CN108531453A - A method of converting non-neuronal cell to neuronal cell - Google Patents

A method of converting non-neuronal cell to neuronal cell Download PDF

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CN108531453A
CN108531453A CN201710117871.5A CN201710117871A CN108531453A CN 108531453 A CN108531453 A CN 108531453A CN 201710117871 A CN201710117871 A CN 201710117871A CN 108531453 A CN108531453 A CN 108531453A
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sirna
extracellular matrix
sequence identity
gene expressions
strikes
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CN108531453B (en
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周琪
李伟
胡宝洋
何正泉
王柳
郝捷
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Institute of Zoology of CAS
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Priority to CN202010322427.9A priority Critical patent/CN111471653B/en
Priority to CN201710117871.5A priority patent/CN108531453B/en
Priority to CN202010322435.3A priority patent/CN111500538A/en
Priority to PCT/CN2018/077192 priority patent/WO2018157769A1/en
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Abstract

It is the method for neuronal cell that the present invention provides a kind of by non-neuronal cell transdifferentiation comprising carries out interference processing to the extracellular matrix shell system of non-neuronal cell, wherein interference processing is selected from：It is handled using cytoskeletal protein micromolecular inhibitor, the specific gene expression of extracellular matrix shell system is carried out striking reduction process using siRNA (siRNA), low adhesion process and directed differentiation culture are carried out to extracellular matrix.The method application of the present invention is simple, only needs single small molecule or single factor test processing, and can efficiently carry out in vitro in vivo, has major application in regeneration, reparation and oncotherapy.

Description

A method of converting non-neuronal cell to neuronal cell
Technical field
The present invention relates to biotechnologies, and in particular to passes through extracellular matrix-shell system to non-neuronal cell Carry out method of the interference processing to convert the non-neuronal cell of humans and animals to functional nerve member.
Background technology
Regulating cell destiny, to generate the particular cell types with different function, in cell replacement therapy and regeneration There is important application foreground in treatment.Cell fate depends on the specific expressed of genome, and expression regulation mode both included Common biology regulation and control, such as signal transduction, transcription regulatory network, epigenetic modification, also by the physicochemical property of cell with And the regulation and control of the chemical factors in cell local environment.Therefore the method for cell fate transformation is also classified into two kinds, that is, changes cell Biological characteristics and physicochemical property.
The method of functioning cell is generated primarily directed to several important gene regulatory pathway by regulating cell destiny at present And epigenetic modification, it is completed by science of heredity means or chemical small molecule processing means.Science of heredity means include research Personnel are using the transcription factors such as ectopic expression Oct4, Sox2, c-Myc, Klf4 by mouse and people at fiber reprogramming of somatic cells For inducing pluripotent stem cells；Using transcription factors such as ectopic expression Adcl1, Brn2 and Myt1l by fibroblast transdifferentiation For functional nerve member；And the functional cardiac muscle cell of tool, islet cells are obtained in the way of ectopic expression specific gene Deng.Chemical small molecule means include that l cell reprogramming is by researcher using chemical small molecule combination VC6TFZ Multipotent stem cells；The fibroblast of people is converted into nerve by the combination (VCRFSGY) formed using 7 kinds of small molecules Member；It is neural stem cell to be reprogrammed l cell using the combination M9 of 9 kinds of small molecules, and then is divided into functional god Through member；People's gastric epithelial cell is transformed into the entoderm progenitor cells of pluripotency using small molecule combinatorial；It will using small molecule combinatorial People is converted to cardiac muscle cell etc. by small molecule at fiber.
Invention content
The present invention mainly carries out interference processing by extracellular matrix-shell system to non-neuronal cell, to realize Cell fate is regulated and controled, particularly, in terms of being neuronal cell by the non-neuronal cell transdifferentiation of human or animal More easy-to-use approach is proposed, initiative, unexpected technique effect is achieved.
The present invention provides a kind of methods converting non-neuronal cell to neuronal cell, it is characterised in that the side Method includes carrying out interference processing to extracellular matrix-shell system of non-neuronal cell.
The interference of the present invention handles at least one selected from the following：It is handled, is adopted using cytoskeletal protein inhibitor The gene expression of extracellular matrix-shell system is carried out striking reduction process with siRNA (siRNA), extracellular matrix is carried out low Adhesion process is simultaneously preferably further trained for a certain area.
According to embodiment of the present invention, wherein the cytoskeletal protein inhibitor selected from the following at least one Kind：Myosin (myosin) inhibitor, actin (actin) assemble inhibitor.
Preferably, wherein the myosin (myosin) inhibitor at least one selected from the following：(-)- Blebbistatin, myosin light chain kinase (MLCK) inhibitor ML-7, a concentration of 10 μM or more, preferably 20 μM or more, more It is preferred that 10-30 μM, wherein final in a concentration of inhibitor inducing culture used in processing non-neuronal cell Concentration.
Preferably, wherein the actin (actin) assembles inhibitor at least one selected from the following： Cytochalasin B, Latrunculin B, wherein a concentration of 1.5 μM or more of Cytochalasin B, preferably 2 μM or more, More preferably 2-3 μM, a concentration of 0.15 μM of Latrunculin B or more, preferably 0.2 μM, more preferable 0.2-0.3 μM, wherein described Ultimate density in a concentration of inhibitor inducing culture used in processing non-neuronal cell.
According to embodiment of the present invention, the method includes non-neuronal cell is placed in inducing culture to train Support 3-7 days, optionally 4 days, 5 days or 6 days, then using maturation medium culture 7-14 days, optionally 8 days, 9 days, 10 days, 11 It, 12 days or 13 days.
Preferably, wherein the inducing culture includes：Cytoskeletal protein inhibitor, N2 cell culture based additives, B27 cell culture based additives, glutamine, β mercaptoethanols.
Preferably, wherein the maturation medium includes：N2 cell culture based additives, B27 cell culture based additives, Glutamine, β mercaptoethanols, neurenergen (NT3), brain-derived neurotrophic factor (BDNF), derived from glial cell line Neurotrophic factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
According to embodiment of the present invention, non-neuronal cell is preferably fibroblast and/or god in the present invention Through spongiocyte.
The present invention also provides cytoskeletal protein inhibitor to be used to non-neuronal cell transdifferentiation be neuronal cell Purposes.
The present invention also provides a kind of kit converting non-neuronal cell to neuronal cell, the kit packet Inducing culture is included, the inducing culture includes cytoskeletal protein inhibitor.
According to embodiment of the present invention, wherein the cytoskeletal protein inhibitor selected from the following at least one Kind：Myosin (myosin) inhibitor, actin (actin) assemble inhibitor.
Preferably, wherein the myosin (myosin) inhibitor at least one selected from the following：(-)- Blebbistatin, myosin light chain kinase (MLCK) inhibitor ML-7, a concentration of 10 μM or more, preferably 20 μM or more, more It is preferred that 10-30 μM, wherein concentration of a concentration of inhibitor in inducing culture.
Preferably, wherein the actin (actin) assembles inhibitor at least one selected from the following： Cytochalasin B, Latrunculin B, wherein a concentration of 1.5 μM or more of Cytochalasin B, preferably 2 μM or more, More preferably 2-3 μM, a concentration of 0.15 μM of Latrunculin B or more, preferably 0.2 μM, more preferable 0.2-0.3 μM, wherein described Concentration of a concentration of inhibitor in inducing culture.
According to embodiment of the present invention, the kit further includes maturation medium.
Preferably, wherein the inducing culture includes：Cytoskeletal protein inhibitor, N2 cell culture based additives, B27 cell culture based additives, glutamine, β mercaptoethanols.
Preferably, wherein the maturation medium includes：N2 cell culture based additives, B27 cell culture based additives, Glutamine, β mercaptoethanols, neurenergen (NT3), brain-derived neurotrophic factor (BDNF), derived from glial cell line Neurotrophic factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
The present invention also provides cytoskeletal protein inhibitor to be used to prepare antitumor drug, regeneration and/or reparation Purposes in drug.
According to embodiment of the present invention, reduction process of striking of the invention includes at least one below：
Using with sequence SEQ ID NO:1 has 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the rock1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:2 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the rock2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:3 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the mrlc1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:4 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the mrlc2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:5 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the mrlc3 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:6 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the myh9 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:7 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the myh10 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:8 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the mrck α gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:9 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the mrck beta gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:10 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the lamina/c gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:11 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the lmnb1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:12 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the lbr gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:13 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the sun1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:14 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the sun2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:15 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the cbx1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:16 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the cbx3 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:17 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the cbx5 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:18 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the banf1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:19 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the syne1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:20 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the syne2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:21 have 95%, 96%, 97%, 98%, 99% or 100% sequence identity SiRNA strikes the β-actin gene expressions in low extracellular matrix-shell system.
