WO2018157769A1 - 一种将非神经元细胞转化为神经元细胞的方法 - Google Patents

一种将非神经元细胞转化为神经元细胞的方法 Download PDF

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WO2018157769A1
WO2018157769A1 PCT/CN2018/077192 CN2018077192W WO2018157769A1 WO 2018157769 A1 WO2018157769 A1 WO 2018157769A1 CN 2018077192 W CN2018077192 W CN 2018077192W WO 2018157769 A1 WO2018157769 A1 WO 2018157769A1
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sirna
extracellular matrix
seq
sequence identity
interfering rna
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周琪
李伟
胡宝洋
何正泉
王柳
郝捷
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中国科学院动物研究所
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for transforming non-neuronal cells of humans and animals into functional neurons by interfering with the extracellular matrix-skeletal system of non-neuronal cells.
  • Cell fate depends on the specific expression of the genome, and the expression regulation includes common biological regulation, such as signal transduction, transcriptional regulatory networks, epigenetic modification, etc., as well as the physical and chemical properties of the cells and the environment in which the cells are located. Regulation of physical and chemical factors. Therefore, the method of cell fate transformation is also divided into two types, namely, changing the biological characteristics and physical and chemical properties of cells.
  • the method for producing functional cells by regulating cell fate is mainly directed to several important gene regulatory pathways and epigenetic modifications, which are accomplished by genetic means or chemical small molecule processing.
  • Genetics include the use of ectopic expression of transcription factors such as Oct4, Sox2, c-Myc, and Klf4 to reprogram mouse and human fibroblasts into induced pluripotent stem cells; ectopic expression of Adcl1, Brn2, and Myt1l
  • the transcription factor converts fibroblasts into functional neurons; and ectopically expresses specific genes to obtain functional cardiomyocytes, islet cells, and the like.
  • Chemical small molecule means that the researchers used the chemical small molecule combination VC6TFZ to reprogram mouse fibroblasts into pluripotent stem cells; using seven small molecule combinations (VCRFSGY) to directly convert human fibroblasts into neurons; Mouse fibroblasts were reprogrammed into neural stem cells using a combination of 9 small molecules, and then differentiated into functional neurons; human gastric epithelial cells were transformed into pluripotent endoderm progenitor cells using small molecule combinations; small molecules were utilized The combination converts human fibrils into small cells into cardiomyocytes and the like.
  • VCRFSGY seven small molecule combinations
  • the present invention mainly aims at regulating cell fate by interfering with the extracellular matrix-skeleton system of non-neuronal cells, in particular, in transducing non-neuronal cells of human or animal into neuronal cells. A more simple and easy way to achieve technological, unexpected technical effects.
  • the present invention provides a method of converting non-neuronal cells into neuronal cells, characterized in that the method comprises interference treatment of an extracellular matrix-skeletal system of non-neuronal cells.
  • the interference treatment of the present invention is selected from at least one of the following: treatment with a cytoskeletal protein inhibitor, low-interference RNA (siRNA) for knockdown of the gene expression of the extracellular matrix-skeletal system, and low for the extracellular matrix Adhesion treatment.
  • a cytoskeletal protein inhibitor low-interference RNA (siRNA) for knockdown of the gene expression of the extracellular matrix-skeletal system
  • siRNA low-interference RNA
  • Orientation culture is carried out by medium by treatment with a cytoskeletal protein inhibitor or by knockdown of gene expression of the extracellular matrix-skeletal system with small interfering RNA (siRNA) or after low adhesion treatment to the extracellular matrix.
  • the directional culture can be carried out using the following maturation medium or directional differentiation medium or the like.
  • the cytoskeletal protein inhibitor is at least one selected from the group consisting of a myosin inhibitor and an actin assembly inhibitor.
  • the myosin inhibitor is at least one selected from the group consisting of: (-)-Blebbistatin, myosin light chain kinase (MLCK) inhibitor ML-7, at a concentration of 10 ⁇ M or more, preferably 20 ⁇ M. Above, more preferably 10-30 ⁇ M, wherein the concentration is the final concentration of the inhibitor in the induction medium used for treating non-neuronal cells.
  • MLCK myosin light chain kinase
  • the actin assembly inhibitor is at least one selected from the group consisting of Cytochalasin B, Latrunculin B, wherein the concentration of Cytochalasin B is 1.5 ⁇ M or more, preferably 2 ⁇ M or more, more preferably 2-3 ⁇ M, Latrunculin The B concentration is 0.15 ⁇ M or more, preferably 0.2 ⁇ M, more preferably 0.2 to 0.3 ⁇ M, wherein the concentration is the final concentration of the inhibitor in the induction medium for treating non-neuronal cells.
  • the method comprises culturing the non-neuronal cells in an induction medium for 3-7 days, optionally 4 days, 5 days or 6 days, and then culturing with the maturation medium 7-14 Days, optionally 8 days, 9 days, 10 days, 11 days, 12 days or 13 days.
  • the induction medium comprises: a cytoskeletal protein inhibitor, an N2 cell culture medium additive, a B27 cell culture medium additive, glutamine, ⁇ -mercaptoethanol.
  • the maturation medium comprises: N2 cell culture medium additive, B27 cell culture medium additive, glutamine, ⁇ -mercaptoethanol, neurotrophin (NT3), brain-derived neurotrophic factor (BDNF), glial cells Derived neurotrophic factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
  • N2 cell culture medium additive B27 cell culture medium additive
  • glutamine ⁇ -mercaptoethanol
  • neurotrophin NT3
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cells Derived neurotrophic factor
  • db-cAMP dibutyryl cyclic adenosine monophosphate
  • the non-neuronal cells of the invention are preferably fibroblasts and/or glial cells.
  • the invention also provides the use of a cytoskeletal protein inhibitor for the transdifferentiation of non-neuronal cells into neuronal cells.
  • the invention also provides a kit for converting non-neuronal cells into neuronal cells, the kit comprising an induction medium comprising a cytoskeletal protein inhibitor.
  • the cytoskeletal protein inhibitor is at least one selected from the group consisting of a myosin inhibitor and an actin assembly inhibitor.
  • the myosin inhibitor is at least one selected from the group consisting of: (-)-Blebbistatin, myosin light chain kinase (MLCK) inhibitor ML-7, at a concentration of 10 ⁇ M or more, preferably 20 ⁇ M. Above, more preferably 10-30 ⁇ M, wherein the concentration is the concentration of the inhibitor in the induction medium.
  • MLCK myosin light chain kinase
  • the actin assembly inhibitor is at least one selected from the group consisting of Cytochalasin B, Latrunculin B, wherein the concentration of Cytochalasin B is 1.5 ⁇ M or more, preferably 2 ⁇ M or more, more preferably 2-3 ⁇ M, Latrunculin The B concentration is 0.15 ⁇ M or more, preferably 0.2 ⁇ M, more preferably 0.2-0.3 ⁇ M, wherein the concentration is the concentration of the inhibitor in the induction medium.
  • the kit further comprises a maturation medium.
  • the induction medium comprises: a cytoskeletal protein inhibitor, an N2 cell culture medium additive, a B27 cell culture medium additive, glutamine, ⁇ -mercaptoethanol.
  • the maturation medium comprises: N2 cell culture medium additive, B27 cell culture medium additive, glutamine, ⁇ -mercaptoethanol, neurotrophin (NT3), brain-derived neurotrophic factor (BDNF), glial cells Derived neurotrophic factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
  • N2 cell culture medium additive B27 cell culture medium additive
  • glutamine ⁇ -mercaptoethanol
  • neurotrophin NT3
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cells Derived neurotrophic factor
  • db-cAMP dibutyryl cyclic adenosine monophosphate
  • the invention also provides the use of a cytoskeletal protein inhibitor for the preparation of an anti-tumor drug, tissue regeneration and/or repair drug.
  • the knockdown processing of the present invention includes at least one of the following:
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 1 knockdown of the rock1 gene expression in the extracellular matrix-skeletal system
  • siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 2 knockdown of the rock2 gene expression in the extracellular matrix-skeletal system
  • Murtc1 gene expression in the extracellular matrix-skeletal system was knocked down by siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO:3,
  • siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 4 knockdown of the mrlc2 gene expression in the extracellular matrix-skeletal system
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 5 knockdown of the mrlc3 gene expression in the extracellular matrix-skeletal system
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 6 knocks down myh9 gene expression in the extracellular matrix-skeletal system
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 7 knockdown myh10 gene expression in the extracellular matrix-skeletal system
  • Mrck ⁇ gene expression in the extracellular matrix-skeleton system was knocked down by siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO:8,
  • Mrck ⁇ gene expression in the extracellular matrix-skeleton system is knocked down by siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO:9,
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 11 knockdown the lmnb1 gene expression in the extracellular matrix-skeletal system
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 12, knockdown the expression of the lbr gene in the extracellular matrix-skeletal system,
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 13 knocks down the expression of the sun1 gene in the extracellular matrix-skeletal system
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 14 knocks down the sun2 gene expression in the extracellular matrix-skeletal system
  • siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 18 knockdown of banf1 gene expression in the extracellular matrix-skeletal system
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 19 knockdown of the syne1 gene expression in the extracellular matrix-skeletal system
  • siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 20 knockdown of the syne2 gene expression in the extracellular matrix-skeletal system
  • the ⁇ -actin gene expression in the extracellular matrix-skeleton system was knocked down by siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO:21.
  • siRNA is used to knock down the rock1 in the extracellular matrix-skeletal system with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 1.
  • Gene expression using the siRNA knockdown of the extracellular matrix-skeleton system with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, using Sequence SEQ ID NO: 8 has a 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the siRNA knockdown of the mrck ⁇ gene expression in the extracellular matrix-skeletal system, and the sequence and SEQ ID NO :9 siRNA knockdown of the mrck ⁇ gene in the extracellular matrix-skeleton system with 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • a siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 3 is used to knock down the mrlc1 in the extracellular matrix-skeletal system Gene expression, knockdown of the mrlc2 gene expression in the extracellular matrix-skeleton system with siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 4, and miRNA knockdown in the extracellular matrix-skeleton system with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 5.
  • siRNA is used to knock down the myh9 in the extracellular matrix-skeletal system with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO:6.
  • a siRNA having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 3 is used to knock down the mrlc1 in the extracellular matrix-skeletal system Gene expression, using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 to knock down mrlc2 gene expression in the extracellular matrix-skeletal system, using Sequence SEQ ID NO: 5 has a 95%, 96%, 97%, 98%, 99% or 100% sequence identity in the siRNA knockdown extracellular matrix-skeletal system for mrlc3 gene expression, and the sequence and SEQ ID NO :6 siRNA knockdown of the extracellular matrix-skeleton system with 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the siRNA of 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO: 13 is used to knock down the sun1 in the extracellular matrix-skeletal system.
  • the knockdown process of the present invention comprises the following steps:
  • the diluted liposome is mixed with the siRNA of the present invention, cultured at room temperature to form a siRNA-liposome mixture, and the mixture is uniformly mixed with a culture solution containing non-neuronal cells to be transfected and cultured.
  • the liposome is employed RNAiMAX Reagent reagent.
  • the siRNA is diluted prior to mixing the liposomes with the siRNA.
  • the liposomes and siRNA are diluted using serum-free Opti-MEM medium.
  • the siRNA is diluted: 12 pmol siRNA duplex (2.5 ul) is diluted in 100 ⁇ L/well of serum-free Opti-MEM medium in an RNase-free EP tube.
  • the dilution transfection reagent RNAiMAX Reagent diluted in 100 ⁇ L/well of serum-free Opti-MEM medium in RNase-free EP tubes RNAiMAX Reagent.
  • the diluted liposome is mixed with the diluted siRNA after incubation for 1-5 min, preferably 2 min, preferably 3 min, preferably 4 min, and cultured at room temperature for 15-25 min, preferably 20 min, to form siRNA-liposome. Mixture.
