CN111500538A

CN111500538A - Method for converting non-neuron cells into neuron cells

Info

Publication number: CN111500538A
Application number: CN202010322435.3A
Authority: CN
Inventors: 周琪; 李伟; 胡宝洋; 何正泉; 王柳; 郝捷
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2017-03-01
Filing date: 2017-03-01
Publication date: 2020-08-07
Also published as: CN108531453B; WO2018157769A1; CN111471653A; CN108531453A; CN111471653B

Abstract

The invention provides a method for transdifferentiating non-neuronal cells into neuronal cells, comprising an extracellular matrix-scaffold system interference treatment of the non-neuronal cells, wherein the interference treatment is selected from the group consisting of treatment with a small molecule inhibitor of a cytoskeletal protein, treatment for knocking down specific gene expression of the extracellular matrix-scaffold system with small interfering RNA (siRNA), treatment for low adhesion of the extracellular matrix and directed differentiation culture.

Description

Method for converting non-neuron cells into neuron cells

The application is divisional application of a patent application with Chinese application number of 201710117871.5, entitled "a method for converting non-neuronal cells into neuronal cells" and application date of 2017, 03 and 01.

Technical Field

The invention relates to the field of biotechnology, in particular to a method for converting non-neuronal cells of human and animals into functional neurons by carrying out interference treatment on an extracellular matrix-scaffold system of the non-neuronal cells.

Background

Regulating cell fate to produce specific cell types with different functions, and has important application foreground in cell substituting treatment and regeneration treatment. The fate of the cell depends on the specific expression of the genome, and the expression regulation mode includes common biological regulation, such as signal transduction, transcription regulation network, epigenetic modification, etc., and is also regulated by the physicochemical characteristics of the cell and the physicochemical factors in the environment where the cell is located. Therefore, the methods for cell fate transition are also divided into two methods, namely, the modification of biological and physicochemical properties of cells.

At present, the method for generating functional cells by regulating cell fate is mainly completed by genetic means or chemical micromolecule processing means aiming at a plurality of important gene regulation and control paths and epigenetic modification. Genetic means include researchers reprogramming mouse and human fibroblasts into induced pluripotent stem cells by ectopically expressing transcription factors such as Oct4, Sox2, c-Myc, Klf4 and the like; the fibroblast is transdifferentiated into a functional neuron by using transcription factors such as ectopic expression Adcl1, Brn2 and Myt1 l; and functional myocardial cells, islet cells and the like are obtained by ectopically expressing specific genes. The chemical small molecule approach includes that researchers reprogram mouse fibroblasts into pluripotent stem cells by using a chemical small molecule combination VC6 TFZ; the combination (VCRFSGY) composed of 7 small molecules is used for directly converting human fibroblasts into neurons; reprogramming mouse fibroblasts into neural stem cells by using a combination M9 of 9 small molecules, and further differentiating the neural stem cells into functional neurons; transforming human gastric epithelial cells into multipotent endodermal progenitor cells by using small molecule combination; the small molecule combination is used to transform human fibroblasts into myocardial cells and the like through small molecules.

Disclosure of Invention

The invention mainly carries out interference treatment on the extracellular matrix-skeleton system of the non-neuron cell, thereby realizing regulation and control on cell fate, and particularly provides a simpler and more convenient way in the aspect of transdifferentiation of the non-neuron cell of human or animal into the neuron cell, thereby obtaining creative and unexpected technical effects.

Specifically, the present invention relates to the following:

1. a method of transdifferentiating non-neuronal cells into neuronal cells, characterized in that the method comprises subjecting the extracellular matrix-scaffold system of the non-neuronal cells to an interference treatment.

2. The method of item 1, wherein the interference processing is selected from at least one of: treatment with cytoskeletal protein inhibitors, knock-down of gene expression from the extracellular matrix-scaffold system with small interfering RNA (siRNA), and low adhesion of the extracellular matrix.