According to embodiment of the present invention, using with sequence SEQ ID NO:1 have 95%, 96%, 97%, 98%, the siRNA of 99% or 100% sequence identity strikes the rock1 gene expressions in low extracellular matrix-shell system, uses With sequence SEQ ID NO:2 siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strike low extracellular Rock2 gene expressions in matrix-shell system, using with sequence SEQ ID NO:8 have 95%, 96%, 97%, 98%, The siRNA of 99% or 100% sequence identity strikes the mrck α gene expressions in low extracellular matrix-shell system, and using with Sequence SEQ ID NO:9 siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strike low extracellular base Mrck beta gene expressions in matter-shell system.
According to embodiment of the present invention, using with sequence SEQ ID NO:3 have 95%, 96%, 97%, 98%, the siRNA of 99% or 100% sequence identity strikes the mrlc1 gene expressions in low extracellular matrix-shell system, uses With sequence SEQ ID NO:4 siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strike low extracellular Mrlc2 gene expressions in matrix-shell system, and use and sequence SEQ ID NO:5 have 95%, 96%, 97%, 98%, the siRNA of 99% or 100% sequence identity strikes the mrlc3 gene expressions in low extracellular matrix-shell system.
According to embodiment of the present invention, using with sequence SEQ ID NO:6 have 95%, 96%, 97%, 98%, the siRNA of 99% or 100% sequence identity strikes the myh9 gene expressions in low extracellular matrix-shell system, and adopts With with sequence SEQ ID NO:7 siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strike low born of the same parents Myh10 gene expressions in epimatrix-shell system.
According to embodiment of the present invention, using with sequence SEQ ID NO:3 have 95%, 96%, 97%, 98%, the siRNA of 99% or 100% sequence identity strikes the mrlc1 gene expressions in low extracellular matrix-shell system, uses With sequence SEQ ID NO:4 siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strike low extracellular Mrlc2 gene expressions in matrix-shell system, using with sequence SEQ ID NO:5 have 95%, 96%, 97%, 98%, The siRNA of 99% or 100% sequence identity strikes the mrlc3 gene expressions in low extracellular matrix-shell system, and using with Sequence SEQ ID NO:6 siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strike low extracellular base Myh9 gene expressions in matter-shell system.
According to embodiment of the present invention, using with sequence SEQ ID NO:13 have 95%, 96%, 97%, 98%, the siRNA of 99% or 100% sequence identity strikes the sun1 gene expressions in low extracellular matrix-shell system, and adopts With with sequence SEQ ID NO:14 siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strike low Sun2 gene expressions in extracellular matrix-shell system.
According to embodiment of the present invention, reduction process of striking of the invention includes the following steps：
Diluted liposome and the siRNA of the present invention are mixed, incubated at room temperature is to form siRNA- liposome mixtures；It will The mixture is mixed evenly with the culture solution containing non-neuronal cell to be transfected, is cultivated.
Preferably, liposome usesRNAiMAX Reagent reagents.
Preferably, before liposome is mixed with siRNA, it is diluted siRNA.
Preferably, liposome and siRNA are diluted, using serum-free Opti-MEM culture mediums.
Preferably, siRNA is diluted：It is managed in the EP of no RNA enzyme, the serum-free Opti-MEM culture mediums with 100 μ L/ per hole are dilute Release 12pmol siRNA double-strands (2.5ul).
Preferably, transfection reagent is dilutedRNAiMAX Reagent：It manages, uses in the EP of no RNA enzyme 100 μ L/ are per the dilution of hole of serum-free Opti-MEM culture mediumsRNAiMAX Reagent。
Preferably, the liposome diluted passes through 1-5min, preferably 2min, preferably 3min, preferably after the incubation of 4min, with The good siRNA of above-mentioned dilution is mixed, incubated at room temperature 15-25min, preferably 20min, to form siRNA- liposome mixtures.
Preferably, siRNA- liposome mixtures are added in the tissue culture plate containing cell and culture solution and are allowed to mixed With.
Preferably, culture plate is placed in 37 DEG C of CO2Cultivated in incubator, transfect every 6-8 hours, preferably 7 hours, after change At neuronal cultured solution.
Preferably, after neuronal cultured solution culture 48-72 hours, preferably 60 hours.
Preferably, neuronal cultured solution includes：N2 cell culture based additives, B27 cell culture based additives, glutamy Amine, β mercaptoethanols.
Preferably, neuronal cultured solution includes：N2 cell culture based additives, B27 cell culture based additives, glutamy Amine, β mercaptoethanols, neurenergen (NT3), brain-derived neurotrophic factor (BDNF), nerve battalion derived from glial cell line Support the factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
According to embodiment of the present invention, low adhesion process includes suspending to cultivate non-neuron using agarose DMEM Cell.Preferably, suspension incubation time is 6-8 days, preferably 7 days.Preferably, the dosage of the agarose is 0.5g/100 millis Rise the agarose DMEM.
Preferably, 1% agarose solution being configured with aseptic double-distilled water first, micro-wave oven is heated to boiling, be then added etc. 2 × DMEM of volume, (Gibco, 12800-017) are made 0.5% agarose solution being dissolved in DMEM, are subsequently poured into 6cm Culture dish in, it is spare after cooled and solidified.
Preferably, half amount changes liquid every other day during suspension incubation.
Preferably, after non-neuronal cell is using agarose DMEM suspension cultures, then directed differentiation culture solution culture is used.
Preferably, the directed differentiation culture solution include GDNF (neurotrophic factor of Deiter's cells source property, Peprotech, 450-10), BDNF (brain-derived neurotrophic factor, peprotech, 450-02), NT3 (neurotrophic factors- 3, peprotech, 450-03) and Forskolin (forskolin, stemgent, 04-0025).
Preferably, GDNF (peprotech, 450-10) a concentration of 15-25ng/ml in the directed differentiation culture solution, it is excellent Select 20ng/ml, BDNF (peprotech, 450-02) a concentration of 15-25ng/ml, preferably 20ng/ml, NT3 (peprotech, The Forskolin (stemgent, 04-0025) of 450-03) a concentration of 15-25ng/ml, preferably 20ng/ml, 4ng/ml are a concentration of 2-6ng/ml, preferably 3,4 or 5ng/ml.
According to the present invention, reduction process is struck, at low adherency by using the micromolecular inhibitor of cytoskeletal protein, siRNA Human or animal's non-neuronal cell, can efficiently be transformed by extracellular matrix-shell system of reason interference non-neuronal cell Neuronal cell.Method through the invention realizes that cell fate change is not yet reported to obtain neuron, same to phase Cell fate than reporting before regulates and controls method, and this method application is more simple, only needs single small molecule processing, or do not need Only cell fate change can be realized by changing cell culture substrate in small molecule and specific gene expression regulation, and in body It is interior efficiently to carry out in vitro, there is major application in oncotherapy, regeneration/reparation.
The method of the present invention has the function of broad-spectrum anti-tumor activity.The outer embryo of nerve is betided for example, glioma is one kind The tumour of layer, it is most of to originate from different types of Deiter's cells.Glioma is most common intracranial tumors according to statistics, Account for 45 or so the percent of all intracranial tumors.It is number two in children malignant tumors position, in recent years, the evil of primary Property intracranial tumors incidence cumulative year after year, annual growth rate is about 1.2%, particularly evident in mid-aged population.According to the literature, In Chinese glioma, to be every 100,000 people have a 3-6 people to incidence every year on average, year death toll be up to 30,000 people.At present both at home and abroad Treatment means to glioma include mainly：Operation, chemotherapy, radiotherapy, X knives and γ knives etc..It, can be by cell bone based on the present invention The micromolecular inhibitor of frame albumen is used to prepare anti-glioma drugs.By using this drug, colloid can be significantly inhibited The proliferation of oncocyte, to reach anticancer effect.