  • the siRNA-liposome mixture is added to a cell culture plate containing cells and a culture solution to be mixed.
  • the culture plate is cultured in a CO 2 incubator at 37 ° C, transfected for 6-8 hours, preferably 7 hours, and then replaced with a neuron culture solution.
  • the neuronal culture medium is cultured for 48-72 hours, preferably 60 hours.
  • the neuron culture solution comprises: an N2 cell culture medium additive, a B27 cell culture medium additive, glutamine, and ⁇ -mercaptoethanol.
  • the neuron culture solution comprises: N2 cell culture medium additive, B27 cell culture medium additive, glutamine, ⁇ -mercaptoethanol, neurotrophin (NT3), brain-derived neurotrophic factor (BDNF), glial cell line derived Neurotrophic factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).
  • N2 cell culture medium additive B27 cell culture medium additive
  • glutamine ⁇ -mercaptoethanol
  • neurotrophin NT3
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cell line derived Neurotrophic factor
  • db-cAMP dibutyryl cyclic adenosine monophosphate
  • the low adhesion treatment comprises suspension culture of non-neuronal cells using agarose DMEM.
  • the suspension culture time is from 6 to 8 days, preferably 7 days.
  • the amount of the agarose is 0.5 g / 100 ml of the agarose DMEM.
  • the 1% agarose solution is first placed in sterile double distilled water, heated to boiling in a microwave oven, and then an equal volume of 2 x DMEM, (Gibco, 12800-017) is added to make 0.5% agarose dissolved in DMEM. The solution was then poured into a 6 cm Petri dish, cooled and solidified, and set aside.
  • 2 x DMEM Gibco, 12800-017
  • the suspension culture process is exchanged for half a day at intervals.
  • the non-neuronal cells are cultured in suspension culture in agarose DMEM and then in a directed differentiation culture medium.
  • the directed differentiation culture medium comprises GDNF (glial cell-derived neurotrophic factor, peprotech, 450-10), BDNF (brain-derived neurotrophic factor, peprotech, 450-02), NT3 (neurotrophic) Factor-3, peprotech, 450-03) and Forskolin (forskolin, stemgent, 04-0025).
  • GDNF glial cell-derived neurotrophic factor, peprotech, 450-10
  • BDNF brain-derived neurotrophic factor, peprotech, 450-02
  • NT3 neurotrophic Factor-3
  • peprotech 450-03
  • Forskolin forskolin, stemgent, 04-0025.
  • the concentration of GDNF (peprotech, 450-10) in the directed differentiation culture medium is 15-25 ng/ml, preferably 20 ng/ml
  • the concentration of BDNF (peprotech, 450-02) is 15-25 ng/ml, preferably 20 ng/ M
  • NT3 peprotech, 450-03
  • 4 ng/ml of Forskolin (stemgent, 04-0025) has a concentration of 2-6 ng/ml, preferably 3, 4 or 5 ng/ Ml.
  • human or animal non-neuronal cells can be efficiently transformed into a multi-neuronal cell-skeletal system that interferes with non-neuronal cells by using a small molecule inhibitor of cytoskeletal protein, siRNA knockdown treatment, and low adhesion treatment.
  • a small molecule inhibitor of cytoskeletal protein siRNA knockdown treatment
  • low adhesion treatment a small molecule inhibitor of cytoskeletal protein
  • the realization of cell fate changes by the method of the present invention has not been reported yet, and the method is simpler to apply than the previously reported cell fate regulation method, requiring only a single small molecule treatment, or requiring no small molecules and specific Regulation of gene expression can achieve cell fate changes only by changing the cell culture medium, and can be efficiently performed in vitro and in vivo, and has great applications in tumor treatment, tissue regeneration/repair.
  • gliomas are tumors that occur in the neuroectoderm, and most originate from different types of glial cells.
  • glioma is the most common intracranial tumor, accounting for about 45 percent of all intracranial tumors. Ranked second in children's malignant tumors.
  • the incidence of primary malignant intracranial tumors has increased year by year, with an annual growth rate of about 1.2%, especially in the middle-aged and elderly population.
  • the average annual incidence of glioma in China is 3-6 per 100,000 people, and the annual death toll is as high as 30,000.
  • the treatment methods for glioma at home and abroad mainly include: surgery, chemotherapy, radiotherapy, X-knife and gamma knife.
  • small molecule inhibitors of cytoskeletal proteins can be used to prepare anti-glioma drugs. By using this drug, the proliferation of glioma cells can be significantly inhibited, thereby achieving an anticancer effect.
  • glial cells mainly astrocytes
  • astrocytes mainly astrocytes
  • glial scars mainly glial scars
  • the present invention utilizes a cytoskeletal protein inhibitor to efficiently efficiently transduce human-derived primary astrocytes into neurons. This provides a new approach to the treatment of aging and pathological damage leading to neurodegenerative diseases.
  • the invention has the following features: 1. The operation is simple, and a single small molecule is added to the induced culture solution or the single factor treatment can be used for fate conversion. 2. Efficient and rapid, in the process of transdifferentiation of non-neuronal cells into neurons by myosin inhibitor/actin assembly inhibitor, on the 7th day, cells with nearly 100% Tuj1-positive neuron fate can be obtained, suspension culture solution The conversion of non-neuronal cells to neurons can be achieved in only 7 days. 3. Universality, the method is universally adaptable to different types of starting cells of different species to neuronal transdifferentiation, and can be used to transform mouse TTF, MEF, setoli and human dorsal fibroblasts, foreskin into fibers.
  • Figure 1A is a photomicrograph (left) of adult foreskin fibroblasts (HFF20y, Beijing stem cell bank) and an immunofluorescence staining image of Tuj1 (right).
  • Fig. 1B is a photomicrograph (left) and Tuj1 immunofluorescence staining image (right) of the fibroblast of Fig. 1A after incubation with the induction medium containing (-)-Blebbistatin for 7 days in Example 1-1.
  • Fig. 1C shows Marker:Map2 expressing mature neurons in Example 1-1 for the fibroblasts of Fig. 1A after 7 days of culture using an induction medium containing (-)-Blebbistatin, and then cultured for 14 days in the mature medium.
  • Figure 1D shows the expression of matured neurons expressing mature cells in Example 1-1 against the fibroblasts of Figure 1A after 7 days of culture with induction medium containing (-)-Blebbistatin, followed by 14 days of culture in mature medium.
  • Figure 1E is a graph showing the results of a mature neuron patch clamp experiment after culturing for 7 days in the induction medium containing (-)-Blebbistatin for the fibroblasts of Fig. 1A in Example 1-1, and then culturing for 14 days in the mature medium. .
  • Fig. 1F shows that the fibroblasts of Fig. 1A were cultured for 7 days in the induction medium containing (-)-Blebbistatin, and then cultured for 14 days in the mature medium for expression of GABA.
  • Neuron marker protein GABA (sigma, SAB4501067), GAD65/67 (santa cruz biotechnology, sc-7513).
  • Fig. 1G shows the expression of midbrain dopaminergic energy in neurons of Example 1-1 for the fibroblasts of Fig. 1A after 7 days of culture in an induction medium containing (-)-Blebbistatin, and then cultured for 14 days in the mature medium.
  • TH antigena cruz biotechnology, sc-14007
  • glutamatergic marker vGlut1 glutamatergic marker vGlut1
  • Figure 1H is a graph showing the results of the induction of GABA neurons in response to high potassium buffer treatment to release gamma aminobutyric acid by ultra performance liquid chromatography-ultra-high resolution mass spectrometry in Example 1-1.
  • Figure 1I shows neuronal survival results after transplantation of GFP-fluorescent-labeled neurons induced in Example 1-1 into the hippocampus of immunodeficient mice for 1 month.
  • Figure 2A is a photograph of the untreated pre-neuron marker protein Tuj1 and Map2 staining of mouse tail tip fibroblasts in Examples 1-3.
  • 2B is a Marker: Tuj1, Map2, NF200, and NeuN expressing neurons in mouse apical fibroblasts after 7 days of culture using a neuroinduction medium containing (-)-Blebbistatin in Examples 1-3.
  • Fig. 2C is a graph showing the results of a mature neuron patch clamp experiment in which the mouse tail tip fibroblasts were cultured for 7 days in a nerve induction medium containing (-)-Blebbistatin in Examples 1-3.
  • Figure 2D shows neuronal survival results after transplantation of GFP-fluorescent-labeled neurons induced in Examples 1-3 into the hippocampus of immunodeficient mice for 1 month.
  • Figure 3A is a photomicrograph of the cell morphology after incubation in DMSO medium without any inhibitor in Example 2 for 7 days.
  • Fig. 3B is a photomicrograph of the cell morphology after incubation in a medium containing 0.4 ⁇ M Cytochalasin B for 7 days in Example 2.
  • Figure 3C is a photomicrograph of the cell morphology after incubation in a medium containing 2 ⁇ M Cytochalasin B for 3 days in Example 2.
  • Fig. 3D is a photomicrograph of the cell morphology after culturing for 7 days in the medium containing 0.2 ⁇ M Latrunculin B in Example 2.
  • Fig. 3E is a comparative diagram of cell neuron-expressing neuronal marker protein Map2 and control DMSO after cultured for 7 days in a medium containing 2 ⁇ M Cytochalasin B in Example 2, and cultured for 10 days in neuron maturation medium.
  • Figure 4A is a graph showing the comparison of changes in neuronal morphology at day 3 in the treatment of glioma cells treated with (-)-Blebbistatin in the same manner as in the control group.
  • Fig. 4B is a graph showing the inhibitory effect of the antitumor drug of Example 3 on glioma cells U87 and U251.
  • Figure 4C shows the expression of the neuronal marker protein Tuj1 by the antitumor drug of Example 3 and the control DMSO for glioma cell U87.
  • Fig. 5A is a graph showing changes in cell morphology on the 7th day after partial knockdown of the target in Example 4.
  • Fig. 5B is a graph showing the results of immunofluorescence staining of Tuj1 cells on the 7th day after partial knockdown of the target in Example 4.
  • Fig. 6A is a photomicrograph of the fibroblasts in Example 5 after suspension culture in a neural stem cell culture medium for 7 days.
  • Fig. 6B is a diagram showing immunofluorescence staining of fibroblasts in Example 5 after suspension culture in a neural stem cell culture medium for 7 days.
  • Fig. 6C is a diagram showing immunofluorescence staining of fibroblasts in Example 5 after suspension culture in a neural stem cell culture medium for 7 days and then cultured in a random differentiation medium for 7 days.
  • Fig. 6D is a diagram showing the immunofluorescence staining of fibroblasts in Example 5 after suspension culture in a neural stem cell culture medium for 7 days and then cultured for 7 days in a directed differentiation culture medium.
  • Figure 7A shows the morphology of primary astrocytes (left panel) and the marker protein GFAP (right panel) expressing astrocytes.
  • Figure 7B is a morphological view of (-)-Blebbistatin induced astrocytes in Example 6 for 13 days.
  • Fig. 7C is a diagram showing the expression of the classical neuronal marker protein Tuj1 by (-)-Blebbistatin induction of astrocytes in Example 6 for 20 days.
  • each well was prepared with 20 ug/ml fibronectin solution (millipore, fc010) 1 ⁇ PBS, coated for 6 hours, or first with 0.1 mg/ml polylysine solution ( The sigma, P6407) was prepared in sterile water, coated in a petri dish for 2 hours, washed three times with sterile water, and further prepared with 10 ⁇ g/ml laminin solution (sigma, L6274) 1 ⁇ PBS, and coated for 6 hours. Remove the coating solution and wash it with 1 ⁇ PBS.
  • the cell transformation kit of the present invention is selected, and the kit includes the following induction medium and maturation medium.
  • N2B27 medium DMEM/F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1:1 mixed with N2 additive (100 ⁇ , Gibco) , 17502084), B27 additive (50 ⁇ , Gibco, 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), Glutamax (200 ⁇ , Gibco, 35050) -061), 1 ⁇ g/ml insulin (Roche, 11376497001), double antibody).