3. The method of item 2, wherein the cytoskeletal protein inhibitor is selected from at least one of: myosin (myostatin) inhibitors, actin (actin) assembly inhibitors;

preferably, wherein the myosin inhibitor is selected from at least one of (-) -Blebbistatin, myosin light chain kinase (M L CK) inhibitor M L-7, at a concentration of 10 μ M or more, preferably 20 μ M or more, more preferably 10-30 μ M, wherein the concentration is the concentration of the myosin inhibitor in the induction medium used to treat the non-neuronal cells;

preferably, wherein the actin (actin) assembly inhibitor is selected from at least one of Cytochaisin B, L atroncin B, wherein the concentration of Cytochaisin B is above 1.5. mu.M, preferably above 2. mu.M, more preferably 2-3. mu.M, and the concentration of L atroncin B is above 0.15. mu.M, preferably 0.2. mu.M, more preferably 0.2-0.3. mu.M, wherein said concentration is the concentration of the inhibitor Cytochaisin B or L atroncin B in the induction medium used to treat non-neuronal cells.

4. The method of item 3, wherein,

when the interference treatment is treatment by cytoskeletal protein inhibitor or knockdown treatment by small interfering RNA (siRNA) on the gene expression of the extracellular matrix-skeleton system,

the method also comprises placing the non-neuronal cells in an induction medium for culturing for 3-7 days, and then culturing for 7-14 days with a maturation medium;

preferably, wherein the induction medium comprises the cytoskeletal protein inhibitor, N2 cell culture medium additive, B27 cell culture medium additive, glutamine, β mercaptoethanol;

preferably, wherein the maturation medium comprises N2 cell culture medium additives, B27 cell culture medium additives, glutamine, β mercaptoethanol, neurotrophins (NT3), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).

5. The method of any one of items 1 to 4, wherein the non-neuronal cells are fibroblasts and/or glial cells.

6. Use of a cytoskeletal protein inhibitor for transdifferentiating non-neuronal cells into neuronal cells.

7. A kit for transdifferentiating non-neuronal cells into neuronal cells, characterized in that the kit comprises an induction medium comprising a cytoskeletal protein inhibitor.

8. The kit of item 7, wherein the cytoskeletal protein inhibitor is selected from at least one of: myosin (myostatin) inhibitors, actin (actin) assembly inhibitors;

preferably, wherein the myosin inhibitor is selected from at least one of (-) -Blebbistatin, myosin light chain kinase (M L CK) inhibitor M L-7 at a concentration of 10 μ M or more, preferably 20 μ M or more, more preferably 10-30 μ M, wherein the concentration is the concentration of the myosin inhibitor in the induction medium;

preferably, wherein the actin (actin) assembly inhibitor is selected from at least one of Cytochaisin B, L atroncin B, wherein the concentration of Cytochaisin B is above 1.5. mu.M, preferably above 2. mu.M, more preferably 2-3. mu.M, and the concentration of L atroncin B is above 0.15. mu.M, preferably 0.2. mu.M, more preferably 0.2-0.3. mu.M, wherein said concentration is the concentration of the inhibitor Cytochaisin B or L atroncin B in the induction medium.

9. The kit according to item 7 or 8, wherein the kit further comprises a maturation medium,

preferably, wherein the induction medium further comprises N2 cell culture media additives, B27 cell culture media additives, glutamine, β mercaptoethanol;

10. The kit of item 7 or 8, wherein the non-neuronal cells are fibroblasts and/or glial cells.

11. Use of cytoskeletal protein inhibitors in the preparation of anti-tumor medicaments, tissue regeneration and/or repair medicaments.

12. The method of item 2, wherein the knock-down treatment comprises at least one of:

knockdown of rock1 gene expression in extracellular matrix-scaffold systems using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 1,

knockdown of rock2 gene expression in extracellular matrix-scaffold systems using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 2,

knockdown of mrlc1 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 3,

knockdown of mrlc2 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 4,

knockdown of mrlc3 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 5,

knockdown of myh9 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 6,

knockdown of myh10 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 7,

knockdown of mrck α gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 8,

knockdown of mrck β gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 9,

uses small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence SEQ ID NO 10 to knock down the expression of the lamina/c gene in the extracellular matrix-skeleton system,

knockdown of lmnb1 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 11,

uses small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence SEQ ID NO 12 to knock down the lbr gene expression in the extracellular matrix-skeleton system,

knockdown of the expression of the sun1 gene in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 13,

knockdown of the expression of the sun2 gene in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO. 14,

using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO. 15 to knock down cbx1 gene expression in the extracellular matrix-scaffold system,

knock down cbx3 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 16,

knockdown of cbx5 gene expression in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 17,

knockdown of the expression of banf1 gene in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 18,

knockdown of the gene expression of syne1 in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 19,

knockdown of the expression of the syne2 gene in the extracellular matrix-scaffold system using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 20,

β -actin gene expression in the extracellular matrix-scaffold system was knocked down using small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 21.