Physiological aging or case cerebral injury lead to a large amount of neuronal deaths, lead to Deiter's cells (predominantly star glue Cell plastid) a large amount of proliferation, occupy original cerebral injury region, inhibit the regeneration of neuron, form intracerebral glial scars, cause god Through degenerative disease, the quality of life of people, such as Parkinson, Ai Erhaimo diseases are seriously affected.Although people attempt a large amount of side Method, the still effectively generation of method control glial scars without simple, promotes neuron regeneration reparation at present.Profit of the invention Can be efficiently neuron by the efficient transdifferentiation of the primary astroglial cells of people source with cytoskeletal protein inhibitor.This Neurodegenerative disease is caused to provide a kind of new method to treat aging and pathology damage in body.
The method have the characteristics that：1. easy to operate, induction broth is added in single small molecule or single factor test processing is It can carry out destiny conversion.2. efficiently quickly, handling non-neuronal cell Godwards in myosin inhibitor/actin assembling inhibitor During first transdifferentiation, the cell of the neuron fates close to the 100% Tuj1 positives was achieved at the 7th day, suspend training Nutrient solution only needs 7 day time that the neuronotropic conversion of non-neuronal cell can be realized.3. universality, this method is in different plant species Different type initiator cell to having universal adaptability during neuron transdifferentiation, can by mouse TTF, MEF, setoli and Efficiently transdifferentiation is neuron to butt fibroblast, human foreskin fibroblasts and the muscle cell of people.4. safety, this hair Bright method has higher safety compared to the science of heredity means that traditional viral vectors mediates.5. controllability, single small The slow-released system of molecule has more feasibility compared to the transdifferentiation that multiple small molecule combinatorials mediate, and is convenient for measure control.
Description of the drawings
Figure 1A is immune at the microphoto (left side) and Tuj1 of human foreskin fibroblasts (HFF20y, Beijing stem cell bank) Fluorescent staining picture (right side).
Figure 1B is that the fibroblast in embodiment 1-1 for Figure 1A is trained using the induction comprising (-)-Blebbistatin The microphoto (left side) and Tuj1 immunofluorescence dyeings picture (right side) of cell after foster base culture 7 days.
Fig. 1 C show that the fibroblast in embodiment 1-1 for Figure 1A uses luring comprising (-)-Blebbistatin After leading medium culture 7 days, then with the Marker of the expression mature neuron after maturation medium culture 14 days：Map2(santa Cruz biotechnology, sc-20172), NF200 (Abcam, ab4680) and NeuN (chemicon, MAB377).
Fig. 1 D show that the fibroblast in embodiment 1-1 for Figure 1A uses luring comprising (-)-Blebbistatin After leading medium culture 7 days, then with the presynaptic of the ripe neuron expression classics of expression after maturation medium culture 14 days Labelled protein Syn1 (millipore, AB1543P) and Syt1 (abcam, ab133856) and postsynaptic labelled protein PSD95 (abcam, ab18258).
Fig. 1 E are that the fibroblast in embodiment 1-1 for Figure 1A is trained using the induction comprising (-)-Blebbistatin After supporting base culture 7 days, then with the ripe neuron patch-clamp experimental result picture after maturation medium culture 14 days.
Fig. 1 F show that the fibroblast in embodiment 1-1 for Figure 1A uses luring comprising (-)-Blebbistatin After leading medium culture 7 days, then with the neuron after maturation medium culture 14 days carry out type mark protein staining expression GABA serotonergic neuron labelled protein GABA (sigma, SAB4501067), GAD65/67 (santa cruz biotechnology, sc-7513)。
Fig. 1 G show that the fibroblast in embodiment 1-1 for Figure 1A uses luring comprising (-)-Blebbistatin After leading medium culture 7 days, then with the dopaminergic marker TH of neuron expression midbrain after maturation medium culture 14 days (santa cruz biotechnology, sc-14007) and Glutamatergic marker vGlut1 (santa cruz Biotechnology, sc-377425).
Fig. 1 H show that ultra high efficiency liquid phase-ultrahigh resolution Mass Spectrometer Method measures induction GABA neurons in embodiment 1-1 Respond high potassium buffer solution processing release gamma aminobutyric acid result figure.
Fig. 2A is Mouse Tail-tip fibroblast untreated preceding neuronal marker protein Tuj1 and Map2 dyes in embodiment 1-3 Chromatic graph piece.
Fig. 2 B are to use the nerve for including (-)-Blebbistatin for Mouse Tail-tip fibroblast in embodiment 1-3 The Marker of neuron is expressed in inducing culture culture after 7 days：Tuj1, Map2, NF200 and NeuN.
Fig. 2 C are to use the nerve for including (-)-Blebbistatin for Mouse Tail-tip fibroblast in embodiment 1-3 The neuron patch-clamp experimental result picture of inducing culture culture after ripening in 7 days.
Fig. 3 A are micro- using cellular morphology of the DMSO medium cultures without any inhibitor after 7 days in embodiment 2 Photo.
Fig. 3 B are that cell shape of the medium culture after 7 days containing 0.4 μM of Cytochalasin B is used in embodiment 2 State microphoto.
Fig. 3 C are that cellular morphology of the medium culture after 3 days containing 2 μM of Cytochalasin B is used in embodiment 2 Microphoto.
Fig. 3 D are that cellular morphology of the medium culture after 7 days containing 0.2 μM of Latrunculin B is used in embodiment 2 Microphoto.
Fig. 3 E are to use the medium culture containing 2 μM of Cytochalasin B after 7 days in embodiment 2, and use nerve Pair for the DMSO that cellular neural member expression neuronal marker protein Map2 after first maturation medium culture 10 days is compareed with use Than figure.
Fig. 4 A are shown in embodiment 3 handles colloid using containing (-)-Blebbistatin processing compared to control group DMSO The variation comparison diagram of oncocyte neuron morphology on day 3.
Fig. 4 B show the antitumor drug of embodiment 3 to glioma cell U87 and U251 inhibition comparison diagram.
Fig. 4 C show the antitumor drug of embodiment 3 with control group DMSO to cell table after glioma cell U87 processing Up to neuronal marker protein Tuj1 situations.
Fig. 5 A are that part target spot strikes low latter 7th day cellular morphology variation diagram in embodiment 4.
Fig. 5 B are that part target spot strikes low rear 7th day cell Tuj1 immunofluorescence dyeing result figure in embodiment 4.
Fig. 6 A are fibroblast light microscopic photo after Culture of neural stem cells liquid suspends and cultivates 7 days in embodiment 5.
Fig. 6 B are immunofluorescence dye of the fibroblast after the suspension of Culture of neural stem cells liquid is cultivated 7 days in embodiment 5 Chromatic graph.
Fig. 6 C are that fibroblast is after Culture of neural stem cells liquid suspends culture 7 days in embodiment 5, then through random differentiation Immunofluorescence dyeing figure after culture solution culture 7 days.
Fig. 6 D are that fibroblast is after Culture of neural stem cells liquid suspends culture 7 days in embodiment 5, then oriented differentiation Immunofluorescence dyeing figure after culture solution culture 7 days.
Fig. 7 A primary astroglial cells forms (left figure) and the labelled protein GFAP (right figure) for expressing astroglia.
Fig. 7 B are (-)-Blebbistatin 13 days aspect graphs of inducing astrocytes in embodiment 6.
Fig. 7 C are (-)-Blebbistatin inducing astrocytes 20 days in embodiment 6, express classical neuron mark Remember albumen Tuj1 figures.
Specific implementation mode
The embodiment and following embodiment are for exemplary purposes, and to have no intention to limit claimed range. Other modifications, purposes or combination in relation to composition of the present invention will be aobvious and easy for those of ordinary skills See, without departing from the spirit and scope of claimed theme.
Embodiment 1-1
(corning, 3335) is per hole 20ug/ml Fibronectin solutions (millipore, fc010) 1 by taking 12 orifice plates as an example × PBS is prepared, and is coated with 6 hours, or first 0.1mg/ml Poly-L-Lysine Solutions (sigma, P6407) sterile water is used to prepare, coating Culture dish 2 hours, sterile washing three times, then with 10 μ g/ml laminin solutions (sigma, L6274) 1 × PBS are prepared, packet By 6 hours.Remove coating buffer to be washed one time with 1 × PBS fliud flushings.
Remove washing lotion, it will be at uniform kind of human foreskin fibroblasts (HFF20y, Beijing stem cell bank) in each hole, often Hole 1 × 104A cell, with basic culture solution (DMEM in high glucose (Gibco, C12430500BT), 1 × Sodium Pyruvate (100 ×, Gibco, 11360-070), 1 × nonessential amino acid (100 ×, Gibco, 11140-050), dual anti-) plus 10% fetal calf serum (Gibco, 16000-044) is cultivated 24 hours.Culture solution is removed, PBS is washed one time.