  • N2B27 medium DMEM/F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1:1 mixed with N2 additive (100 ⁇ , Gibco) , 17502084), B27 additive (50 ⁇ , Gibco, 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco,
  • the cells cultured in the above induction medium were added to a neuronal maturation medium (N2B27 medium, 100 ⁇ M N6, 2'-O-dibutyryl adenosine 3', 5'-cyclophosphate sodium salt (sigma, D0627), 20 ng /ml Recombinant Human NT-3 (Peprotech, 450-03), 20 ng/ml brain-derived neurotrophic factor (peprotech, 450-02), 20 ng/ml (peprotech, 450-10) glial cell-derived nerve Nutritional factors) mature culture for 7-14 days.
  • N2B27 medium 100 ⁇ M N6, 2'-O-dibutyryl adenosine 3', 5'-cyclophosphate sodium salt (sigma, D0627)
  • 20 ng /ml Recombinant Human NT-3 Peprotech, 450-03
  • 20 ng/ml brain-derived neurotrophic factor peprotech, 450-02
  • the myosin inhibitor (-)-Blebbistatin was used to induce the transition of fibroblasts to neuronal cells, and when it was 15 ⁇ M relative to the neuron-inducing medium, although good neurons were obtained.
  • the cell transformation effect was not optimal; when the concentration was 20 ⁇ M-30 ⁇ M, the neuronal cell conversion rate reached the highest.
  • Figure 1A shows the cell morphology and Tuj1 immunofluorescence staining of adult foreskin fibroblasts (HFF20y, Beijing stem cell bank).
  • FIG. 1B shows photomicrographs (left) and Tuj1 immunofluorescence stained images (right) of cells fibrocytes cultured for 7 days in a neural induction medium containing 20 ⁇ M (-)-Blebbistatin for the fibroblasts of Figure 1A.
  • the cultured cells have obvious neuronal morphology, large cell bodies and long axons, and neuronal fate marker Tuj1 staining, and the Tuj1 positive rate (Tuj1 positive cell/nucleus ratio), Tuj1 positive rate Close to 100%.
  • Figure 1C shows Marker:Map2 (santa cruz) expressing mature neurons after incubation with fibroblasts of Figure 1A for 7 days in a neural induction medium containing 20 ⁇ M (-)-Blebbistatin and then cultured for 14 days in neuronal maturation medium.
  • Figure 1D shows that mature fibroblasts expressing the classical presynaptic markers after cultured for 7 days in the neural induction medium containing 20 ⁇ M (-)-Blebbistatin for the fibroblasts of Fig. 1A, and then cultured for 14 days in the neuronal maturation medium.
  • Fig. 1E is a graph showing the results of a mature neuron patch clamp experiment after the culture of the fibroblast of Fig. 1A was carried out for 7 days in an induction medium containing (-)-Blebbistatin, and then cultured for 14 days in the mature medium. It shows that the cells have sodium current, potassium current of mature neurons and have certain action potential activity.
  • Fig. 1F shows that the type of marker protein staining is nearly 100% expressed in the neurons of Fig. 1A after cultured for 7 days in a nerve induction medium containing 20 ⁇ M (-)-Blebbistatin, and then cultured for 14 days in the neuronal maturation medium.
  • GABAergic marker protein GABA (sigma, SAB4501067), GAD65/67 (santa cruz biotechnology, sc-7513).
  • Figure 1G shows that the fibroblasts of Figure 1A were cultured for 7 days in culture medium containing (-)-Blebbistatin, and then cultured for 14 days in mature medium to express midbrain dopaminergic marker TH (santa cruz biotechnology). , sc-14007) and the glutamatergic marker vGlut1 (santa cruz biotechnology, sc-377425). It shows that the induced neurons do not substantially express the midbrain dopaminergic marker TH (santa cruz biotechnology, sc-14007) and the glutamatergic marker vGlut1 (santa cruz biotechnology, sc-377425).
  • Figure 1H shows ultra-high performance liquid-ultra-high resolution mass spectrometry detection showing induction of GABA neurons in response to high potassium buffer treatment to release gamma aminobutyric acid.
  • Figure 1I shows neuronal survival results after transplantation of GFP-fluorescent-labeled neurons induced in Example 1-1 into the hippocampus of immunodeficient mice for 1 month.
  • Example 1-1 The experimental method of Example 1-1 was employed except that the myosin inhibitor used was myosin light chain kinase (MLCK) inhibitor ML-7.
  • MLCK myosin light chain kinase
  • the myosin inhibitor myosin light chain kinase (MLCK) inhibitor ML-7 was used to induce the transition of fibroblasts to neuronal cells when the concentration relative to the neuronal induction medium was 15 ⁇ M. Although the better neuronal cell transformation effect was obtained, it was not optimal; when the concentration was 20 ⁇ M-30 ⁇ M, the neuronal cell conversion rate reached the highest.
  • Example 1-1 The experimental method of Example 1-1 was employed except that the treated subject was mouse tail tip fibroblasts.
  • Figure 2A shows the untreated pre-neuronal marker protein Tuj1 and Map2 stained images of mouse tail tip fibroblasts.
  • Figure 2B shows Marker: Tuj1, Map2, NF200 and NeuN expressing neurons after incubation with mouse tail-tip fibroblasts for 7 days in a neural induction medium containing 20 ⁇ M (-)-Blebbistatin.
  • mice tail-tip fibroblasts were cultured for 7 days in a nerve induction medium containing 20 ⁇ M (-)-Blebbistatin, and then cultured for 14 days in the mature medium, the positive rates of Tuj1, Tuj1/Map2 and Map2/NeuN were measured. 96%, 96%, 97%.
  • Fig. 2C shows that the cells of Example 1-3 have a neuronal maturation after cultured for 7 days in a nerve-inducing medium containing 20 ⁇ M (-)-Blebbistatin, and then cultured for 14 days in a mature medium.
  • the sodium current, potassium current and a certain action potential activity are examples of the cells of Example 1-3.
  • Figure 2D shows neuronal survival results after transplantation of GFP-fluorescent-labeled neurons induced in Examples 1-3 into the hippocampus of immunodeficient mice for 1 month.
  • mice tail tip fibroblasts treated with the myosin inhibitor (-)-Blebbistatin used in the present invention can be converted into neuronal cells with high conversion.
  • Example 1-1 The experimental method of Example 1-1 was used, except that the actin assembly inhibitor 2 ⁇ M cytochalasin B (CB, sigma, C6762) and 0.2 ⁇ M Latrunculin B (BioVision, 2182-1) were used to induce myosin inhibitors, respectively.
  • the actin assembly inhibitor Cytochalasin B was used to induce the transition of fibroblasts to neuronal cells, and when it was 1.5 ⁇ M relative to the neuron-inducing medium, good neuronal cells were obtained.
  • the conversion effect was not optimal; when the concentration was 2 ⁇ M-3 ⁇ M, the neuronal cell conversion rate reached the highest.
  • the actin assembly inhibitor Latrunculin B was used as a transition factor for inducing fibroblasts to neuronal cells, and when it was 0.15 ⁇ M relative to the neuron-inducing medium, although a good neuron was obtained.
  • the cell transformation effect was not optimal; when the concentration was 0.2 ⁇ M and 0.3 ⁇ M, the neuronal cell conversion rate reached the highest.
  • Figures 3A-3E show neural induction of cell morphology using Cytochalasin B and Latrunculin B against adult foreskin fibroblasts (HFF20y, Beijing Stem Cell Bank).
  • Figure 3A shows cell morphology after 7 days of culture in DMSO medium without any inhibitor. It shows that the treated fibroblasts did not have any neuronal morphology.
  • Fig. 3B shows the morphology of cells after 7 days of culture using a medium containing 0.4 ⁇ M Cytochalasin B, which shows a slight change in neuronal morphology of the treated fibroblasts.
  • Figure 3C shows the morphology of cells after 3 days of culture in a medium containing 2 ⁇ M Cytochalasin B, which shows that the treated fibroblasts already have significant changes in neuronal morphology.
  • Figure 3D shows the morphology of cells after 7 days of culture in a medium containing 0.2 ⁇ M Latrunculin B, which shows that the treated fibroblasts almost completely transformed into neuronal morphology.
  • Figure 3E shows the expression of neuronal marker protein Map2, which was expressed in cells cultured with 2 ⁇ M Cytochalasin B for 7 days, and cultured for 10 days in neuronal maturation medium, showing a marked change in neuronal fat morphology of the treated cells.
  • This embodiment provides a specific embodiment for using a cytoskeletal protein inhibitor for the preparation of an anticancer drug, which is capable of transdifferentiating glioma cells into neuronal cells.
  • Antitumor drug I DMEM/F12 (gibco, 10565018) was mixed with Neurobasal (Gibco, 21103-049) 1:1 with the following reagents, N2 additive (100 ⁇ , Gibco, 17500248), B27 additive (50 ⁇ , Gibco) , 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), Glutamax (200 ⁇ , Gibco, 35050-061), 1 ⁇ g/ml insulin (Roche, 11376497001), double-antibody), and (-)-Blebbistatin (dimethyl sulfoxide dissolved (sigma, D2650) in 100 mM storage, stored at -20 ° C in the dark for 1 month). The concentration of (-)-Blebbistatin was adjusted to 20 ⁇ M relative to the entire drug I.
  • Antitumor Drug II was formulated with the following reagents: 100 ⁇ M N6,2'-O-dibutyryladenosine 3',5'-cyclophosphate sodium salt (sigma, D0627), 20 ng/ml Recombinant Human NT based on antitumor drug I -3 (Peprotech, 450-03), 20 ng/ml brain-derived neurotrophic factor (peprotech, 450-02), 20 ng/ml glial cell-derived neurotrophic factor (peprotech, 450-10).
  • antitumor drugs I and II were administered in combination, and the antitumor drug II was administered 3-7 days after the application of the antitumor drug I.
  • the anti-tumor drugs I and II of the present embodiment were used to treat human malignant glioma cells (U87), astroglioma cells (U251), human malignant glioma cells (LN229) and human glioblastoma, respectively.
  • Cells (T98G) The morphology, growth curve and transformation results of the final glioma cells were compared with those of the control group (i.e., the drug reagent did not contain (-)-Blebbistatin) as shown in Figs. 4A and 4B.
  • human malignant glioma cells U87
  • astroglioma cells U251
  • human malignant glioma cells LN229
  • human glia were treated with 20 ⁇ M (-)-Blebbistatin and control DMSO, respectively.
  • Maternal tumor cells T98G
  • the starting cells were the same in each experimental group.
  • Figure 4A shows that there was a significant change in neuronal morphology on day 3 with (-)-Blebbistatin containing 20 ⁇ M compared to the control DMSO, and the cells were significantly less abundant than the control group.
  • Figure 4B shows that the antitumor drug of this example significantly inhibited the proliferation of glioma cell cells, and the inhibition efficiencies of the optional glioma cells U87 and U251 were 50% and 99%, respectively, in the first 5 days.
  • Fig. 4C shows that the antitumor drug of the present embodiment transdifferentiates the optionally rapidly proliferating glioma cell U87 into differentiated neuronal cells, and expresses the neuronal marker protein Tuj1 with a positive rate close to 95%.
  • RNAiMAX (13778150, invitrogen) kit was transfected with target sites rock1, rock2, mrlc1, mrlc2, mrlc3, myh9, myh10, mrck ⁇ , mrck ⁇ , lamina/c, lmnb1, lbr, sun1, sun2, cbx1, cbx3, cbx5, banf1, respectively. , syne1, synne2, ⁇ -actin.
  • Table 5 shows the correspondence of siRNA sequences to gene expressions that are knocked down.
  • Mrck ⁇ 8476 SEQ ID NO:8 Cgagaagacu uugaaauaa Mrck ⁇ 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 Ugacaacac agaggauuu Cbx5 23468 SEQ ID NO: 17 Uaaacccagg gagaaguca Banf1 8815 SEQ
  • FIG. 5A shows the specific protein Tuj1 expressing the fate of neurons on the 7th day by immunofluorescence staining.