13. The method of item 12, wherein the knockdown process preferably:

knockdown of rock1 gene expression in extracellular matrix-scaffold system using small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 1, knockdown of rock2 gene expression in extracellular matrix-scaffold system using small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 2, knockdown of mrck α gene expression in extracellular matrix-scaffold system using small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 8, and knockdown of mrck β gene expression in extracellular matrix-scaffold system using small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 9;

knocking down mrlc1 gene expression in extracellular matrix-scaffold system with small interfering rna (sirna) having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 3, knocking down mrlc2 gene expression in extracellular matrix-scaffold system with small interfering rna (sirna) having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 4, and knocking down mrlc3 gene expression in extracellular matrix-scaffold system with small interfering rna (sirna) having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 5;

knockdown of myh9 gene expression in extracellular matrix-scaffold system with small interfering RNA (siRNA) having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 6 and of myh10 gene expression in extracellular matrix-scaffold system with small interfering RNA (siRNA) having 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 7;

the sequence of the polypeptide is similar to the sequence SEQ ID NO:3 small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to knock down mrlc1 gene expression in the extracellular matrix-scaffold system using a sequence identical to the sequence SEQ ID NO:4 small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to knock down mrlc2 gene expression in the extracellular matrix-scaffold system using a sequence identical to the sequence SEQ ID NO:5 Small interfering RNA (siRNA) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to knock down mrlc3 gene expression in an extracellular matrix-scaffold system, and the use of a peptide having the sequence of SEQ ID NO:6 small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity knockdown myh9 gene expression in the extracellular matrix-scaffold system; or is

Knockdown of the sun1 gene expression in the extracellular matrix-scaffold system using small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 13 and knockdown of the sun2 gene expression in the extracellular matrix-scaffold system using small interfering rna (sirna) with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 14.

14. The method of item 12 or 13, wherein the knock-down process comprises:

mixing diluted liposomes with said small interfering RNA (siRNA) to form a mixture;

and uniformly mixing the mixture with a culture solution containing non-neuronal cells, and performing transfection and culture.

15. The method of item 2, wherein the low adhesion treatment comprises culturing non-neuronal cells in suspension with agarose DMEM;

preferably, the suspension culture time is 6 days, 7 days or 8 days;

preferably, the agarose is used in an amount of 0.5g/100 ml of the agarose DMEM;

preferably, the non-neuronal cells are cultured by using an oriented differentiation culture solution after agarose DMEM suspension culture;

preferably, the directed differentiation medium comprises: glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT3), and Forskolin (Forskolin);

preferably, the concentration of glial cell line-derived neurotrophic factor (GDNF) in the directed differentiation medium is 15-25ng/ml, preferably 20ng/ml, the concentration of brain-derived neurotrophic factor (BDNF) is 15-25ng/ml, preferably 20ng/ml, the concentration of neurotrophic factor-3 (NT3) is 15-25ng/ml, preferably 20ng/ml, and the concentration of Forskolin (Forskolin) is 2-6ng/ml, preferably 3, 4 or 5 ng/ml.

The invention provides a method for converting non-neuronal cells into neuronal cells, characterized in that the method comprises an interference treatment of the extracellular matrix-scaffold system of the non-neuronal cells.

The interference handling of the present invention is selected from at least one of: treating with cytoskeletal protein inhibitor, knocking down the gene expression of the extracellular matrix-scaffold system with small interfering RNA (siRNA), performing low adhesion treatment on the extracellular matrix and preferably further performing directional culture.