It includes following inducing culture and maturation culture to select the cell transformation kit of the present invention, the kit Base.
Neuronal induction culture medium (N2B27 culture solutions will be added through treated fibroblast：DMEM/F12 (gibco, 10565018) is mixed with Neurobasal (Gibco, 21103-049) 1 to 1, addition N2 additives (100 ×, Gibco, 17502048), B27 additives (50 ×, Gibco, 17504044), 2% bovine serum albumin (1000 ×, sigma, A8022), beta -mercaptoethanol (1000 ×, Gibco, 21985023), Glutamax (200 ×, Gibco, 35050-061), 1 μ g/ Ml insulin (Roche, 11376497001), dual anti-).Myosin inhibitor (-)-Blebbistatin (dimethyl sulfoxide (DMSO)s are added The dense storages of 100mM of (sigma, D2650) are dissolved, -20 degrees Celsius are kept in dark place January), (-)-Blebbistatin is in Fiber differentiation Final concentration of 15-30 μM in base.The cell of culture 3-7 days, culture has apparent neuron morphology, big cell space and long aixs cylinder Deng and neuron fates marker Tuj1 dyeing, and count Tuj1 positive rates (Tuj1 positive cells/nucleus ratio).
By the cell Jing Guo above-mentioned inducing culture culture be added neuronal maturation culture medium (N2B27 culture mediums, 100 μM N6,2 '-O- dibutyryl 3 ', 5 '-cycli phosphate of adenosine sodium salts (sigma, D0627), 20ng/ml Recombinant Human NT-3 (Peprotech, 450-03), 20ng/ml brain-derived neurotrophic factor (peprotech, 450-02), 20ng/ml The neurotrophic factor of (peprotech, 450-10) Deiter's cells source property) maturation culture 7-14 days.
Above-mentioned experimental implementation is repeated, is handled respectively from 21 weeks, 8 years old, the human foreskin fibroblasts of 13 years old people, 21 weeks back ofs the body of people The mouse of skin fibroblast, monkey tail point fibroblast, mouse fetal fibroblast and non-increment cell mitomycin C processing Fetal fibroblast, the results are shown in Table 1 for final gained Tuj1 positive rates and MAP2 positive rates.
Table 1
As shown in Table 1, using myosin inhibitor (-)-Blebbistatin induced fibroblasts to neuronal cell Transformation situation, when its a concentration of 15 μM relative to Neuronal induction culture medium, although it is thin to achieve good neuron Born of the same parents' changing effect, but and it is not up to best；And when using a concentration of 20 μM -30 μM, neuronal cell conversion ratio reaches highest.
In order to further illustrate the effect of myosin inhibitor (-)-Blebbistatin, attached drawing 1A- is applicant provided Fig. 1 H.
Figure 1A shows into the cellular morphology of human foreskin fibroblasts (HFF20y, Beijing stem cell bank) and Tuj1 is immunized Fluorescent staining picture.
Figure 1B shows the fibroblast for Figure 1A, using the nerve-inducing for including 20 μM of (-)-Blebbistatin The microphoto (left side) and Tuj1 immunofluorescence dyeings picture (right side) of cell of the medium culture after 7 days.As shown in the drawing, it cultivates Cell have an apparent neuron morphology, aixs cylinder of big cell space and length etc. and the Tuj1 dyeing of neuron fates marker, through statistics Tuj1 positive rates (Tuj1 positive cells/nucleus ratio), Tuj1 positive rates are close to 100%.
Fig. 1 C show that the fibroblast for Figure 1A uses the nerve-inducing for including 20 μM of (-)-Blebbistatin After medium culture 7 days, then with the Marker of the expression mature neuron after neuronal maturation medium culture 14 days：Map2 (santa cruz biotechnology, sc-20172), Nf200 (Abcam, ab4680) and NeuN (chemicon, MAB377)。
Fig. 1 D show that the fibroblast for Figure 1A uses the nerve-inducing for including 20 μM of (-)-Blebbistatin After medium culture 7 days, then with the presynaptic of the ripe neuron expression classics after neuronal maturation medium culture 14 days Labelled protein Syn1 (millipore, AB1543P) and Syt1 (abcam, ab133856) and postsynaptic labelled protein PSD95 (abcam, ab18258).
Fig. 1 E show the fibroblast for Figure 1A using the inducing culture training comprising (-)-Blebbistatin After supporting 7 days, then with the ripe neuron patch-clamp experimental result picture after maturation medium culture 14 days.It shows that cell has Sodium current, potassium current and the activity for having certain action potential of mature neuron.
Fig. 1 F show that the fibroblast for Figure 1A uses the nerve-inducing for including 20 μM of (-)-Blebbistatin After medium culture 7 days, then carries out type mark protein staining with the neuron after neuronal maturation medium culture 14 days and connect The labelled protein GABA (sigma, SAB4501067), GAD65/67 (santa cruz of nearly 100% expression GABA energy Biotechnology, sc-7513).
Fig. 1 G show the fibroblast for Figure 1A using the inducing culture training comprising (-)-Blebbistatin After supporting 7 days, then with dopaminergic marker TH (the santa cruz of neuron expression midbrain after maturation medium culture 14 days Biotechnology, sc-14007) and Glutamatergic marker vGlut1 (santa cruz biotechnology, sc- 377425).Which show the neurons of the induction not to express dopaminergic marker TH (the santa cruz of midbrain substantially Biotechnology, sc-14007) and Glutamatergic marker vGlut1 (santa cruz biotechnology, sc- 377425)。
Fig. 1 H show that ultra high efficiency liquid phase-ultrahigh resolution Mass Spectrometer Method display induction GABA neurons respond high potassium buffering Liquid processing release gamma aminobutyric acid.
Figure 1B-Fig. 1 H clearly show that treated by myosin inhibitor (-)-Blebbistatin used in the present invention Fibroblast has high neuron transformation efficiency.
Embodiment 1-2
Using the experimental method of embodiment 1-1, the difference is that the myosin inhibitor used is myosin light chain Kinases (MLCK) inhibitor ML-7.
Human foreskin fibroblasts of the processing from 20 years old HFF (people's foreskin cells) P15,21 weeks/8 years old/13 years old people respectively, people At 21 weeks butt fibroblasts, monkey tail point fibroblast, mouse fetal fibroblast and non-increment cell mitomycin C The mouse fetal fibroblast of reason, the results are shown in Table 2 for final gained Tuj1 positive rates and MAP2 positive rates.
Table 2
As shown in Table 2, using the ML-7 inductions of myosin inhibitor myosin light chain kinase (MLCK) inhibitor at fiber Transformation situation from cell to neuronal cell, when its a concentration of 15 μM relative to Neuronal induction culture medium, although obtain Preferable neuronal cell changing effect, but and it is not up to best；And when using a concentration of 20 μM -30 μM, neuron is thin Dysuria with lower abdominal colic rate reaches highest.
Embodiment 1-3
Using the experimental method of embodiment 1-1, the difference is that process object is Mouse Tail-tip fibroblast.
Fig. 2A shows Mouse Tail-tip fibroblast untreated preceding neuronal marker protein Tuj1 and Map2 dyeing picture. Fig. 2 B are shown uses the Neuronal induction media for including 20 μM of (-)-Blebbistatin for Mouse Tail-tip fibroblast The Marker of neuron is expressed in culture after 7 days：Tuj1, Map2, NF200 and NeuN.
The Neuronal induction media training comprising 20 μM of (-)-Blebbistatin is used for Mouse Tail-tip fibroblast Support 7 days after, then with after maturation medium culture 14 days, measure expression Tuj1, Tuj1/Map2 and Map2/NeuN positive rate difference It is 96%, 96%, 97%.
Fig. 2 C show in embodiment 1-3 and are used comprising 20 μM (-)-for Mouse Tail-tip fibroblast After the Neuronal induction media culture 7 days of Blebbistatin, then with the cell after maturation medium culture 14 days there is nerve Sodium current, potassium current and the activity for having certain action potential that first maturation shows.
This example demonstrates that by myosin inhibitor (-)-Blebbistatin treated mouse tails used in the present invention Sharp fibroblast can be converted into neuronal cell with high conversion.