  • Neuron medium N2B27 medium: DMEM/F12 (gibco, 10565018) mixed with Neurobasal (Gibco, 21103-049) 1:1, N2 additive (100 ⁇ , Gibco, 17500248), B27 additive (50 ⁇ , Gibco) , 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), Glutamax (200 ⁇ , Gibco, 35050-061), 1 ⁇ g/ml insulin (Roche, 11376497001), double antibody).
  • N2B27 medium DMEM/F12 (gibco, 10565018) mixed with Neurobasal (Gibco, 21103-049) 1:1, N2 additive (100 ⁇ , Gibco, 17500248), B27 additive (50 ⁇ , Gibco) , 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco
  • a 1% agarose solution was prepared with sterile double distilled water, and the microwave was heated to boiling. Then, an equal volume of 2 ⁇ DMEM (Gibco, 12800-017) was added to prepare a 0.5% agarose solution dissolved in DMEM, and then Pour into a 6 cm Petri dish, cool and solidify, and set aside.
  • 2 ⁇ DMEM Gibco, 12800-017
  • Human foreskin fibroblasts were seeded in the above-mentioned culture dish at a density of 1 ⁇ 105, and cultured in suspension with a neural stem cell culture solution for 7 days, and half a day was changed.
  • the photomicrograph after the 7th day of suspension culture is shown in Fig. 6A. Among them, neuronal morphology has been shown.
  • the circular slides placed in a four-well plate were treated with 1 x PBS containing 1 ug/ml of fibronectin (Millipore, FC010) and placed in an incubator overnight for use.
  • Human fibroblasts on the 7th day of suspension culture were centrifuged and digested with Tryple (Gibco, A1285901) in a 37 ° C incubator for 3 min, diluted with PBS, centrifuged, and the supernatant was discarded and resuspended in neural stem cell culture medium at 5 per well.
  • ⁇ 104 was inoculated into a four-well plate, and the culture solution was removed the next day for immunofluorescence staining, as follows:
  • the cells were fixed with PBS solution containing 4% paraformaldehyde (Sigma, 158127) for 20 min at room temperature, and washed three times with PBS for 5 min each time. It was then permeabilized for 1 h in a PBS solution containing 0.3% Triton X-100 (Solarbio, T8200) and 0.2% BSA (Sigma, A3803).
  • mouse anti-Nestin (Millipore, MAB353, 1:100), goat anti-Sox2 (Santa, sc-17320, 1:100), mouse anti-Nkx2 .2 (abcam, ab187375,) rabbit anti-En1 (abcam, ab70993, 1:50), rabbit anti-N-cadherin (abcam, ab12221, 1:100), rabbit anti-Pax6 (abcam, ab5790, 1:50 Incubate overnight in PBS solution.
  • the cells were washed three times with PBS solution, and incubated with anti-rabbit, anti-mouse, or anti-goat secondary antibody, Alexa Fluor-488 or Alexa Fluor-561 (1:500, Invitrogen) for 1 hour at room temperature.
  • the cells were then washed three times with PBS solution, and the nuclei were stained with Hoechst (Inventgen, H3570, 1:1000) in PBS for 5 min.
  • the round slide was then inverted on a glass slide with anti-fluorescence quencher and the nail polish was sealed.
  • the cells were then observed using a Leica two-photon confocal microscope.
  • the test results are shown in Fig. 6B, where Merge represents a coincident graph.
  • the above identified neural stem cells were similarly inoculated into a four-well plate on the 7th day according to the above method, and the randomly differentiated group was cultured in a random differentiation culture medium, and the differentiated differentiation group was cultured in a directed differentiation culture medium, and the liquid was changed halfway every other day, the 14th day. Immunofluorescence staining was performed.
  • the primary antibodies were rabbit anti-Tuj1 (abcam, ab18207, 1:2000), mouse anti-GFAP (sigma, G3893, 1:100), chicken anti-NF2000 (abcam, ab4680, 1:1000).
  • the test results are shown in 6C and 6D, respectively.
  • Fig. 6C indicate that the neural stem cells obtained by low adhesion treatment can obtain GFAP-positive astrocytes through random differentiation, and further these cells are negative for Tuj1.
  • Fig. 6D indicate that the neural stem cells obtained by the low adhesion treatment can obtain the Tuj1 and NF2000 double positive neurons by directed differentiation.
  • DMEM/F12 Made of DMEM/F12 (Gibco, 12400-024) and Neurobasal (Gibco, 21103-049) in a volume ratio of 1:1, and added 100 ⁇ N2 (Gibco, 17502-048), 50 ⁇ B27 (Gibco, 17504- 044), 100 ⁇ GlutaMAX (Gibco, 35050-079, 1000 ⁇ -mercaptoethanol (Gibco, 21985), 1000 ⁇ 2% BSA (sigma, A7906-100G), 1000 ⁇ Insulin (Roche applied science, 11376497001, 10 mg/mL ), 100 x SP (Gibco, 15140-122).
  • bFGF epidermal growth factor
  • FBS fetal calf serum, Gibco, 16000-044
  • Base medium was added 20 ng/ml GDNF (peprotech, 450-10), 20 ng/ml BDNF (peprotech, 450-02), 20 ng/ml NT3 (peprotech, 450-03), 4 ng/ml Forskolin (stemgent) , 04-0025) formulated.
  • each well was prepared with 20 ug/ml fibronectin solution (millipore, fc010) 1 ⁇ PBS, coated for 6 hours, or first with 0.1 mg/ml polylysine solution ( The sigma, P6407) was prepared in sterile water, coated in a petri dish for 2 hours, washed three times with sterile water, and further prepared with 10 ⁇ g/ml laminin solution (sigma, L6274) 1 ⁇ PBS, and coated for 6 hours. Remove the coating solution and wash it with 1 ⁇ PBS.
  • the cell transformation kit of the present invention is selected, and the kit includes the following induction medium and maturation medium.
  • the glial cells treated as described above were added to a neuron induction medium (N2B27 culture solution: DMEM/F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1 to 1 mixed, and N2 additive (100 ⁇ , Gibco, 17502084), B27 additive (50 ⁇ , Gibco, 17504044), 2% bovine serum albumin (1000 ⁇ , sigma, A8022), ⁇ -mercaptoethanol (1000 ⁇ , Gibco, 21985023), Glutamax (200 ⁇ , Gibco, 35050-061), 1 ⁇ g/ml insulin (Roche, 11376497001), double antibody).
  • N2B27 culture solution DMEM/F12 (gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1 to 1 mixed
  • N2 additive 100 ⁇ , Gibco, 17502084)
  • B27 additive 50 ⁇ , Gibco, 17504044
  • 2% bovine serum albumin 1000 ⁇ ,
  • Figure 7A shows the morphology of primary astrocytes (left panel) and the marker protein GFAP (right panel) expressing astrocytes.
  • Figure 7B (-)-Blebbistatin induced 13-day astrocytes to appear as classical neuronal morphology.
  • Figure 7C (-)-Blebbistatin induced astrocytes for 20 days and nearly 100% expressed the classical neuronal marker protein Tuj1.

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Abstract

提供了一种将非神经元细胞转分化为神经元细胞的方法，包括对非神经元细胞的胞外基质-骨架系统进行干扰处理，该干扰处理选自：采用细胞骨架蛋白小分子抑制剂进行处理，采用小干扰RNA(siRNA)对胞外基质-骨架系统的特定基因表达进行敲低处理，对胞外基质进行低粘附处理并定向分化培养。该转分化的方法可用于组织再生、修复和肿瘤治疗。