According to one embodiment of the invention, wherein the cytoskeletal protein inhibitor is selected from at least one of the following: myosin (myostatin) inhibitors, actin (actin) assembly inhibitors.

Preferably, wherein the myosin inhibitor is selected from at least one of (-) -Blebbistatin, myosin light chain kinase (M L CK) inhibitor M L-7 at a concentration of 10 μ M or more, preferably 20 μ M or more, more preferably 10-30 μ M, wherein the concentration is the final concentration of the inhibitor in the induction medium used to treat the non-neuronal cells.

Preferably, wherein the actin (actin) assembly inhibitor is selected from at least one of Cytochaisin B, L atronculin B, wherein the concentration of Cytochaisin B is 1.5. mu.M or more, preferably 2. mu.M or more, more preferably 2-3. mu.M, and L atronculin B is 0.15. mu.M or more, preferably 0.2. mu.M, more preferably 0.2-0.3. mu.M, wherein the concentration is the final concentration of the inhibitor in the induction medium used to treat the non-neuronal cells.

According to one embodiment of the invention, the method comprises placing the non-neuronal cells in an induction medium for 3-7 days, optionally 4 days, 5 days or 6 days, followed by culture with maturation medium for 7-14 days, optionally 8 days, 9 days, 10 days, 11 days, 12 days or 13 days.

Preferably, wherein the induction medium comprises a cytoskeletal protein inhibitor, N2 cell culture medium additives, B27 cell culture medium additives, glutamine, β mercaptoethanol.

According to one embodiment of the invention, 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 transdifferentiating 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.

Preferably, wherein the myosin inhibitor is selected from at least one of (-) -Blebbistatin, myosin light chain kinase (M L CK) inhibitor M L-7 at a concentration of 10. mu.M or more, preferably 20. mu.M or more, more preferably 10-30. mu.M, wherein the concentration is the concentration of the inhibitor in the induction medium.

Preferably, wherein the actin (actin) assembly inhibitor is selected from at least one of Cytochaisin B, L atronculin B, wherein the concentration of Cytochaisin B is 1.5. mu.M or more, preferably 2. mu.M or more, more preferably 2-3. mu.M, and the concentration of L atronculin B is 0.15. mu.M or more, preferably 0.2. mu.M, more preferably 0.2-0.3. mu.M, wherein the concentration is the concentration of the inhibitor in the induction medium.

According to one embodiment of the invention, the kit further comprises a maturation medium.

The invention also provides application of the cytoskeletal protein inhibitor in preparing anti-tumor drugs and drugs for tissue regeneration and/or repair.

According to one embodiment of the present invention, the inventive knock-down process comprises at least one of:

siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 1 is used to knock down rock1 gene expression in the extracellular matrix-scaffold system,

siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 2 is used to knock down rock2 gene expression in the extracellular matrix-scaffold system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 3 to knock down the expression of mrlc1 gene in the extracellular matrix-scaffold system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO. 4 to knock down the expression of mrlc2 gene in the extracellular matrix-scaffold system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 5 to knock down the expression of mrlc3 gene in the extracellular matrix-scaffold system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with sequence SEQ ID NO 6 to knock down myh9 gene expression in extracellular matrix-skeleton system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO. 7 to knock down myh10 gene expression in the extracellular matrix-scaffold system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 8 to knock down the expression of the mrck α gene in the extracellular matrix-scaffold system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with sequence SEQ ID NO 9 to knock down the expression of mrck β gene in extracellular matrix-skeleton system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 10 to knock down the expression of the lamina/c gene in the extracellular matrix-skeleton system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with sequence SEQ ID NO. 11 to knock down lmnb1 gene expression in extracellular matrix-skeleton system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence SEQ ID NO. 12 to knock down the lbr gene expression in the extracellular matrix-skeleton system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with sequence SEQ ID NO 13 to knock down the expression of sun1 gene in extracellular matrix-skeleton system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence SEQ ID NO. 14 to knock down the expression of the sun2 gene in the extracellular matrix-skeleton system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 15 to knock down cbx1 gene expression in the extracellular matrix-scaffold system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 16 to knock down cbx3 gene expression in the extracellular matrix-scaffold system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with sequence SEQ ID NO 17 to knock down cbx5 gene expression in extracellular matrix-skeleton system,

adopting siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity with sequence SEQ ID NO 18 to knock down the expression of banf1 gene in extracellular matrix-skeleton system,

using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 19 to knock down the syne1 gene expression in the extracellular matrix-scaffold system,

the expression of syne2 gene in extracellular matrix-scaffold system was knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 20,

β -actin gene expression in the extracellular matrix-scaffold system was knocked down using siRNAs with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID NO 21.