Embodiment 2
Using the experimental method of embodiment 1-1, the difference is that actin assembling 2 μM of cell pines of inhibitor are respectively adopted Relax element B (CB, sigma, C6762) and 0.2 μM of Latrunculin B (BioVision, 2182-1) replacement myosin inhibitor Induced respectively from people 21 weeks, 8 years old, the human foreskin fibroblasts of 13 years old people, 21 weeks butt fibroblasts of people, monkey tail point at The mouse fetal fibroblast of fibrocyte, mouse fetal fibroblast and non-increment cell mitomycin C processing, final gained Tuj1 positive rates and Map2 positive rate results are as shown in Table 3 and Table 4.
Table 3
As shown in Table 3, inhibitor C ytochalasin B induced fibroblasts are assembled to neuronal cell using actin Transformation situation, when its a concentration of 1.5 μM relative to Neuronal induction culture medium, although it is thin to achieve good neuron Born of the same parents' changing effect, but and it is not up to best；And when using a concentration of 2 μM -3 μM, neuronal cell conversion ratio reaches highest.
Table 4
As shown in Table 4, using actin assembling inhibitor Latrunculin B as induced fibroblast to neuron The transformation situation of cell, when its a concentration of 0.15 μM relative to Neuronal induction culture medium, although achieving good god Through first cell transformation effect, but and it is not up to best；And when using a concentration of 0.2 μM and 0.3 μM, neuronal cell conversion ratio Reach highest.
The effect that inhibitor is assembled in order to further illustrate actin, applicant provides attached drawing 3A-3E.Fig. 3 B-3E are shown For at human foreskin fibroblasts (HFF20y, Beijing stem cell bank), using comprising Cytochalasin B and Latrunculin B carry out the cellular morphology of nerve-inducing.Fig. 3 A are shown using the DMSO culture mediums without any inhibitor Cellular morphology after cultivating 7 days.Wherein showing processed fibroblast not has any neuron morphology to generate.Fig. 3 B are aobvious Show using cellular morphology of the medium culture after 7 days containing 0.4 μM of Cytochalasin B, wherein showing processed Fibroblast slightly has the variation of neuron morphology.Fig. 3 C are shown using the culture medium training containing 2 μM of Cytochalasin B Cellular morphology after supporting 3 days, wherein showing that processed fibroblast has had the variation of apparent neuron morphology.Fig. 3 D are aobvious Shown using cellular morphology of the medium culture after 7 days containing 0.2 μM of Latrunculin B, wherein show it is processed at Fibrocyte almost converts position neuron morphology.Fig. 3 E are shown using the culture medium containing 2 μM of Cytochalasin B After culture 7 days, and cellular neural member of the neuronal maturation medium culture after 10 days is used to express neuronal marker protein Map2, Wherein show the change of the apparent neural destiny form of processed cell.
Embodiment 3
A kind of specific implementation mode that cytoskeletal protein inhibitor is used to prepare to anticancer drug is present embodiments provided, Neuroglial cytoma transdifferentiation can be neuronal cell by the anticancer drug.
Using following reagent configuration antitumor drug I：DMEM/F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1 to 1 is mixed, addition N2 additives (100 ×, Gibco, 17502048), B27 additives (50 ×, Gibco, 17504044), 2% bovine serum albumin (1000 ×, sigma, A8022), beta -mercaptoethanol (1000 ×, Gibco, 21985023), Glutamax (200 ×, Gibco, 35050-061), 1 μ g/ml insulin (Roche, 11376497001) is double It is anti-), and (the dense storages of 100mM of dmso solution (sigma, D2650), -20 degrees Celsius are protected from light (-)-Blebbistatin Preserve January).It is 20 μM that (-)-Blebbistatin concentration, which is adjusted, relative to entire drug I.
Using following reagent configuration antitumor drug II：100 μM of N6,2 '-O- dibutyryls are prepared based on antitumor drug I 3 ', 5 '-cycli phosphate of adenosine sodium salt (sigma, D0627), 20ng/ml Recombinant Human NT-3 (Peprotech, 450-03), 20ng/ml brain-derived neurotrophic factor (peprotech, 450-02), 20ng/ml Deiter's cells source property Neurotrophic factor (peprotech, 450-10).
Above-mentioned antitumor drug I and II priority compounding applications, antitumor drug II is 3-7 days after antitumor drug I applies It is administered again.
People's Malignant glioma cells (U87), star glue are handled using antitumor drug I and the II cooperation of the present embodiment respectively Matter oncocyte (U251), people's Malignant glioma cells (LN229) and people's glioblastoma cells (T98G).Final glioma is thin The form of born of the same parents, growth curve and conversion results are compared to control group (not containing (-)-Blebbistatin i.e. in pharmaceutical agent) Comparative situation is as illustrated in figures 4 a and 4b.Wherein, located respectively using (-)-Blebbistatin containing 20 μM and control group DMSO Manage people's Malignant glioma cells (U87), DBT cell (U251), people's Malignant glioma cells (LN229) and people's colloid Blastoma cell (T98G), each experimental group initiator cell amount are identical.
Fig. 4 A show have on day 3 obviously compared to control group DMSO using (-)-Blebbistatin containing 20 μM Neuron morphology variation, and cell is obviously rare compared with control group.
Further influences of research (-)-Blebbistatin to glioma rate.Optional two plants of colloids respectively Oncocyte U87 and U251 are planted respectively in 48 orifice plates, and cell count is respectively adopted (-)-containing 20 μM per 5000, hole cell Blebbistatin and DMSO processing utilized Cell Counting Kit (CCK-8) per 3 parallel holes of sample at interval of 24 hours CCK-8 kits (sigma, 96992) detect cell viability (cell Proliferation vigor), survey 5 days in total.Specific steps are such as：(1) exist Inoculating cell suspension (100 holes μ L/) in 48 orifice plates；(2) by culture plate put in the incubator preculture for a period of time (37 DEG C, 5% CO2)；(3) 10 μ l CCK solution are added to every hole；(4) absorbance at 450nm is measured with microplate reader.
Fig. 4 B show that the antitumor drug of the present embodiment significantly inhibits the proliferation of glioma cell cell, right at first 5 days It is respectively 50% and 99% that optional glioma cell U87 and U251, which inhibits efficiency,.
Fig. 4 C show that optional fast breeding glioma cell U87 transdifferentiations are point by the antitumor drug of the present embodiment The neuronal cell of change, expresses neuronal marker protein Tuj1, and positive rate is close to 95%.
Embodiment 4
(siRNA strikes the transformation experiment and effect of low cytoskeleton system protein ingredient)
By human foreskin fibroblasts with 1 × 105Density be inoculated in 12 orifice plates, with the Fibroblast cell-culture of 1ml Liquid (DMEM+10%FBS) is cultivated.It is utilized when cell reaches 50-70% degree of convergingRNAiMAX (13778150, invitrogen) kit transfect respectively target position rock1, rock2, mrlc1, mrlc2, mrlc3, myh9, myh10、mrckα、mrckβ、lamina/c、lmnb1、lbr、sun1、sun2、cbx1、cbx3、cbx5、banf1、syne1、 syne2、β-actin.It is as follows：(1) dilution such as sequence SEQ ID NO respectively:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、 SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO: 15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21 siRNA：It is managed in the EP of no RNA enzyme, the serum-free Opti-MEM culture mediums with 100 μ L/ per hole dilute 12pmol SiRNA double-strand (2.5ul), mild mixing；(2) transfection reagent is dilutedRNAiMAX Reagent：In nothing The EP of RNA enzyme is managed, with 100 μ L/ per the dilution of hole of serum-free Opti-MEM culture mediumsRNAiMAX Reagent, mild mixing；(3) it has dilutedRNAiMAX Reagent after the incubation of 2min, The siRNA diluted with above-mentioned (2) is gently mixed, and incubated at room temperature 20min is to form RNAiMAX Reagent mixtures, solution might have muddiness, but not interfere with.(4) mixed liquor of above-mentioned (3) is added It in tissue culture plate containing cell and culture solution, jiggles, is allowed to mix；(5) culture plate is placed in 37 DEG C of CO2Training It supports and is cultivated in case, transfection changes neuronal cultured solution into after 6-8 hours.(6) it transfects 48-72 hours.
Table 5 shows that siRNA sequence strikes the correspondence of low gene expression with it.