Description

一种将非神经元细胞转化为神经元细胞的方法 技术领域
本发明涉及生物技术领域，具体涉及通过对非神经元细胞的胞外基质-骨架系统进行干扰处理从而将人和动物的非神经元细胞转化为功能性神经元的方法。
背景技术
调控细胞命运，从而产生具有不同功能的特定细胞类型，在细胞替代治疗和再生治疗中具有重要应用前景。细胞命运取决于基因组的特异性表达，而表达调控方式既包括常见的生物学调控，如信号转导、转录调控网络、表观遗传修饰等，也受细胞的理化特性以及细胞所处环境中的理化因素的调控。因此细胞命运转变的方法也分为两种，即改变细胞的生物学特性和理化特性。
目前通过调控细胞命运产生功能细胞的方法主要是针对若干重要基因调控通路和表观遗传修饰，通过遗传学手段或者化学小分子处理手段来完成。遗传学手段包括研究人员利用异位表达Oct4、Sox2、c-Myc、Klf4等转录因子将小鼠和人的成纤维体细胞重编程为诱导多能性干细胞；利用异位表达Adcl1、Brn2和Myt1l等转录因子将成纤维细胞转分化为功能性神经元；以及利用异位表达特定基因的方式获得具有功能的心肌细胞、胰岛细胞等。化学小分子手段包括研究人员利用化学小分子组合VC6TFZ将小鼠成纤维细胞重编程为多能性干细胞；利用7种小分子组成的组合(VCRFSGY)将人的成纤维细胞直接转化为神经元；利用9种小分子的组合M9将小鼠成纤维细胞重编程为神经干细胞，进而分化为功能性神经元；利用小分子组合将人胃上皮细胞转变成多潜能的内胚层祖细胞；利用小分子组合将将人的成纤维通过小分子转化为心肌细胞等。
发明内容
本发明主要通过对非神经元细胞的胞外基质-骨架系统进行干扰处理，从而实现对细胞命运进行调控，特别地，在将人或动物的非神经元细胞转分化为神经元细胞的方面提出了更为简便易行的途径，取得了开创性的、预料不到的技术效果。
本发明提供了一种将非神经元细胞转化为神经元细胞的方法，其特征在于所述方法包括对非神经元细胞的胞外基质-骨架系统进行干扰处理。
本发明的干扰处理选自以下的至少一种：采用细胞骨架蛋白抑制剂进行处理，采用小干扰RNA(siRNA)对胞外基质-骨架系统的基因表达进行敲低处理，对胞外基质进行低粘附处理。
采用细胞骨架蛋白抑制剂进行处理或采用小干扰RNA(siRNA)对胞外基质-骨架系统的基因表达进行敲低处理或在对胞外基质进行低粘附处理之后，通过培养基进行定向培养。该定向培养可以采用下述成熟培养基或定向分化培养基等来进行。
根据本发明的一个实施方式，其中所述细胞骨架蛋白抑制剂选自以下的至少一种：肌球蛋白(myosin)抑制剂、肌动蛋白(actin)组装抑制剂。
优选地，其中所述肌球蛋白(myosin)抑制剂选自以下的至少一种：(-)-Blebbistatin、肌球蛋白轻链激酶(MLCK)抑制剂ML-7，浓度为10μM以上，优选20μM以上，更优选10-30μM，其中所述浓度为所述抑制剂在处理非神经元细胞所用诱导培养基中的最终浓度。
优选地，其中所述肌动蛋白(actin)组装抑制剂选自以下的至少一种：Cytochalasin B、Latrunculin B，其中Cytochalasin B的浓度为1.5μM以上，优选2μM以上，更优选2-3μM，Latrunculin B浓度为0.15μM以上，优选0.2μM，更优选0.2-0.3μM，其中所述浓度为所述抑制剂在处理非神经元细胞所用诱导培养基中的最终浓度。
根据本发明的一个实施方式，所述方法包括将非神经元细胞置于诱导培养基中培养3-7天，任选地4天、5天或6天，然后采用成熟培养基培养7-14天，任选地8天、9天、10天、11天、12天或13天。
优选地，其中所述诱导培养基包含：细胞骨架蛋白抑制剂、N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇。
优选地，其中所述成熟培养基包含：N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇，神经营养素(NT3)，脑源性神经营养因子(BDNF)，胶质细胞系衍生的神经营养因子(GDNF)，二丁酰环腺苷酸(db-cAMP)。
根据本发明的一个实施方式，本发明中非神经元细胞优选为成纤维细胞和/或神经胶质细胞。
本发明还提供了细胞骨架蛋白抑制剂用于将非神经元细胞转分化为神经元细胞的用途。
本发明还提供了一种将非神经元细胞转化为神经元细胞的试剂盒，所述试剂盒包括诱导培养基，所述诱导培养基包含细胞骨架蛋白抑制剂。
根据本发明的一个实施方式，其中所述细胞骨架蛋白抑制剂选自以下的至少一种：肌球蛋白(myosin)抑制剂、肌动蛋白(actin)组装抑制剂。
优选地，其中所述肌球蛋白(myosin)抑制剂选自以下的至少一种：(-)-Blebbistatin、肌球蛋白轻链激酶(MLCK)抑制剂ML-7，浓度为10μM以上，优选20μM以上，更优选10-30μM，其中所述浓度为所述抑制剂在诱导培养基中的浓度。
优选地，其中所述肌动蛋白(actin)组装抑制剂选自以下的至少一种：Cytochalasin B、Latrunculin B，其中Cytochalasin B的浓度为1.5μM以上，优选2μM以上，更优选2-3μM，Latrunculin B浓度为0.15μM以上，优选0.2μM，更优选0.2-0.3μM，其中所述浓度为所述抑制剂在诱导培养基中的浓度。
根据本发明的一个实施方式，所述试剂盒还包括成熟培养基。
优选地，其中所述诱导培养基包含：细胞骨架蛋白抑制剂、N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇。
优选地，其中所述成熟培养基包含：N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇，神经营养素(NT3)，脑源性神经营养因子(BDNF)，胶质细胞系衍生的神经营养因子(GDNF)，二丁酰环腺苷酸(db-cAMP)。
本发明还提供了细胞骨架蛋白抑制剂用于制备抗肿瘤药物、组织再生和/或修复药物中的用途。
根据本发明的一个实施方式，本发明的敲低处理包括以下的至少一种：
采用与序列SEQ ID NO:1具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的rock1基因表达，
采用与序列SEQ ID NO:2具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的rock2基因表达，
采用与序列SEQ ID NO:3具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc1基因表达，
采用与序列SEQ ID NO:4具有95％、96％、97％、98％、99％或100％序列同 一性的siRNA敲低胞外基质-骨架系统中的mrlc2基因表达，
采用与序列SEQ ID NO:5具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc3基因表达，
采用与序列SEQ ID NO:6具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的myh9基因表达，
采用与序列SEQ ID NO:7具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的myh10基因表达，
采用与序列SEQ ID NO:8具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrckα基因表达，
采用与序列SEQ ID NO:9具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrckβ基因表达，
采用与序列SEQ ID NO:10具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的lamina/c基因表达，
采用与序列SEQ ID NO:11具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的lmnb1基因表达，
采用与序列SEQ ID NO:12具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的lbr基因表达，
采用与序列SEQ ID NO:13具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的sun1基因表达，
采用与序列SEQ ID NO:14具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的sun2基因表达，
采用与序列SEQ ID NO:15具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的cbx1基因表达，
采用与序列SEQ ID NO:16具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的cbx3基因表达，
采用与序列SEQ ID NO:17具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的cbx5基因表达，
采用与序列SEQ ID NO:18具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的banf1基因表达，
采用与序列SEQ ID NO:19具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的syne1基因表达，
采用与序列SEQ ID NO:20具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的syne2基因表达，
采用与序列SEQ ID NO:21具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的β-actin基因表达。
根据本发明的一个实施方式，采用与序列SEQ ID NO:1具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的rock1基因表达，采用与序列SEQ ID NO:2具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的rock2基因表达，采用与序列SEQ ID NO:8具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrckα基因表达，以及采用与序列SEQ ID NO:9具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrckβ基因表达。
根据本发明的一个实施方式，采用与序列SEQ ID NO:3具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc1基因表达，采用与序列SEQ ID NO:4具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc2基因表达，以及采用与序列SEQ ID NO:5具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc3基因表达。
根据本发明的一个实施方式，采用与序列SEQ ID NO:6具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的myh9基因表达，以及采用与序列SEQ ID NO:7具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的myh10基因表达。
根据本发明的一个实施方式，采用与序列SEQ ID NO:3具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc1基因表达，采用与序列SEQ ID NO:4具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc2基因表达，采用与序列SEQ ID NO:5具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的mrlc3基因表达，以及采用与序列SEQ ID NO:6具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的myh9基因表达。
根据本发明的一个实施方式，采用与序列SEQ ID NO:13具有95％、96％、 97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的sun1基因表达，以及采用与序列SEQ ID NO:14具有95％、96％、97％、98％、99％或100％序列同一性的siRNA敲低胞外基质-骨架系统中的sun2基因表达。
根据本发明的一个实施方式，本发明的敲低处理包括如下步骤：
将稀释的脂质体与本发明的siRNA混和，室温培养以形成siRNA-脂质体混和物；将所述混合物与含有非神经元细胞的培养液混和均匀进行转染、培养。
优选地，脂质体采用
Figure PCTCN2018077192-appb-000001
RNAiMAX Reagent试剂。
优选地，在脂质体与siRNA混合之前，进行稀释siRNA。
优选地，稀释脂质体和siRNA，采用无血清Opti-MEM培养基。
优选地，稀释siRNA：在无RNA酶的EP管，用100μL/每孔的无血清Opti-MEM培养基稀释12pmol siRNA双链(2.5ul)。
优选地，稀释转染试剂
Figure PCTCN2018077192-appb-000002
RNAiMAX Reagent：在无RNA酶的EP管，用100μL/每孔of无血清Opti-MEM培养基稀释
Figure PCTCN2018077192-appb-000003
RNAiMAX Reagent。
优选地，稀释好的脂质体经过1-5min，优选2min，优选3min，优选4min的孵育后，与上述稀释好的siRNA混和，室温培养15-25min，优选20min，以形成siRNA-脂质体混和物。
优选地，将siRNA-脂质体混和物加入含有细胞以及培养液的细胞培养板中使之混和。
优选地，将培养板置于37℃的CO 2培养箱中培养，转染隔6-8小时，优选7小时，后换成神经元培养液。
优选地，神经元培养液培养48-72小时后，优选60小时。
优选地，神经元培养液包含：N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇。
优选地，神经元培养液包含：N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇，神经营养素(NT3)，脑源性神经营养因子(BDNF)，胶质细胞系衍生的神经营养因子(GDNF)，二丁酰环腺苷酸(db-cAMP)。
根据本发明的一个实施方式，低粘附处理包括采用琼脂糖DMEM悬浮培养非神经元细胞。优选地，悬浮培养时间为6-8天，优选地7天。优选地，所述琼脂糖的用量为0.5g/100毫升所述琼脂糖DMEM。