According to one embodiment of the invention, the expression of rock1 gene in the extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 1, the expression of rock2 gene in the extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 2, the expression of mrck α gene in the extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 8, and the expression of mrck β gene in the extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 9.

According to one embodiment of the invention, the expression of mrlc1 gene in extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 3, the expression of mrlc2 gene in extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 4, and the expression of mrlc3 gene in extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 5.

According to one embodiment of the invention, the expression of myh9 gene in the extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 6 and the expression of myh10 gene in the extracellular matrix-scaffold system is knocked down using siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID NO 7.

According to one embodiment of the invention, mrlc1 gene expression in the extracellular matrix-scaffold system is knocked down using sirnas with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 3, mrlc2 gene expression in the extracellular matrix-scaffold system is knocked down using sirnas with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 4, mrlc3 gene expression in the extracellular matrix-scaffold system is knocked down using sirnas with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 5, and myh9 gene expression in the extracellular matrix-scaffold system is knocked down using sirnas with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence SEQ ID No. 6.

According to one embodiment of the invention, siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 13 is used to knock down the expression of the sun1 gene in the extracellular matrix-scaffold system, and siRNA with 95%, 96%, 97%, 98%, 99% or 100% sequence identity to sequence SEQ ID No. 14 is used to knock down the expression of the sun2 gene in the extracellular matrix-scaffold system.

According to one embodiment of the present invention, the knock-down process of the present invention comprises the steps of:

mixing the diluted liposomes with the siRNA of the present invention, and culturing at room temperature to form an siRNA-liposome mixture; and uniformly mixing the mixture with a culture solution containing non-neuronal cells, and performing transfection and culture.

Preferably, liposomes are used

RNAiMAX Reagent.

Preferably, the siRNA is diluted prior to mixing the liposome with the siRNA.

Preferably, the liposomes and siRNA are diluted using serum-free Opti-MEM medium.

Preferably, siRNA is diluted 12pmol siRNA duplexes (2.5ul) in RNase free EP tubes in 100. mu. L/per well of serum free Opti-MEM medium.

Preferably, the transfection reagent is diluted

RNAimax Reagent in RNase-free EP tubes, diluted with 100. mu. L/well of serum-free Opti-MEM medium

RNAiMAX Reagent。

Preferably, the diluted liposome is mixed with the diluted siRNA after 1-5min, preferably 2min, preferably 3min, preferably 4min of incubation, and cultured at room temperature for 15-25min, preferably 20min, to form siRNA-liposome mixture.

Preferably, the siRNA-liposome mixture is added to and mixed with a cell culture plate containing the cells and culture medium.

Preferably, the plates are placed in CO at 37 ℃₂Culturing in an incubator, transfecting every 6-8 hours, preferably 7 hours, and then replacing with neuron culture solution.

Preferably, the neuronal culture fluid is cultured for 48 to 72 hours, preferably 60 hours.

Preferably, the neuron culture solution comprises N2 cell culture medium additive, B27 cell culture medium additive, glutamine, β mercaptoethanol.

Preferably, the neuron culture fluid comprises N2 cell culture medium additives, B27 cell culture medium additives, glutamine, β mercaptoethanol, neurotrophins (NT3), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), dibutyryl cyclic adenosine monophosphate (db-cAMP).

According to one embodiment of the invention, the low adhesion treatment comprises culturing non-neuronal cells in suspension with agarose DMEM. Preferably, the suspension culture time is 6-8 days, preferably 7 days. Preferably, the agarose is used in an amount of 0.5g/100 ml of the agarose DMEM.