Table 5
Gene name NCBI ID It is numbered in sequence table Sequence
rock1 6093 SEQ ID NO:1 gguuagaaca agagguaaa
rock2 9475 SEQ ID NO:2 ggaucgaacc cauggauca
mrlc1 10398 SEQ ID NO:3 ucgcaauggu ugaccaguc
mrlc2 103910 SEQ ID NO:4 gccucuucuu uuugaugua
mrlc3 10627 SEQ ID NO:5 ggucuauaca gagucaaua
myh9 4627 SEQ ID NO:6 ggguaucaau gugaccgau
myh10 4628 SEQ ID NO:7 gggcaacucu acaaagaau
mrckα 8476 SEQ ID NO:8 cgagaagacu uugaaauaa
mrckβ 9578 SEQ ID NO:9 cgagaagacu uugaaauaa
lamina/c 4000 SEQ ID NO:10 gaaggagggu gaccugaua
lmnb1 4001 SEQ ID NO:11 cgagcauccu caagucgua
lbr 3930 SEQ ID NO:12 ggccgacauu aaggaagca
sun1 23353 SEQ ID NO:13 cagcuuuuag uaucaacca
sun2 25777 SEQ ID NO:14 gacucagaag accucuuca
cbx1 10951 SEQ ID NO:15 ggaagggauu cucagauga
cbx3 11335 SEQ ID NO:16 ugacaaacca agaggauuu
cbx5 23468 SEQ ID NO:17 uaaacccagg gagaaguca
banf1 8815 SEQ ID NO:18 uggccaguuu cuggugcua
syne1 23345 SEQ ID NO:19 gguagaacgu caaccucaa
syne2 23224 SEQ ID NO:20 gaacgagucu gauuugaua
β-actin 60 SEQ ID NO:21 gcauccacga aacuaccuu
Any target spot strike it is low after the variation for having apparent neuromorphic in the 7th day, which part target spot strike it is low after cell shape State variation diagram is as shown in Figure 5A.The differential protein of Fig. 5 B immunofluorescence dyeing results display the 7th day expression neuron fates of induction Tuj1。
Neuronal culture (N2B27 culture solutions：DMEM/F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1 to 1 mixing, addition N2 additives (100 ×, Gibco, 17502048), B27 additives (50 ×, Gibco, 17504044), 2% bovine serum albumin (1000 ×, sigma, A8022), beta -mercaptoethanol (1000 ×, Gibco, 21985023), Glutamax (200 ×, Gibco, 35050-061), 1 μ g/ml insulin (Roche, 11376497001) is double It is anti-).
Embodiment 5
(interference extracellular matrix handles the low transformation experiment sticked and effect)
1% agarose solution is configured with aseptic double-distilled water first, micro-wave oven is heated to boiling, and isometric 2 are then added × DMEM, (Gibco, 12800-017) are made 0.5% agarose solution being dissolved in DMEM, are subsequently poured into the culture dish of 6cm In, it is spare after cooled and solidified.
Human foreskin fibroblasts are inoculated in 1 × 105 density in above-mentioned culture dish, with Culture of neural stem cells liquid Suspend culture 7 days, and half amount changes liquid every other day.The light microscopic photo after cultivating the 7th day that suspends is shown in Fig. 6 A.Wherein have shown that neuron Form.
It is placed in the circle glass in four orifice plates with 1 × PBS processing of the fibronectin (Millipore, FC010) containing 1ug/ml Piece, be placed in incubator be coated with overnight it is spare.It suspends after the human fibroblasts cultivated the 7th day centrifuge and uses Tryple (Gibco, A1285901) digests 3min in 37 DEG C of incubators, is centrifuged after being diluted with PBS, is trained with neural stem cell after abandoning supernatant Nutrient solution is inoculated in every hole 5 × 104 in four orifice plates after being resuspended, and next day removes culture solution, carries out immunofluorescence dyeing, specifically such as Under：
In under room temperature, 20min is fixed with the PBS solution containing 4% paraformaldehyde (Sigma, 158127), PBS washes 3 It is secondary, each 5min.Then containing 0.3%TritonX-100 (Solarbio, T8200) and 0.2%BSA (Sigma, A3803) PBS solution in penetrating 1h.Then at 4 DEG C, containing 2% BSA and primary antibody：The anti-Nestin of mouse (Millipore, MAB353,1:100), goat-anti-Sox2 (Santa, sc-17320,1:100), the anti-Nkx2.2 of mouse (abcam, ab187375) rabbit Anti- En1 (abcam, ab70993,1:50), rabbit-anti-N-cadherin (abcam, ab12221,1:100), rabbit-anti-Pax6 (abcam,ab5790,1:50) it is incubated overnight in PBS solution.Then, cell is washed three times with PBS solution, and in room temperature item Under part, with anti-rabbit, anti-mouse or anti-sheep secondary antibody, Alexa Fluor-488 or Alexa Fluor-561 (1:500, Invitrogen it) is incubated 1 hour.Then wash cell three times with PBS solution, then with containing Hoechst (Inventgen, H3570,1:1000) PBS solution dye nucleus 5min.Then circle slide is tipped upside down on drop has the glass slide of anti-fluorescence quenching On, nail sheet for oil seal.Followed by Leica two-photon confocal microscopy cells.Testing result is shown in Fig. 6 B, wherein Merge indicates coincidence pattern.
Fig. 6 B's the result shows that, through it is too low stick suspend culture human foreskin fibroblasts can be converted into Nestin and The neural stem cell of the bis- positives of Sox2.Further, these double positive cells are to Nkx2,2, En1, N-cad and Pax6 be also in sun Property.
The neural stem cell of above-mentioned identification was similarly inoculated at the 7th day in four orifice plates according to the method described above, random differentiation Group random differentiation culture solution culture, the directed differentiation culture solution culture of directed differentiation group, partly amount changes liquid every other day, carries out within the 14th day Immunofluorescence dyeing.Wherein primary antibody is rabbit-anti-Tuj1 (abcam, ab18207,1:2000), the anti-GFAP of mouse (sigma, G3893, 1:100), chicken anti-NF2000 (abcam, ab4680,1:1000).Testing result is shown in 6C and 6D respectively.
Fig. 6 C's the result shows that, it is low to stick the obtained neural stem cell of processing by random differentiation GFAP to can be obtained positive Star spongiocyte, these further cells are negative to Tuj1.
Fig. 6 D's the result shows that, it is low stick the obtained neural stem cell of processing by directed differentiation can be obtained Tuj1 with The neuron of the bis- positives of NF2000.
Basic culture solution
By DMEM/F12 (Gibco, 12400-024) and Neurobasal (Gibco, 21103-049) by volume 1:1 system At, and add 100 × N2 (Gibco, 17502-048), 50 × B27 (Gibco, 17504-044), 100 × GlutaMAX (Gibco, 35050-079,1000 × β-mercaptoethanol (Gibco, 21985), 1000 × 2%BSA (sigma, A7906-100G), 1000 × Insulin (Roche applied science, 11376497001,10mg/mL), 100 × SP (Gibco,15140-122)。
Culture of neural stem cells liquid
The bFGF (epidermal growth factor, R＆D, 233-FB-001MG/CF) that 20ng/ml is added by basic culture solution is prepared.
Random differentiation culture solution
It is prepared by basic culture solution addition 1%FBS (fetal calf serum, Gibco, 16000-044).
Directed differentiation culture solution
Basic culture solution adds the GDNF (peprotech, 450-10), the BDNF of 20ng/ml of 20ng/ml (peprotech, 450-02), the NT3 (peprotech, 450-03) of 20ng/ml, 4ng/ml Forskolin (stemgent, 04-0025) prepare.
Embodiment 6
(primary astroglial cells transdifferentiation is neuron)
(corning, 3335) is per hole 20ug/ml Fibronectin solutions (millipore, fc010) 1 by taking 12 orifice plates as an example × PBS is prepared, and is coated with 6 hours, or first 0.1mg/ml Poly-L-Lysine Solutions (sigma, P6407) sterile water is used to prepare, coating Culture dish 2 hours, sterile washing three times, then with 10 μ g/ml laminin solutions (sigma, L6274) 1 × PBS are prepared, packet By 6 hours.Remove coating buffer to be washed one time with 1 × PBS fliud flushings.
Remove washing lotion, by the original of primary astroglial cells (ScienCell, 1800) or Cerebral Cortex separation of being grown up For uniform kind of Deiter's cells in each hole, with basic culture solution (DMEM in high glucose (Gibco, C12430500BT), 1 × the third Ketone acid sodium (100 ×, gibco, 11360-070), 1 × nonessential amino acid (100 ×, Gibco, 11140-050), dual anti-) plus 10% fetal calf serum (Gibco, 16000-044) is cultivated 24 hours.Culture solution is removed, PBS is washed one time.
It includes following inducing culture and maturation culture to select the cell transformation kit of the present invention, the kit Base.