优选地，首先用无菌双蒸水配置1％琼脂糖溶液，微波炉加热至沸腾，随后 加入等体积的2×DMEM，(Gibco,12800-017)制成溶于DMEM中的0.5％的琼脂糖溶液，然后倒入6cm的培养皿中，冷却凝固后备用。
优选地，悬浮培养过程期间隔天半量换液。
优选地，非神经元细胞采用琼脂糖DMEM悬浮培养后，再采用定向分化培养液培养。
优选地，所述定向分化培养液包含GDNF(神经胶质细胞源性的神经营养因子，peprotech,450-10)、BDNF(脑源性神经营养因子，peprotech,450-02)、NT3(神经营养因子-3，peprotech,450-03)和Forskolin(毛喉素，stemgent,04-0025)。
优选地，所述定向分化培养液中GDNF(peprotech,450-10)浓度为15-25ng/ml，优选20ng/ml，BDNF(peprotech,450-02)浓度为15-25ng/ml，优选20ng/ml，NT3(peprotech,450-03)浓度为15-25ng/ml，优选20ng/ml，4ng/ml的Forskolin(stemgent,04-0025)浓度为2-6ng/ml，优选3、4或5ng/ml。
根据本发明，通过采用细胞骨架蛋白的小分子抑制剂、siRNA敲低处理、低粘附处理干扰非神经元细胞的胞外基质-骨架系统，可高效地将人或动物非神经元细胞转变成为神经元细胞。通过本发明的方法实现细胞命运改变从而获得神经元尚未有所报道，同时相比之前报道的细胞命运调控方法，本方法应用更为简单，只需单个小分子处理，或者不需要小分子及特定基因表达调控，仅通过改变细胞培养基质即可实现细胞命运改变，并且在体内体外均可高效进行，在肿瘤治疗、组织再生/修复中具有重大应用。
本发明的方法具有广谱抗肿瘤活性的作用。比如，胶质瘤是一种发生于神经外胚层的肿瘤，大多数起源于不同类型的神经胶质细胞。据统计脑胶质瘤是最常见的颅内肿瘤，占所有颅内肿瘤的百分之四十五左右。在儿童恶性肿瘤中排名第二位，近年来，原发性的恶性颅内肿瘤发生率逐年递增，每年增长率约为1.2％，在中老年人群尤为明显。据文献报道，在中国脑胶质瘤平均每年发病率为每10万人有3-6人，年死亡人数高达3万人。目前国内外对胶质瘤的治疗手段主要包括：手术、化疗、放疗、X刀和γ刀等。基于本发明，可以将细胞骨架蛋白的小分子抑制剂用于制备抗胶质瘤的药物。通过采用这种药物，能够显著抑制胶质瘤细胞的增殖，从而达到抗癌效果。
生理衰老或病例脑损伤导致大量神经元死亡，导致神经胶质细胞(主要为星形胶质细胞)的大量增殖，占据原有脑损伤区域，抑制神经元的再生，形成脑内 胶质疤痕，引起神经退行性疾病，严重影响人的生活质量，如帕金森、艾尔海默症等。尽管人们尝试大量的方法，目前仍然没有简单而有效的方法控制胶质疤痕的产生，促进神经元再生修复。本发明利用细胞骨架蛋白抑制剂能高效地将人来源的原代星形胶质细胞高效的转分化为神经元。这为在体治疗衰老和病理损伤导致神经退行性疾病提供一种新的方法。
本发明的特点在于：1.操作简单，单一的小分子加入诱导培养液或单因素处理即可进行命运转换。2.高效快速，在myosin抑制剂/actin组装抑制剂处理非神经元细胞向神经元转分化过程中，在第7天就可获得接近100％的Tuj1阳性的神经元命运的细胞，悬浮培养液仅需7天时间即可实现非神经元细胞向神经元的转化。3.普适性，该方法在不同物种的不同类型起始细胞向神经元转分化过程中具有普遍适应性，可以将小鼠TTF、MEF、setoli和人的背皮成纤维细胞、包皮成纤维细胞和肌肉细胞高效地转分化为神经元。4.安全性，本发明的方法相比于传统的病毒载体介导的遗传学手段具有更高的安全性。5.可控性，单个小分子的缓释系统相比于多个小分子组合介导的转分化更具有可行性，便于计量控制。
附图说明
图1A为成人包皮成纤维细胞(HFF20y，北京干细胞库)的显微照片(左)和Tuj1免疫荧光染色图片(右)。
图1B为实施例1-1中针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后细胞的显微照片(左)和Tuj1免疫荧光染色图片(右)。
图1C显示了实施例1-1中针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后，再用成熟培养基培养14天后的表达成熟神经元的Marker：Map2(santa cruz biotechnology，sc-20172)、NF200(Abcam，ab4680)和NeuN(chemicon，MAB377)。
图1D显示了实施例1-1中针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后，再用成熟培养基培养14天后的表达成熟的神经元表达经典的突触前标记蛋白Syn1(millipore，AB1543P)和Syt1(abcam，ab133856)及突触后标记蛋白PSD95(abcam，ab18258)。
图1E为实施例1-1中针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后，再用成熟培养基培养14天后的成熟的神经元膜片钳实验结果图。
图1F显示了实施例1-1中针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后，再用成熟培养基培养14天后的神经元进行类型标记蛋白染色表达GABA能神经元标记蛋白GABA(sigma，SAB4501067)，GAD65/67(santa cruz biotechnology，sc-7513)。
图1G显示了实施例1-1中针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后，再用成熟培养基培养14天后的神经元表达中脑多巴胺能的标记物TH(santa cruz biotechnology，sc-14007)和谷氨酸能标记物vGlut1(santa cruz biotechnology，sc-377425)。
图1H显示了实施例1-1中超高效液相-超高分辨率质谱检测测得诱导GABA神经元响应高钾缓冲液处理释放γ氨基丁酸结果图。
图1I显示了实施例1-1中诱导的带GFP荧光标记的神经元移植到免疫缺陷小鼠海马区1月后神经元存活结果。
图2A为实施例1-3中小鼠尾尖成纤维细胞未处理前神经元标记蛋白Tuj1和Map2染色图片。
图2B为实施例1-3中针对小鼠尾尖成纤维细胞采用包含(-)-Blebbistatin的神经诱导培养基培养7天后表达神经元的Marker：Tuj1、Map2、NF200和NeuN。
图2C为实施例1-3中针对小鼠尾尖成纤维细胞采用包含(-)-Blebbistatin的神经诱导培养基培养7天后成熟的神经元膜片钳实验结果图。
图2D显示了实施例1-3中诱导的带GFP荧光标记的神经元移植到免疫缺陷小鼠海马区1月后神经元存活结果。
图3A为实施例2中采用不含任何抑制剂的DMSO培养基培养7天后的细胞形态显微照片。
图3B为实施例2中采用含有0.4μM Cytochalasin B的培养基培养7天后的细胞形态显微照片。
图3C为实施例2中采用含有2μM Cytochalasin B的培养基培养3天后的细胞形态显微照片。
图3D为实施例2中采用含有0.2μM Latrunculin B的培养基培养7天后的细胞形态显微照片。
图3E为实施例2中采用含有2μM Cytochalasin B的培养基培养7天后，又采用神经元成熟培养基培养10天后的细胞神经元表达神经元标记蛋白Map2与采用对照的DMSO的对比图。
图4A显示实施例3中采用含有(-)-Blebbistatin处理相比于对照组DMSO处理胶质瘤细胞在第3天神经元形态的变化对比图。
图4B显示了实施例3的抗肿瘤药物对胶质瘤细胞U87和U251抑制效果对比图。
图4C显示了实施例3的抗肿瘤药物与对照组DMSO对胶质瘤细胞U87处理后细胞表达神经元标记蛋白Tuj1情况。
图5A为实施例4中部分靶点敲低后第7天的细胞形态变化图。
图5B为实施例4中部分靶点敲低后第7天细胞Tuj1免疫荧光染色结果图。
图6A为实施例5中成纤维细胞经神经干细胞培养液悬浮培养7天后光镜照片。
图6B为实施例5中成纤维细胞经神经干细胞培养液悬浮培养7天后的免疫荧光染色图。
图6C为实施例5中成纤维细胞经神经干细胞培养液悬浮培养7天后，再经随机分化培养液培养7天后的免疫荧光染色图。
图6D为实施例5中成纤维细胞经神经干细胞培养液悬浮培养7天后，再经定向分化培养液培养7天后的免疫荧光染色图。
图7A原代星形胶质细胞形态(左图)及表达星形胶质细胞的标记蛋白GFAP(右图)。
图7B为实施例6中(-)-Blebbistatin诱导星形胶质细胞13天的形态图。
图7C为实施例6中(-)-Blebbistatin诱导星形胶质细胞20天，表达经典神经元标记蛋白Tuj1图。
具体实施方式
所述实施方式以及以下实施例是用于示例目的，且无意于限制请求保护的范围。有关本发明所述组合物的其它修饰、用途或组合对于本领域普通技术人员而言将是显而易见的，而不脱离请求保护的主题的精神和范围。
实施例1-1
以12孔板为例(corning，3335)每孔用20ug/ml纤连蛋白溶液(millipore，fc010)1×PBS配制，包被6小时，或先用0.1mg/ml多聚赖氨酸溶液(sigma，P6407)无菌水配制，包被培养皿2小时，无菌水洗三次，再用10μg/ml层粘连蛋白溶液 (sigma，L6274)1×PBS配制，包被6小时。去掉包被液用1×PBS冲液洗一遍。
去掉洗液，将成人包皮成纤维细胞(HFF20y，北京干细胞库)均匀种于每一个孔，每孔1×10 4个细胞，用基础培养液(高糖DMEM(Gibco，C12430500BT)，1×丙酮酸钠(100×，gibco，11360-070)，1×非必须氨基酸(100×，Gibco，11140-050)，双抗)加10％胎牛血清(Gibco，16000-044)培养24小时。去除培养液，PBS洗一遍。
选用本发明的细胞转化试剂盒，所述试剂盒包括如下的诱导培养基和成熟培养基。
将经上述处理后的成纤维细胞加入神经元诱导培养基(N2B27培养液：DMEM/F12(gibco，10565018)与Neurobasal(Gibco，21103-049)1比1混合，加入N2添加剂(100×，Gibco，17502048)，B27添加剂(50×，Gibco，17504044)，2％牛血清蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，Glutamax(200×，Gibco，35050-061),1μg/ml胰岛素(Roche，11376497001)，双抗)。加入myosin抑制剂(-)-Blebbistatin(二甲基亚砜溶解(sigma，D2650)的100mM浓储，-20摄氏度避光保存1月)，(-)-Blebbistatin在诱导培养基中的终浓度为15-30μM。培养3-7天，培养的细胞有明显神经元形态，大的胞体和长的轴突等及神经元命运标记物Tuj1染色，并统计Tuj1阳性率(Tuj1阳性细胞/细胞核比值)。
将经过上述诱导培养基培养的细胞加入神经元成熟培养基(N2B27培养基，100μM N6,2′-O-二丁酰基腺苷3′,5′-环磷酸钠盐(sigma，D0627)，20ng/ml Recombinant Human NT-3(Peprotech，450-03)，20ng/ml脑源性神经营养因子(peprotech，450-02)，20ng/ml(peprotech，450-10)神经胶质细胞源性的神经营养因子)成熟培养7-14天。
重复上述实验操作，分别处理来自21周、8岁、13岁人的包皮成纤维细胞，人21周背皮成纤维细胞，猴子尾尖成纤维细胞，鼠胎儿成纤维细胞及非增值细胞丝裂霉素C处理的鼠胎儿成纤维细胞，最终所得Tuj1阳性率和MAP2阳性率结果如表1所示。
表1
Figure PCTCN2018077192-appb-000004
Figure PCTCN2018077192-appb-000005
由表1所示，采用myosin抑制剂(-)-Blebbistatin诱导成纤维细胞向神经元细胞的转变情况，当其相对于神经元诱导培养基的浓度为15μM时，虽然取得了很好的神经元细胞转化效果，但并未达到最佳；而当采用浓度为20μM-30μM时，神经元细胞转化率达到最高。
为了进一步说明myosin抑制剂(-)-Blebbistatin的效果，申请人提供了附图1A-图1I。
图1A显示了成人包皮成纤维细胞(HFF20y，北京干细胞库)的细胞形态和Tuj1免疫荧光染色图片。
图1B显示了针对图1A的成纤维细胞，采用包含20μM(-)-Blebbistatin的神经诱导培养基培养7天后的细胞的显微照片(左)和Tuj1免疫荧光染色图片(右)。如该图所示，培养的细胞有明显神经元形态，大的胞体和长的轴突等及神经元命运标记物Tuj1染色，经统计Tuj1阳性率(Tuj1阳性细胞/细胞核比值)，Tuj1 阳性率接近100％。
图1C显示了针对图1A的成纤维细胞采用包含20μM(-)-Blebbistatin的神经诱导培养基培养7天后，再用神经元成熟培养基培养14天后的表达成熟神经元的Marker：Map2(santa cruz biotechnology，sc-20172)、Nf200(Abcam，ab4680)和NeuN(chemicon，MAB377)。
图1D显示了针对图1A的成纤维细胞采用包含20μM(-)-Blebbistatin的神经诱导培养基培养7天后，再用神经元成熟培养基培养14天后的成熟的神经元表达经典的突触前标记蛋白Syn1(millipore，AB1543P)和Syt1(abcam，ab133856)及突触后标记蛋白PSD95(abcam，ab18258)。
图1E显示了针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后，再用成熟培养基培养14天后的成熟的神经元膜片钳实验结果图。其显示细胞具有成熟神经元的钠电流、钾电流并有一定的动作电位的活性。
图1F显示了针对图1A的成纤维细胞采用包含20μM(-)-Blebbistatin的神经诱导培养基培养7天后，再用神经元成熟培养基培养14天后的神经元进行类型标记蛋白染色接近100％表达GABA能的标记蛋白GABA(sigma，SAB4501067)，GAD65/67(santa cruz biotechnology，sc-7513)。
图1G显示了针对图1A的成纤维细胞采用包含(-)-Blebbistatin的诱导培养基培养7天后，再用成熟培养基培养14天后的神经元表达中脑多巴胺能的标记物TH(santa cruz biotechnology，sc-14007)和谷氨酸能标记物vGlut1(santa cruz biotechnology，sc-377425)。其显示了该诱导的神经元基本不表达中脑多巴胺能的标记物TH(santa cruz biotechnology，sc-14007)和谷氨酸能标记物vGlut1(santa cruz biotechnology，sc-377425)。
图1H显示了超高效液相-超高分辨率质谱检测显示诱导GABA神经元响应高钾缓冲液处理释放γ氨基丁酸。
图1I显示了实施例1-1中诱导的带GFP荧光标记的神经元移植到免疫缺陷小鼠海马区1月后神经元存活结果。
图1B-图1I明显表明，经过本发明所用myosin抑制剂(-)-Blebbistatin处理后的成纤维细胞具有极高的神经元转化效率。