Preferably, the 1% agarose solution is first prepared with sterile double distilled water, heated to boiling in a microwave oven, and then added with an equal volume of 2 × DMEM, (Gibco,12800-017) to make a 0.5% agarose solution in DMEM, and then poured into a 6cm petri dish, cooled and solidified for use.

Preferably, the medium is changed half a day apart during the suspension culture process.

Preferably, the non-neuronal cells are cultured in agarose DMEM suspension and then in a directional differentiation medium.

Preferably, the directed differentiation medium comprises GDNF (glial cell line-derived neurotrophic factor, peprotech,450-10), BDNF (brain-derived neurotrophic factor, peprotech,450-02), NT3 (neurotrophic factor-3, peprotech,450-03) and Forskolin (Forskolin, stement, 04-0025).

Preferably, the concentration of GDNF (peprotech,450-10) in the directed differentiation culture solution is 15-25ng/ml, preferably 20ng/ml, the concentration of BDNF (peprotech,450-02) is 15-25ng/ml, preferably 20ng/ml, the concentration of NT3(peprotech,450-03) is 15-25ng/ml, preferably 20ng/ml, and the concentration of Forskolin (stemgent,04-0025) at 4ng/ml is 2-6ng/ml, preferably 3, 4 or 5 ng/ml.

According to the invention, non-neuronal cells of humans or animals can be efficiently converted into neuronal cells by interfering the extracellular matrix-scaffold system of the non-neuronal cells with small molecule inhibitors of cytoskeletal proteins, siRNA knock-down treatments, low adhesion treatments. The method realizes the change of cell fate so as to obtain the neuron which is not reported yet, and simultaneously compared with the previously reported cell fate regulation and control method, the method has simpler application, only needs single micromolecule treatment or does not need the regulation and control of micromolecule and specific gene expression, can realize the change of cell fate only by changing a cell culture substrate, can be efficiently carried out in vivo and in vitro, and has important application in tumor treatment and tissue regeneration/repair.

The method of the invention has the function of broad-spectrum antitumor activity. For example, glioma is a tumor that occurs in neuroectoderm, mostly originating from different types of glial cells. Brain gliomas are statistically the most common intracranial tumors, accounting for about forty-five percent of all intracranial tumors. The Chinese medicinal composition ranks the second place in malignant tumors of children, in recent years, the incidence rate of primary malignant intracranial tumors is increased year by year, the annual growth rate is about 1.2 percent, and the Chinese medicinal composition is particularly obvious for middle-aged and elderly people. According to the literature, the average annual incidence rate of the brain glioma in China is 3-6 in 10 ten thousand, and the number of annual deaths is as high as 3 ten thousand. At present, domestic and foreign treatment means for glioma mainly comprise: surgery, chemotherapy, radiotherapy, X knife, gamma knife, etc. Based on the invention, the small molecule inhibitor of cytoskeletal protein can be used for preparing the anti-glioma drug. By adopting the medicine, the proliferation of glioma cells can be obviously inhibited, thereby achieving the anticancer effect.

The physiological aging or brain injury of a patient causes a large number of neurons to die, leads a large number of glia cells (mainly astrocytes) to proliferate, occupies the original brain injury area, inhibits the regeneration of the neurons, forms colloid scars in the brain, causes neurodegenerative diseases, and seriously affects the life quality of people, such as Parkinson's disease, Alzheimer's disease and the like. Despite the numerous attempts, there is currently no simple yet effective way to control glial scar production and promote neuronal regenerative repair. The invention utilizes the cytoskeletal protein inhibitor to efficiently transdifferentiate primary astrocytes from human sources into neurons. It provides a new method for treating neurodegenerative diseases caused by aging and pathological injuries in vivo.