Neuronal induction culture medium (N2B27 culture solutions will be added through treated Deiter's cells：DMEM/F12 (gibco, 10565018) is mixed with Neurobasal (Gibco, 21103-049) 1 to 1, addition N2 additives (100 ×, Gibco, 17502048), B27 additives (50 ×, Gibco, 17504044), 2% bovine serum albumin (1000 ×, sigma, A8022), beta -mercaptoethanol (1000 ×, Gibco, 21985023), Glutamax (200 ×, Gibco, 35050-061), 1 μ g/ Ml insulin (Roche, 11376497001), dual anti-).Myosin inhibitor (-)-Blebbistatin (dimethyl sulfoxide (DMSO)s are added The dense storages of 100mM of (sigma, D2650) are dissolved, -20 degrees Celsius are kept in dark place January), (-)-Blebbistatin is in Fiber differentiation Final concentration of 15-30 μM in base.The cell of culture 3-7 days, culture has apparent neuron morphology, big cell space and long aixs cylinder Deng and neuron fates marker Tuj1 dyeing, and count Tuj1 positive rates (Tuj1 positive cells/nucleus ratio).
Fig. 7 A show primary astroglial cells form (left figure) and express the labelled protein GFAP of astroglia (right figure).As shown in Figure 7 B, (-)-Blebbistatin induces 13 days astroglias to show as classical neuron morphology. As seen in figure 7 c, (-)-Blebbistatin inducing astrocytes 20 days, close to the classical neural meta-tag egg of 100% expression White Tuj1.
Sequence table
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Claims (15)

1. it is a kind of by non-neuronal cell transdifferentiation be neuronal cell method, it is characterised in that the method includes to described Extracellular matrix-shell system of non-neuronal cell carries out interference processing.
2. according to the method described in claim 1, the wherein described interference handles at least one selected from the following：Using cytoskeleton Protein inhibitor is handled, and to the gene expression of extracellular matrix-shell system strike using siRNA (siRNA) low Processing carries out low adhesion process to extracellular matrix and trains for a certain area.
3. according to the method described in claim 2, the wherein described cytoskeletal protein inhibitor at least one selected from the following：Flesh Globulin (myosin) inhibitor, actin (actin) assemble inhibitor；
Preferably, wherein the myosin (myosin) inhibitor at least one selected from the following：(-)-Blebbistatin、 Myosin light chain kinase (MLCK) inhibitor ML-7, myosin (myosin) inhibitor concentration is 10 μM or more, excellent Select 20 μM or more, more preferable 10-30 μM, wherein a concentration of myosin (myosin) inhibitor is handling non-nerve Concentration in inducing culture used in first cell；
Preferably, wherein the actin (actin) assembles inhibitor at least one selected from the following：Cytochalasin B, Latrunculin B, wherein a concentration of 1.5 μM or more of Cytochalasin B, preferably 2 μM or more, more preferable 2-3 μM, A concentration of 0.15 μM of Latrunculin B or more, preferably 0.2 μM, more preferable 0.2-0.3 μM, wherein a concentration of suppression Concentration in formulation C ytochalasin B or Latrunculin B inducing cultures used in processing non-neuronal cell.
4. according to the method described in claim 3, wherein the method includes non-neuronal cell is placed in inducing culture Then culture 3-7 days uses maturation medium culture 7-14 days；
Preferably, wherein the inducing culture includes：The cytoskeletal protein inhibitor, N2 cell culture based additives, B27 cell culture based additives, glutamine, β mercaptoethanols；
Preferably, wherein the maturation medium includes：N2 cell culture based additives, B27 cell culture based additives, paddy ammonia Amide, β mercaptoethanols, neurenergen (NT3), brain-derived neurotrophic factor (BDNF), nerve derived from glial cell line Trophic factors (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
5. method according to any one of claims 1 to 4, wherein the non-neuronal cell be fibroblast and/or Deiter's cells.
6. cytoskeletal protein inhibitor is used for the purposes for neuronal cell by non-neuronal cell transdifferentiation.
7. it is a kind of by non-neuronal cell transdifferentiation be neuronal cell kit, it is characterised in that the kit include lure Culture medium is led, the inducing culture includes cytoskeletal protein inhibitor.
8. kit according to claim 7, wherein the cytoskeletal protein inhibitor at least one selected from the following： Myosin (myosin) inhibitor, actin (actin) assemble inhibitor；
Preferably, wherein the myosin (myosin) inhibitor at least one selected from the following：(-)-Blebbistatin、 Myosin light chain kinase (MLCK) inhibitor ML-7, a concentration of 10 μM or more, preferably 20 μM or more, more preferable 10-30 μM, Described in concentration of a concentration of myosin (myosin) inhibitor in inducing culture；
Preferably, wherein the actin (actin) assembles inhibitor at least one selected from the following：Cytochalasin B, Latrunculin B, wherein a concentration of 1.5 μM or more of Cytochalasin B, preferably 2 μM or more, more preferable 2-3 μM, A concentration of 0.15 μM of Latrunculin B or more, preferably 0.2 μM, more preferable 0.2-0.3 μM, wherein a concentration of suppression Concentration of formulation C ytochalasin B or the Latrunculin B in inducing culture.
9. kit according to claim 7 or 8, wherein the kit further includes maturation medium,
Preferably, wherein the inducing culture also includes：N2 cell culture based additives, B27 cell culture based additives, paddy Glutamine, β mercaptoethanols；
Preferably, wherein the maturation medium includes：N2 cell culture based additives, B27 cell culture based additives, paddy ammonia Amide, β mercaptoethanols, neurenergen (NT3), brain-derived neurotrophic factor (BDNF), nerve derived from glial cell line Trophic factors (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
10. method according to claim 7 or 8, wherein the non-neuronal cell is fibroblast and/or neuroglia Cell plastid.
11. purposes of the cytoskeletal protein inhibitor in preparing antitumor drug, regeneration and/or repair medicine.
12. according to the method described in claim 2, it includes at least one below to strike reduction process described in wherein：
Using with sequence SEQ ID NO:1 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes rock1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:2 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes rock2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:3 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes mrlc1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:4 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes mrlc2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:5 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes mrlc3 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:6 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes myh9 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:7 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes myh10 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:8 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes mrck α gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:9 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes mrck beta gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:10 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes lamina/c gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:11 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes lmnb1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:12 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes lbr gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:13 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes sun1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:14 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes sun2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:15 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes cbx1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:16 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes cbx3 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:17 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes cbx5 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:18 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes banf1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:19 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes syne1 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:20 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes syne2 gene expressions in low extracellular matrix-shell system,
Using with sequence SEQ ID NO:21 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity It disturbs RNA (siRNA) and strikes β-actin gene expressions in low extracellular matrix-shell system.
13. according to the method for claim 12, wherein described to strike reduction process preferred：
Using with sequence SEQ ID NO:1 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity Disturb RNA (siRNA) and strike rock1 gene expressions in low extracellular matrix-shell system, using with sequence SEQ ID NO:2 have 95%, the siRNA (siRNA) of 96%, 97%, 98%, 99% or 100% sequence identity strikes low extracellular matrix-skeleton Rock2 gene expressions in system, using with sequence SEQ ID NO:8 have 95%, 96%, 97%, 98%, 99% or The siRNA (siRNA) of 100% sequence identity strikes the mrck α gene expressions in low extracellular matrix-shell system, and Using with sequence SEQ ID NO:9 siRNAs with 95%, 96%, 97%, 98%, 99% or 100% sequence identity (siRNA) the mrck beta gene expressions in low extracellular matrix-shell system are struck；
Using with sequence SEQ ID NO:3 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity Disturb RNA (siRNA) and strike mrlc1 gene expressions in low extracellular matrix-shell system, using with sequence SEQ ID NO:4 have 95%, the siRNA (siRNA) of 96%, 97%, 98%, 99% or 100% sequence identity strikes low extracellular matrix-skeleton Mrlc2 gene expressions in system, and use and sequence SEQ ID NO:5 have 95%, 96%, 97%, 98%, 99% or The siRNA (siRNA) of 100% sequence identity strikes the mrlc3 gene expressions in low extracellular matrix-shell system；
Using with sequence SEQ ID NO:6 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity Disturb RNA (siRNA) and strike myh9 gene expressions in low extracellular matrix-shell system, and using and sequence SEQ ID NO:7 tools There is the siRNA (siRNA) of 95%, 96%, 97%, 98%, 99% or 100% sequence identity to strike low extracellular matrix-bone Myh10 gene expressions in frame system；
Using with sequence SEQ ID NO:3 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity Disturb RNA (siRNA) and strike mrlc1 gene expressions in low extracellular matrix-shell system, using with sequence SEQ ID NO:4 have 95%, the siRNA (siRNA) of 96%, 97%, 98%, 99% or 100% sequence identity strikes low extracellular matrix-skeleton Mrlc2 gene expressions in system, using with sequence SEQ ID NO:5 have 95%, 96%, 97%, 98%, 99% or The siRNA (siRNA) of 100% sequence identity strikes the mrlc3 gene expressions in low extracellular matrix-shell system, and Using with sequence SEQ ID NO:6 siRNAs with 95%, 96%, 97%, 98%, 99% or 100% sequence identity (siRNA) the myh9 gene expressions in low extracellular matrix-shell system are struck；Or
Using with sequence SEQ ID NO:13 is small dry with 95%, 96%, 97%, 98%, 99% or 100% sequence identity Disturb RNA (siRNA) and strike sun1 gene expressions in low extracellular matrix-shell system, and using and sequence SEQ ID NO:14 SiRNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity strikes low extracellular matrix- Sun2 gene expressions in shell system.