实施例1-2
采用实施例1-1的实验方法，不同之处在于采用的myosin抑制剂为肌球蛋白 轻链激酶(MLCK)抑制剂ML-7。
分别处理来自20岁HFF(人包皮细胞)P15、21周/8岁/13岁人的包皮成纤维细胞，人21周背皮成纤维细胞，猴子尾尖成纤维细胞，鼠胎儿成纤维细胞及非增值细胞丝裂霉素C处理的鼠胎儿成纤维细胞，最终所得Tuj1阳性率和MAP2阳性率结果如表2所示。
表2
Figure PCTCN2018077192-appb-000006
由表2所示，采用myosin抑制剂肌球蛋白轻链激酶(MLCK)抑制剂ML-7诱导成纤维细胞向神经元细胞的转变情况，当其相对于神经元诱导培养基的浓度为15μM时，虽然取得了较好的神经元细胞转化效果，但并未达到最佳；而当采用浓度为20μM-30μM时，神经元细胞转化率达到最高。
实施例1-3
采用实施例1-1的实验方法，不同之处在于处理对象为小鼠尾尖成纤维细胞。
图2A显示了小鼠尾尖成纤维细胞未处理前神经元标记蛋白Tuj1和Map2染色图片。图2B显示了针对小鼠尾尖成纤维细胞采用包含20μM(-)-Blebbistatin的神经诱导培养基培养7天后表达神经元的Marker：Tuj1、Map2、NF200和NeuN。
针对小鼠尾尖成纤维细胞采用包含20μM(-)-Blebbistatin的神经诱导培养基培养7天后，再用成熟培养基培养14天后，测得表达Tuj1、Tuj1/Map2和Map2/NeuN阳性率分别为96％、96％、97％。
图2C显示了实施例1-3中针对小鼠尾尖成纤维细胞采用包含20μM(-)-Blebbistatin的神经诱导培养基培养7天后，再用成熟培养基培养14天后的细胞具有神经元成熟展现的钠电流、钾电流并有一定的动作电位的活性。
图2D显示了实施例1-3中诱导的带GFP荧光标记的神经元移植到免疫缺陷小鼠海马区1月后神经元存活结果。
本实施例表明经过本发明所用myosin抑制剂(-)-Blebbistatin处理后的小鼠尾尖成纤维细胞能够以高转化率转化为神经元细胞。
实施例2
采用实施例1-1的实验方法，不同之处在于分别采用actin组装抑制剂2μM细胞松弛素B(CB，sigma，C6762)和0.2μM Latrunculin B(BioVision，2182-1)替换myosin抑制剂分别诱导来自人21周、8岁、13岁人的包皮成纤维细胞，人21周背皮成纤维细胞，猴子尾尖成纤维细胞，鼠胎儿成纤维细胞及非增值细胞丝裂霉素C处理的鼠胎儿成纤维细胞，最终所得Tuj1阳性率和Map2阳性率结果如表3和表4所示。
表3
Figure PCTCN2018077192-appb-000007
Figure PCTCN2018077192-appb-000008
由表3所示，采用actin组装抑制剂Cytochalasin B诱导成纤维细胞向神经元细胞的转变情况，当其相对于神经元诱导培养基的浓度为1.5μM时，虽然取得了很好的神经元细胞转化效果，但并未达到最佳；而当采用浓度为2μM-3μM时，神经元细胞转化率达到最高。
表4
Figure PCTCN2018077192-appb-000009
Figure PCTCN2018077192-appb-000010
由表4所示，采用actin组装抑制剂Latrunculin B作为诱导成纤维细胞向神经元细胞的转变情况，当其相对于神经元诱导培养基的浓度为0.15μM时，虽然取得了很好的神经元细胞转化效果，但并未达到最佳；而当采用浓度为0.2μM和0.3μM时，神经元细胞转化率达到最高。
为了进一步说明actin组装抑制剂的效果，申请人提供了附图3A-3E。图3B-3E显示了针对成人包皮成纤维细胞(HFF20y，北京干细胞库)，采用包含Cytochalasin B和Latrunculin B进行神经诱导的细胞形态。图3A显示了采用不含任何抑制剂的DMSO培养基培养7天后的细胞形态。其中显示被处理的成纤维细胞并未有任何神经元形态产生。图3B显示了采用含有0.4μM Cytochalasin B的培养基培养7天后的细胞形态，其中显示被处理的成纤维细胞略有神经元形态的变化。图3C显示了采用含有2μM Cytochalasin B的培养基培养3天后的细胞形态，其中显示被处理的成纤维细胞已经有明显神经元形态的变化。图3D显示了采用含有0.2μM Latrunculin B的培养基培养7天后的细胞形态，其中显示被处理的成纤维细胞几乎完全转化位神经元形态。图3E显示了采用含有2μM Cytochalasin B的培养基培养7天后，又采用神经元成熟培养基培养10天后的细胞神经元表达神经元标记蛋白Map2，其中显示被处理的细胞明显神经命运形态的改变。
实施例3
本实施例提供了一种将细胞骨架蛋白抑制剂用于制备抗癌药物的具体实施方式，所述抗癌药物能够将神经胶质瘤细胞转分化为神经元细胞。
采用如下试剂配置抗肿瘤药物I：DMEM/F12(gibco，10565018)与Neurobasal(Gibco，21103-049)1比1混合，加入N2添加剂(100×，Gibco，17502048)，B27添加剂(50×，Gibco，17504044)，2％牛血清蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，Glutamax(200×，Gibco，35050-061),1μg/ml 胰岛素(Roche，11376497001)，双抗)，以及(-)-Blebbistatin(二甲基亚砜溶解(sigma，D2650)的100mM浓储，-20摄氏度避光保存1月)。调节(-)-Blebbistatin浓度相对于整个药物I为20μM。
采用如下试剂配置抗肿瘤药物II：基于抗肿瘤药物I配制100μM N6,2′-O-二丁酰基腺苷3′,5′-环磷酸钠盐(sigma，D0627)，20ng/ml Recombinant Human NT-3(Peprotech，450-03)，20ng/ml脑源性神经营养因子(peprotech，450-02)，20ng/ml神经胶质细胞源性的神经营养因子(peprotech，450-10)。
上述抗肿瘤药物I和II先后配合施用，抗肿瘤药物II在抗肿瘤药物I施加后3-7天再进行施用。
采用本实施例的抗肿瘤药物I和II配合分别处理人恶性胶质瘤细胞(U87)、星形胶质瘤细胞(U251)、人恶性胶质瘤细胞(LN229)和人胶质母细胞瘤细胞(T98G)。最终胶质瘤细胞的形态，生长曲线及转化结果相比于对照组(即药物试剂中不含有(-)-Blebbistatin)的对比情况如图4A和4B所示。其中，采用含有20μM的(-)-Blebbistatin及对照组DMSO分别处理人恶性胶质瘤细胞(U87)、星形胶质瘤细胞(U251)、人恶性胶质瘤细胞(LN229)和人胶质母细胞瘤细胞(T98G)，各实验组起始细胞量相同。
图4A显示，采用含有20μM的(-)-Blebbistatin相比于对照组DMSO在第3天有明显的神经元形态的变化，且细胞较对照组明显稀少。
进一步研究(-)-Blebbistatin对胶质瘤细胞增殖速率的影响。分别任选两株胶质瘤细胞U87和U251分别种于48孔板，细胞计数每孔5000个细胞，分别采用含有20μM的(-)-Blebbistatin及DMSO处理，每样3个平行孔，每间隔24小时利用Cell Counting Kit(CCK-8)CCK-8试剂盒(sigma，96992)检测细胞活力(细胞增殖活力)，总共测5天。具体步骤如：(1)在48孔板中接种细胞悬液(100μL/孔)；(2)将培养板放在培养箱中预培养一段时间(37℃,5％CO 2)；(3)向每孔加入10μl CCK溶液；(4)用酶标仪测定在450nm处的吸光度。
图4B显示了本实施例的抗肿瘤药物显著抑制胶质瘤细胞细胞的增殖，在前5天对任选的胶质瘤细胞U87和U251抑制效率分别为50％和99％。
图4C显示了本实施例的抗肿瘤药物将任选的快速增殖胶质瘤细胞U87转分化为分化的神经元细胞，表达神经元标记蛋白Tuj1,其阳性率接近95％。
实施例4
(siRNA敲低细胞骨架系统蛋白成分的转化实验及效果)
将人包皮成纤维细胞以1×10 5的密度接种于12孔板中，用1ml的成纤维细胞培养液(DMEM+10％FBS)培养。当细胞达到50–70％汇合度时利用
Figure PCTCN2018077192-appb-000011
RNAiMAX(13778150，invitrogen)试剂盒分别转染靶位rock1、rock2、mrlc1、mrlc2、mrlc3、myh9、myh10、mrckα、mrckβ、lamina/c、lmnb1、lbr、sun1、sun2、cbx1、cbx3、cbx5、banf1、syne1、syne2、β-actin。具体步骤如下：(1)分别稀释如序列SEQ ID NO: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的siRNA：在无RNA酶的EP管，用100μL/每孔的无血清Opti-MEM培养基稀释12pmol siRNA双链(2.5ul)，温和混匀；(2)稀释转染试剂
Figure PCTCN2018077192-appb-000012
RNAiMAX Reagent：在无RNA酶的EP管，用100μL/每孔of无血清Opti-MEM培养基稀释
Figure PCTCN2018077192-appb-000013
RNAiMAX Reagent，温和混匀；(3)稀释好的
Figure PCTCN2018077192-appb-000014
RNAiMAX Reagent经过2min的孵育后，与上述(2)稀释好的siRNA轻轻混和，室温培养20min以形成siRNA-
Figure PCTCN2018077192-appb-000015
RNAiMAX Reagent混和物，溶液可能会有浑浊，不过不会影响。(4)将上述(3)的混合液加入含有细胞以及培养液的细胞培养板中，轻轻摇晃，使之混和；(5)将培养板置于37℃的CO 2培养箱中培养，转染隔6-8小时后换成神经元培养液。(6)转染48-72小时。
表5显示了siRNA序列与其所敲低的基因表达的对应关系。
表5
基因名 NCBI ID 序列表中编号 序列
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
任一靶点敲低后第7天有明显的神经形态的变化，其中部分靶点敲低后的细胞形态变化图如图5A所示。图5B免疫荧光染色结果显示诱导第7天表达神经元命运的特异蛋白Tuj1。
神经元培养基(N2B27培养液：DMEM/F12(gibco，10565018)与Neurobasal(Gibco，21103-049)1比1混合，加入N2添加剂(100×，Gibco，17502048)，B27添加剂(50×，Gibco，17504044)，2％牛血清蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，Glutamax(200×，Gibco，35050-061),1μg/ml胰岛素(Roche，11376497001)，双抗)。
实施例5
(干扰胞外基质处理低黏附的转化实验及效果)
首先用无菌双蒸水配置1％琼脂糖溶液，微波炉加热至沸腾，随后加入等体积的2×DMEM，(Gibco,12800-017)制成溶于DMEM中的0.5％的琼脂糖溶液，然后倒入6cm的培养皿中，冷却凝固后备用。
将人包皮成纤维细胞以1×105的密度接种于上述培养皿中，用神经干细胞 培养液悬浮培养7天，隔天半量换液。悬浮培养第7天后的光镜照片见图6A。其中已经显示了神经元形态。
用含有1ug/ml的纤连蛋白(Millipore,FC010)的1×PBS处理置于四孔板中的圆玻片，并置于培养箱中过夜包被备用。悬浮培养第7天的人成纤维细胞离心后用Tryple(Gibco,A1285901)在37℃培养箱中消化3min，用PBS稀释后离心，弃上清后用神经干细胞培养液重悬后以每孔5×104接种于四孔板中，次日去掉培养液，进行免疫荧光染色，具体如下：
于室温条件下，用含有4％多聚甲醛(Sigma,158127)的PBS溶液固定20min，PBS洗3次，每次5min。然后在含有0.3％TritonX-100(Solarbio,T8200)和0.2％BSA(Sigma,A3803)的PBS溶液中通透1h。接着在4℃下，在含有2％的BSA和一抗：鼠抗-Nestin(Millipore,MAB353,1:100)、羊抗-Sox2(Santa,sc-17320,1:100)、鼠抗-Nkx2.2(abcam,ab187375，)兔抗-En1(abcam,ab70993,1:50)，兔抗-N-cadherin(abcam,ab12221,1:100)、兔抗-Pax6(abcam,ab5790,1:50)的PBS溶液中过夜孵育。然后，用PBS溶液洗涤细胞三次，并在室温条件下，用抗兔、抗鼠、或抗羊二抗，，Alexa Fluor-488或Alexa Fluor-561(1:500,Invitrogen)孵育1小时。然后用PBS溶液洗涤细胞三次，再用含有Hoechst(Inventgen,H3570,1:1000)的PBS溶液染细胞核5min。然后将圆玻片倒扣在滴有防荧光淬灭剂的载玻片上，指甲油封片。随后利用Leica双光子共聚焦显微镜观察细胞。检测结果见图6B，其中Merge表示重合图。
图6B的结果表明，经过低黏附悬浮培养的人包皮成纤维细胞可转化为Nestin和Sox2双阳性的神经干细胞。进一步的，这些双阳性细胞对Nkx2,2、En1、N-cad和Pax6也呈阳性。
上述鉴定的神经干细胞同样的在第7天按照上述方法接种于四孔板中，随机分化组用随机分化培养液培养，定向分化组用定向分化培养液培养，隔天半量换液，第14天进行免疫荧光染色。其中一抗为兔抗-Tuj1(abcam,ab18207,1:2000)、鼠抗-GFAP(sigma,G3893,1:100)、鸡抗-NF2000(abcam,ab4680,1:1000)。检测结果分别见6C和6D。
图6C的结果表明，低黏附处理得到的神经干细胞经过随机分化可得到GFAP阳性的星型胶质细胞，进一步的这些细胞对Tuj1呈阴性。
图6D的结果表明，低黏附处理得到的神经干细胞经过定向分化可得到Tuj1和NF2000双阳性的神经元。
基础培养液
由DMEM/F12(Gibco,12400-024)和Neurobasal(Gibco,21103-049)按体积比1:1制成，并添加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)。
神经干细胞培养液
由基础培养液添加20ng/ml的bFGF(表皮生长因子，R&D,233-FB-001MG/CF)配制。
随机分化培养液
由基础培养液添加1％FBS(胎牛血清，Gibco,16000-044)配制。
定向分化培养液
基础培养液添加20ng/ml的GDNF(peprotech,450-10)，20ng/ml的BDNF(peprotech,450-02)，20ng/ml的NT3(peprotech,450-03)，4ng/ml的Forskolin(stemgent,04-0025)配制。
实施例6
(原代星形胶质细胞转分化为神经元)