The invention is characterized in that: 1. the operation is simple, and fate conversion can be carried out by adding single micromolecule into an induction culture solution or treating by single factor. 2. The method is efficient and rapid, in the process of transdifferentiation of non-neuronal cells into neurons by the myostatin inhibitor/actin assembly inhibitor treatment, nearly 100% of cells with positive neuron fate of Tuj1 can be obtained on the 7 th day, and the transformation of the non-neuronal cells into the neurons can be realized by the suspension culture solution only in 7 days. 3. The method has universal adaptability in the process of transdifferentiation of different types of initial cells of different species into neurons, and can efficiently transdifferentiate mouse TTF, MEF, setoli and human dorsal dermal fibroblasts, foreskin fibroblasts and muscle cells into neurons. 4. Safety, the method of the present invention has higher safety compared to conventional viral vector-mediated genetic approaches. 5. Controllability, compared with the transdifferentiation mediated by the combination of a plurality of small molecules, the slow-release system of a single small molecule has more feasibility and is convenient for metering control.

Drawings

Fig. 1A is a micrograph (left) of adult foreskin fibroblasts (HFF20y, beijing stem cell bank) and a picture (right) of Tuj1 immunofluorescence staining.

FIG. 1B is a photomicrograph (left) and a picture of Tuj1 immunofluorescent staining (right) of the cells of example 1-1 after 7 days of culture with induction medium containing (-) -Blebbistatin for the fibroblasts of FIG. 1A.

FIG. 1C shows Marker expressing mature neurons after culturing the fibroblasts of example 1-1 in induction medium containing (-) -Blebbistatin for 7 days and then in maturation medium for 14 days in example 1-1: map2(santacruz biotechnology, sc-20172), NF200(Abcam, ab4680), and NeuN (chemicon, MAB 377).

FIG. 1D shows the expression of classical presynaptic marker proteins Syn1(millipore, AB1543P) and Syt1(abcam, AB133856) and postsynaptic marker protein PSD95(abcam, AB18258) by mature-expressing neurons 14 days after 7 days of culture in induction medium containing (-) -Blebbistatin for the fibroblasts of FIG. 1A in example 1-1.

FIG. 1E is a graph showing the results of the experiments on mature neuron patch clamp after culturing the fibroblast cells of example 1-1 in the induction medium containing (-) -Blebbistatin for 7 days and then in the maturation medium for 14 days.

FIG. 1F shows the type marker protein staining of neurons after culturing the fibroblasts of FIG. 1A for 7 days in induction medium containing (-) -Blebbistatin and after culturing for 14 days in maturation medium in example 1-1 to express GABAergic neuronal marker proteins GABA (sigma, SAB4501067), GAD65/67(santa cruz biotechnology, sc-7513).

FIG. 1G shows neuronal expression of the mesencephalic dopaminergic marker TH (santa cruz biotechnology, sc-14007) and the glutamatergic marker vGlut1(santa cruz biotechnology, sc-377425) after 7 days of culture in induction medium containing (-) -Blebbistatin and 14 days of culture in maturation medium for the fibroblasts of FIG. 1A in example 1-1.

FIG. 1H is a graph showing the results of detecting the release of GABA in response to treatment with high potassium buffer by the HPLC-MS assay in example 1-1.

FIG. 2A is a photograph showing staining of untreated neuronal marker proteins Tuj1 and Map2 of mouse tail fibroblasts in examples 1 to 3.

FIG. 2B is the Marker expressing neurons after 7 days of culture in neural induction medium containing (-) -Blebbistatin in examples 1-3 for mouse tip fibroblasts: tuj1, Map2, NF200, and NeuN.

FIG. 2C is a graph of the results of the neuronal patch clamp experiments in examples 1-3 that matured 7 days after culture of mouse tip fibroblasts in the neural induction medium containing (-) -Blebbistatin.

FIG. 3A is a photomicrograph of the cell morphology after 7 days of culture in example 2 in DMSO medium without any inhibitor.

FIG. 3B is a photomicrograph of the cell morphology after 7 days of culture in example 2 using medium containing 0.4. mu.M Cytocalasin B.

FIG. 3C is a photomicrograph of the cell morphology after 3 days of culture in example 2 using medium containing 2. mu.M Cytochaisin B.

FIG. 3D is a photomicrograph of the cell morphology after 7 days of culture in example 2 using medium containing 0.2. mu.M L atrunculin B.

FIG. 3E is a graph comparing the expression of the neuronal marker protein Map2 by neurons of cells in example 2 after 7 days of culture in 2. mu.M Cytochaisin B medium and 10 days of culture in neuronal maturation medium with DMSO of the control.