14. method according to claim 12 or 13, the reduction process of striking include：
Diluted liposome and the siRNA (siRNA) mixing are formed into mixture；
The mixture is mixed evenly with the culture solution containing non-neuronal cell and transfected, cultivated.
15. according to the method described in claim 2, the wherein described low adhesion process includes non-using agarose DMEM suspension cultures Neuronal cell；
Preferably, suspension incubation time is 6 days, 7 days or 8 days；
Preferably, the dosage of the agarose is the 0.5g/100 milliliters of agarose DMEM；
Preferably, after non-neuronal cell is using agarose DMEM suspension cultures, then directed differentiation culture solution culture is used；
Preferably, the directed differentiation culture solution includes：The neurotrophic factor (GDNF) of Deiter's cells source property, brain source property Neurotrophic factor (BDNF), neurotrophic factor -3 (NT3) and forskolin (Forskolin)；
Preferably, neurotrophic factor (GDNF) a concentration of 15- of directed differentiation culture solution mesoglia cells source property 25ng/ml, preferably 20ng/ml, brain-derived neurotrophic factor (BDNF) a concentration of 15-25ng/ml, preferably 20ng/ml, nerve Trophic factors -3 (NT3) a concentration of 15-25ng/ml, preferably 20ng/ml, forskolin (Forskolin) a concentration of 2-6ng/ml, It is preferred that 3,4 or 5ng/ml.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110408594A (en) * 2019-07-31 2019-11-05 吉林大学 A method of human fibroblasts are efficiently largely reprogrammed as mature neuron
CN113583962A (en) * 2021-07-30 2021-11-02 新乡医学院 Induction culture medium and method for inducing and obtaining cancer cell persistent infection NDV
WO2022037570A1 (en) * 2020-08-17 2022-02-24 中国科学院动物研究所 Expansion culture medium and culture method for neural cells
WO2022077549A1 (en) * 2020-10-14 2022-04-21 中国科学院动物研究所 Composition and method for transdifferentiating non-neuronal cells into neurons

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2020290509A1 (en) 2019-06-14 2021-11-11 Regeneron Pharmaceuticals, Inc. Models of tauopathy
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101104615A (en) * 2006-07-13 2008-01-16 陈正书 Anticancer cytoskeleton inhibiting and cell elongation inducing compound and synthetic method thereof
CN101125146A (en) * 2007-07-02 2008-02-20 中国药科大学 Medicine target for preventing and treating cardiovascular and cerebrovascular diseases associated with inflammation and its inhibitor
CN102803473A (en) * 2009-05-27 2012-11-28 先进细胞技术公司 Genetically intact induced pluripotent cells or transdifferentiated cells and methods for the production thereof
CN102940631A (en) * 2012-11-02 2013-02-27 清华大学 Application of Blebbistatin in promoting stem cell survival and keeping stem cell dry
CN104178456A (en) * 2013-07-03 2014-12-03 深圳市三启生物技术有限公司 Passage method and application of human induced pluripotent stem cells
CN104195108A (en) * 2014-07-29 2014-12-10 深圳市三启生物技术有限公司 Use of kinase inhibitor for preparing nerve cells from non-nerve cells
CN106574248A (en) * 2014-08-19 2017-04-19 细胞动力国际有限公司 Neural networks formed from cells derived from pluripotent stem cells

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1293924C (en) * 2003-09-02 2007-01-10 中国人民解放军第四军医大学口腔医学院 Tissue engineering peripheral nerve used for repairing peripheral nerve defect and its preparation method
AR057252A1 (en) * 2005-12-27 2007-11-21 Alcon Mfg Ltd INHIBITION OF RHO KINASE MEDIATED BY ARNI FOR THE TREATMENT OF EYE DISORDERS
US9528087B2 (en) * 2009-10-31 2016-12-27 Genesis Technologies Limited Methods for reprogramming cells and uses thereof
CN103865875B (en) * 2012-12-18 2016-08-10 中国科学院遗传与发育生物学研究所 A kind of method preparing neural stem cell with fibroblast
AU2014253960B2 (en) * 2013-04-16 2020-06-18 Memorial Sloan-Kettering Cancer Center Age-modified cells and methods for making age-modified cells
GB201318126D0 (en) * 2013-10-14 2013-11-27 Univ Cardiff Neuronal Stem Cell Differentiation
KR20150062817A (en) * 2013-11-29 2015-06-08 가톨릭대학교 산학협력단 Method of inducing differentiation of mesenchymal stem cell derived from chorion or warthon's jelly isolated from human term placenta into neuron and hair cell
CN105950557B (en) * 2016-04-22 2019-10-25 中国科学院生物物理研究所 It is a kind of to control the signal path and its application that human nerve cell reprograms

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101104615A (en) * 2006-07-13 2008-01-16 陈正书 Anticancer cytoskeleton inhibiting and cell elongation inducing compound and synthetic method thereof
CN101125146A (en) * 2007-07-02 2008-02-20 中国药科大学 Medicine target for preventing and treating cardiovascular and cerebrovascular diseases associated with inflammation and its inhibitor
CN102803473A (en) * 2009-05-27 2012-11-28 先进细胞技术公司 Genetically intact induced pluripotent cells or transdifferentiated cells and methods for the production thereof
CN102940631A (en) * 2012-11-02 2013-02-27 清华大学 Application of Blebbistatin in promoting stem cell survival and keeping stem cell dry
CN104178456A (en) * 2013-07-03 2014-12-03 深圳市三启生物技术有限公司 Passage method and application of human induced pluripotent stem cells
CN104195108A (en) * 2014-07-29 2014-12-10 深圳市三启生物技术有限公司 Use of kinase inhibitor for preparing nerve cells from non-nerve cells
CN106574248A (en) * 2014-08-19 2017-04-19 细胞动力国际有限公司 Neural networks formed from cells derived from pluripotent stem cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ADAM P. CROFT,ET AL.: "Formation of neurons by non-neural adult stem cells: Potential mechanism implicates an artifact of growth in culture", 《STEM CELLS》 *
BIRGIT NEUHUBE，ET AL.: "Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype", 《JOURNAL OF NEUROSCIENCE RESEARCH》 *
KYEONGMI KIM,ET AL.: "Neural Crest Specification by Inhibition of the ROCK/Myosin II Pathway", 《STEM CELLS》 *
SONEELA ANKAM: "GEOMETRY AND SIZE OF SUBSTRATE TOPOGRAPHY GUIDES THE NEURAL DIFFERENTIATION OF HUMAN EMBRYONIC STEMCELLS", 《新加坡国立大学博士论文》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110408594A (en) * 2019-07-31 2019-11-05 吉林大学 A method of human fibroblasts are efficiently largely reprogrammed as mature neuron
WO2022037570A1 (en) * 2020-08-17 2022-02-24 中国科学院动物研究所 Expansion culture medium and culture method for neural cells
CN115916240A (en) * 2020-08-17 2023-04-04 中国科学院动物研究所 Amplification culture medium and culture method of neural cells
WO2022077549A1 (en) * 2020-10-14 2022-04-21 中国科学院动物研究所 Composition and method for transdifferentiating non-neuronal cells into neurons
CN113583962A (en) * 2021-07-30 2021-11-02 新乡医学院 Induction culture medium and method for inducing and obtaining cancer cell persistent infection NDV

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