以12孔板为例(corning，3335)每孔用20ug/ml纤连蛋白溶液(millipore，fc010)1×PBS配制，包被6小时，或先用0.1mg/ml多聚赖氨酸溶液(sigma，P6407)无菌水配制，包被培养皿2小时，无菌水洗三次，再用10μg/ml层粘连蛋白溶液(sigma，L6274)1×PBS配制，包被6小时。去掉包被液用1×PBS冲液洗一遍。
去掉洗液，将成人原代星形胶质细胞(ScienCell，1800)或小鼠大脑皮层分离的原代神经胶质细胞均匀种于每一个孔，用基础培养液(高糖DMEM(Gibco，C12430500BT)，1×丙酮酸钠(100×，gibco，11360-070)，1×非必须氨基酸(100×，Gibco，11140-050)，双抗)加10％胎牛血清(Gibco，16000-044)培养24小时。去除培养液，PBS洗一遍。
选用本发明的细胞转化试剂盒，所述试剂盒包括如下的诱导培养基和成熟培养基。
将经上述处理后的神经胶质细胞加入神经元诱导培养基(N2B27培养液：DMEM/F12(gibco，10565018)与Neurobasal(Gibco，21103-049)1比1混合， 加入N2添加剂(100×，Gibco，17502048)，B27添加剂(50×，Gibco，17504044)，2％牛血清蛋白(1000×，sigma，A8022)，β-巯基乙醇(1000×，Gibco，21985023)，Glutamax(200×，Gibco，35050-061),1μg/ml胰岛素(Roche，11376497001)，双抗)。加入myosin抑制剂(-)-Blebbistatin(二甲基亚砜溶解(sigma，D2650)的100mM浓储，-20摄氏度避光保存1月)，(-)-Blebbistatin在诱导培养基中的终浓度为15-30μM。培养3-7天，培养的细胞有明显神经元形态，大的胞体和长的轴突等及神经元命运标记物Tuj1染色，并统计Tuj1阳性率(Tuj1阳性细胞/细胞核比值)。
图7A显示了原代星形胶质细胞形态(左图)及表达星形胶质细胞的标记蛋白GFAP(右图)。如图7B所示，(-)-Blebbistatin诱导13天的星形胶质细胞表现为经典神经元形态。如图7C所示，(-)-Blebbistatin诱导星形胶质细胞20天，接近100％表达经典神经元标记蛋白Tuj1。

Claims (15)

  1. 一种将非神经元细胞转分化为神经元细胞的方法，其特征在于所述方法包括对所述非神经元细胞的胞外基质-骨架系统进行干扰处理。
  2. 根据权利要求1所述的方法，其中所述干扰处理选自以下的至少一种：采用细胞骨架蛋白抑制剂进行处理，采用小干扰RNA(siRNA)对胞外基质-骨架系统的基因表达进行敲低处理，和对胞外基质进行低粘附处理。
  3. 根据权利要求2所述的方法，其中所述细胞骨架蛋白抑制剂选自以下的至少一种：肌球蛋白(myosin)抑制剂、肌动蛋白(actin)组装抑制剂；
    优选地，其中所述肌球蛋白(myosin)抑制剂选自以下的至少一种：(-)-Blebbistatin、肌球蛋白轻链激酶(MLCK)抑制剂ML-7，所述肌球蛋白(myosin)抑制剂浓度为10μM以上，优选20μM以上，更优选10-30μM，其中所述浓度为所述肌球蛋白(myosin)抑制剂在处理非神经元细胞所用诱导培养基中的浓度；
    优选地，其中所述肌动蛋白(actin)组装抑制剂选自以下的至少一种：Cytochalasin B、Latrunculin B，其中Cytochalasin B的浓度为1.5μM以上，优选2μM以上，更优选2-3μM，Latrunculin B浓度为0.15μM以上，优选0.2μM，更优选0.2-0.3μM，其中所述浓度为所述抑制剂Cytochalasin B或Latrunculin B在处理非神经元细胞所用诱导培养基中的浓度。
  4. 根据权利要求3所述的方法，其中，
    在所述干扰处理为采用细胞骨架蛋白抑制剂进行处理或采用小干扰RNA(siRNA)对胞外基质-骨架系统的基因表达进行敲低处理时，
    所述方法还包括将非神经元细胞置于诱导培养基中培养3-7天，然后采用成熟培养基培养7-14天；
    优选地，其中所述诱导培养基包含：所述细胞骨架蛋白抑制剂、N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇；
    优选地，其中所述成熟培养基包含：N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇，神经营养素(NT3)，脑源性神经营养因子(BDNF)，胶质细胞系衍生的神经营养因子(GDNF)，二丁酰环腺苷酸(db-cAMP)。
  5. 根据权利要求1至4任一项所述的方法，其中所述非神经元细胞为成纤维细胞和/或神经胶质细胞。
  6. 细胞骨架蛋白抑制剂用于将非神经元细胞转分化为神经元细胞的用途。
  7. 一种将非神经元细胞转分化为神经元细胞的试剂盒，其特征在于所述试剂盒包括诱导培养基，所述诱导培养基包含细胞骨架蛋白抑制剂。
  8. 根据权利要求7所述的试剂盒，其中所述细胞骨架蛋白抑制剂选自以下的至少一种：肌球蛋白(myosin)抑制剂、肌动蛋白(actin)组装抑制剂；
    优选地，其中所述肌球蛋白(myosin)抑制剂选自以下的至少一种：(-)-Blebbistatin、肌球蛋白轻链激酶(MLCK)抑制剂ML-7，浓度为10μM以上，优选20μM以上，更优选10-30μM，其中所述浓度为所述所述肌球蛋白(myosin)抑制剂在诱导培养基中的浓度；
    优选地，其中所述肌动蛋白(actin)组装抑制剂选自以下的至少一种：Cytochalasin B、Latrunculin B，其中Cytochalasin B的浓度为1.5μM以上，优选2μM以上，更优选2-3μM，Latrunculin B浓度为0.15μM以上，优选0.2μM，更优选0.2-0.3μM，其中所述浓度为所述抑制剂Cytochalasin B或Latrunculin B在诱导培养基中的浓度。
  9. 根据权利要求7或8所述的试剂盒，其中，所述试剂盒还包括成熟培养基，
    优选地，其中所述诱导培养基还包含：N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇；
    优选地，其中所述成熟培养基包含：N2细胞培养基添加剂，B27细胞培养基添加剂，谷氨酰胺，β巯基乙醇，神经营养素(NT3)，脑源性神经营养因子(BDNF)，胶质细胞系衍生的神经营养因子(GDNF)，二丁酰环腺苷酸(db-cAMP)。
  10. 根据权利要求7或8所述的试剂盒，其中所述非神经元细胞为成纤维细胞和/或神经胶质细胞。
  11. 细胞骨架蛋白抑制剂在制备抗肿瘤药物、组织再生和/或修复药物中的用途。
  12. 根据权利要求2所述的方法，其中所述敲低处理包括以下的至少一种：
    采用与序列SEQ ID NO:1具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的rock1基因表达，
    采用与序列SEQ ID NO:2具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的rock2基因表达，
    采用与序列SEQ ID NO:3具有95％、96％、97％、98％、99％或100％序列同 一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc1基因表达，
    采用与序列SEQ ID NO:4具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc2基因表达，
    采用与序列SEQ ID NO:5具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc3基因表达，
    采用与序列SEQ ID NO:6具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的myh9基因表达，
    采用与序列SEQ ID NO:7具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的myh10基因表达，
    采用与序列SEQ ID NO:8具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrckα基因表达，
    采用与序列SEQ ID NO:9具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrckβ基因表达，
    采用与序列SEQ ID NO:10具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的lamina/c基因表达，
    采用与序列SEQ ID NO:11具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的lmnb1基因表达，
    采用与序列SEQ ID NO:12具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的lbr基因表达，
    采用与序列SEQ ID NO:13具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的sun1基因表达，
    采用与序列SEQ ID NO:14具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的sun2基因表达，
    采用与序列SEQ ID NO:15具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的cbx1基因表达，
    采用与序列SEQ ID NO:16具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的cbx3基因表达，
    采用与序列SEQ ID NO:17具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的cbx5基因表达，
    采用与序列SEQ ID NO:18具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的banf1基因表达，
    采用与序列SEQ ID NO:19具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的syne1基因表达，
    采用与序列SEQ ID NO:20具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的syne2基因表达，
    采用与序列SEQ ID NO:21具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的β-actin基因表达。
  13. 根据权利要求12所述的方法，其中所述敲低处理优选：
    采用与序列SEQ ID NO:1具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的rock1基因表达，采用与序列SEQ ID NO:2具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的rock2基因表达，采用与序列SEQ ID NO:8具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrckα基因表达，以及采用与序列SEQ ID NO:9具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrckβ基因表达；
    采用与序列SEQ ID NO:3具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc1基因表达，采用与序列SEQ ID NO:4具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc2基因表达，以及采用与序列SEQ ID NO:5具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc3基因表达；
    采用与序列SEQ ID NO:6具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的myh9基因表达，以及采用与序列SEQ ID NO:7具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的myh10基因表达；
    采用与序列SEQ ID NO:3具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc1基因表达，采用与序列SEQ ID NO:4具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc2基因表达，采用与序列SEQ ID NO:5具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的mrlc3基因表达，以及采用与序列SEQ ID NO:6具 有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的myh9基因表达；或是
    采用与序列SEQ ID NO:13具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的sun1基因表达，以及采用与序列SEQ ID NO:14具有95％、96％、97％、98％、99％或100％序列同一性的小干扰RNA(siRNA)敲低胞外基质-骨架系统中的sun2基因表达。
  14. 根据权利要求12或13所述的方法，所述敲低处理包括：
    将稀释的脂质体与所述小干扰RNA(siRNA)混和形成混合物；
    将所述混合物与含有非神经元细胞的培养液混和均匀进行转染、培养。
  15. 根据权利要求2所述的方法，其中所述低粘附处理包括采用琼脂糖DMEM悬浮培养非神经元细胞；
    优选地，悬浮培养时间为6天、7天或8天；
    优选地，所述琼脂糖的用量为0.5g/100毫升所述琼脂糖DMEM；
    优选地，非神经元细胞采用琼脂糖DMEM悬浮培养后，再采用定向分化培养液培养；
    优选地，所述定向分化培养液包含：神经胶质细胞源性的神经营养因子(GDNF)、脑源性神经营养因子(BDNF)、神经营养因子-3(NT3)和毛喉素(Forskolin)；
    优选地，所述定向分化培养液中神经胶质细胞源性的神经营养因子(GDNF)浓度为15-25ng/ml，优选20ng/ml，脑源性神经营养因子(BDNF)浓度为15-25ng/ml，优选20ng/ml，神经营养因子-3(NT3)浓度为15-25ng/ml，优选20ng/ml，毛喉素(Forskolin)浓度为2-6ng/ml，优选3、4或5ng/ml。
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