn3c simultaneously；
Perhaps plasmid refers to the episome or matter for carrying Ascl1 to the episome for carrying Ascl1+Isl1+Brn3a
Grain, carrying the episome of Isl1, perhaps plasmid carries the episome or plasmid three's mixture of Brn3a；And/or simultaneously
Carrying the episome of Ascl1, Isl1, perhaps plasmid carries both episome or plasmids of Brn3a mixture；And/or
Carrying the episome of Ascl1, Brn3a simultaneously, perhaps plasmid carries both episome or plasmids of Isl1 mixture；
And/or the episome of Brn3a, Isl1 is carried simultaneously perhaps plasmid carries the mixing of both episome or plasmid of Ascl1
Object；And/or the episome or plasmid of Ascl1, Isl1, Brn3a are carried simultaneously；
Perhaps plasmid refers to the episome or matter for carrying Ascl1 to the episome for carrying Ascl1+Isl1+Brn3b
Grain, carrying the episome of Isl1, perhaps plasmid carries the episome or plasmid three's mixture of Brn3b；And/or simultaneously
Carrying the episome of Ascl1, Isl1, perhaps plasmid carries both episome or plasmids of Brn3b mixture；And/or
Carrying the episome of Ascl1, Brn3b simultaneously, perhaps plasmid carries both episome or plasmids of Isl1 mixture；
And/or the episome of Brn3b, Isl1 is carried simultaneously perhaps plasmid carries the mixing of both episome or plasmid of Ascl1
Object；And/or the episome or plasmid of Ascl1, Isl1, Brn3b are carried simultaneously；
Carrying the episome of Ascl1+Isl1+Brn3c, perhaps plasmid refers to the episome or plasmid for carrying Ascl1, takes
Perhaps plasmid carries the episome or plasmid three's mixture of Brn3c to episome with Isl1；And/or it carries simultaneously
Having the episome of Ascl1, Isl1, perhaps plasmid carries both episome or plasmids of Brn3c mixture；And/or simultaneously
Carrying the episome of Ascl1, Brn3c, perhaps plasmid carries both episome or plasmids of Isl1 mixture；And/or
Carrying the episome of Brn3c, Isl1 simultaneously, perhaps plasmid carries both episome or plasmids of Ascl1 mixture；
And/or the episome or plasmid of Ascl1, Isl1, Brn3c are carried simultaneously.
5. a kind of preparation method of composition as described in claim 1, it is characterised in that:
The composition is when carrying the virion of combination transcription factor, and preparation step is as follows:
1) it constructs viral vectors: combination transcription factor coded sequence clone being entered the carrier of virion, building transfection
Plasmid；
2) preparation virus: culture Viral packaging cell, plasmid prepared by step 1) utilize rouge together with viral packaging plasmid
Plastid or electricity turn or calcium chloride or calcium phosphate transfection Viral packaging cell, are cultivated；
3) virion is collected from culture solution or/and Viral packaging cell, obtains the virion for carrying combination transcription factor.
6. a kind of use composition inducing somatic transdifferentiation as described in claim 1 for sensory neuron and neural ganglia knot
The method of structure, it is characterised in that:
Include the following steps:
(1) acquire and expand body cell；
(2) composition is infected body cell, makes to be overexpressed combination transcription factor: Ascl1+Isl1 or Ascl1+ in body cell
Brn3a or Ascl1+Brn3b or Ascl1+Brn3c or Ascl1+Isl1+Brn3a or Ascl1+Isl1+Brn3b, or
Ascl1+Isl1+Brn3c；
(3) body cell after culture transfection, during which body cell transdifferentiation is sensory neuron and forms netted mind interconnected
Warp knuckle spline structure.
7. inducing somatic transdifferentiation according to claim 6 is the method for sensory neuron and neural ganglia structure,
It is characterized in that:
The process of the step (2) is:
Body cell is added in the coated culture dish of poly-l-ornithine and laminin with culture medium and is cultivated；Remove culture
Base is proportionally added into composition transfected somatic cell.
8. inducing somatic transdifferentiation according to claim 6 is the method for sensory neuron and neural ganglia structure,
It is characterized in that:
The composition is the episome or plasmid for carrying combination transcription factor, or the mRNA/ of combination transcription factor
Protein mixture, the direct infection body cell in the way of liposome or electrotransfer or calcium phosphate are overexpressed combination transcription factor.
9. inducing somatic transdifferentiation according to claim 6 is the method for sensory neuron and neural ganglia structure,
It is characterized in that:
When the step (2) transfects, using CRISPR technology, by dCAS9-VP64 plasmid vector or with the same function melt
Hop protein, and the gRNA of targeting said combination transcription factor activate transcription of these transcription factors in body cell, thus mistake
Express these transcription factors；Or by the nuclease-VP64 fusion protein of targeting said combination transcription factor or with identical
Transcription of the fusion protein activation said combination transcription factor of function in body cell, to be overexpressed these transcription factors；
Nuclease-VP64 the fusion protein includes ZFN, TALEN.
10. the application of a kind of sensory neuron that method as claimed in claim 5 obtains and neural ganglia structure.
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