FIG. 4A is a graph comparing the change in neuronal morphology at day 3 in example 3 of glioma cells treated with DMSO containing (-) -Blebbistatin compared to control.

FIG. 4B is a graph showing the effect of the antitumor agent of example 3 on the inhibition of glioma cells U87 and U251.

FIG. 4C shows the expression of Tuj1 in the cells of glioma cells U87 treated with the anti-tumor drug of example 3 and control DMSO.

FIG. 5A is a graph of the cell morphology change at day 7 after partial target knockdown in example 4.

FIG. 5B is a graph of the results of immunofluorescence staining of Tuj1 cells at day 7 after partial target knockdown in example 4.

FIG. 6A is a photograph of a light microscope showing the fibroblasts of example 5 after being cultured in suspension in the neural stem cell culture medium for 7 days.

FIG. 6B is a photograph of immunofluorescence staining of fibroblasts of example 5 after 7 days of suspension culture in neural stem cell culture fluid.

FIG. 6C is a photograph showing immunofluorescence staining of fibroblasts of example 5 after cultured in neural stem cell culture medium for 7 days in suspension and then in random differentiation culture medium for 7 days.

FIG. 6D is a photograph of immunofluorescence staining of fibroblasts of example 5 after 7 days of suspension culture in neural stem cell culture medium and then 7 days of culture in directed differentiation medium.

FIG. 7A Primary astrocyte morphology (left panel) and the astrocyte-expressing marker protein GFAP (right panel).

FIG. 7B is a 13-day map of the induction of astrocytes by (-) -Blebbistatin in example 6.

FIG. 7C shows that (-) -Blebbistatin induced astrocytes for 20 days in example 6, expressed the classical neuronal marker Tuj 1.

Detailed Description

The described embodiments and the following examples are for illustrative purposes and are not intended to limit the scope of the claims. Other modifications, uses, or combinations of the compositions described herein will be apparent to those of ordinary skill in the art without departing from the spirit and scope of the claimed subject matter.

Examples 1 to 1

Using a 12-well plate as an example (corning, 3335), each well was coated with 20ug/ml fibronectin solution (millipore, fc010)1 × PBS for 6 hours, or first with 0.1mg/ml polylysine solution (sigma, P6407) in sterile water, the plate was coated for 2 hours, washed with sterile water three times, then with 10 ug/ml laminin solution (sigma, 366256 74)1 × PBS, coated for 6 hours, and the coating was removed and washed with 1 × PBS.

Removing the washing solution, and uniformly seeding adult foreskin fibroblast (HFF20y, Beijing stem cell bank) into each well, wherein each well contains 1 × 10 cells⁴The cells were cultured in basal medium (high-glucose DMEM (Gibco, C12430500BT), 1 × sodium pyruvate (100 ×, Gibco, 11360-.

The cell transformation kit provided by the invention is selected, and comprises the following induction culture medium and maturation culture medium.

The fibroblasts treated as described above were added to a neuron induction medium (N2B27 medium: DMEM/F12(Gibco, 10565018) and Neurobasal (Gibco,21103-049) 1:1, mixed with N2 additives (100 ×, Gibco, 17502048), B27 additives (50 ×, Gibco, 17504044), 2% bovine serum albumin (1000 ×, sigma, A8022), β -mercaptoethanol (1000 ×, Gibco, 21985023), Glutamax (200 ×, Gibco, 35050-061), 1. mu.g/ml insulin (Roche, 11376497001), double antibody). 100mM concentrated storage with myostatin inhibitor (-) -Blebbistatin (dimethyl sulfoxide (D2650) dissolved in dimethyl sulfoxide (DMSO, D) was added, stored at-20 ℃ for 1 month), (-) -Blebbistatin culture medium was final concentration of Blebbistatin, cultured at 15-30-day, 23-day, and the ratio of neurite growth of neurons was found to be large in neuron induction medium (Tubco, neuron growth rate, neuron growth was found), and neuron growth was found to be marked by statistics at the same time of neuron growth rate of neuron growth (Tubco.

The cells cultured in the induction medium are added into a neuron maturation medium (N2B27 medium, 100 mu MN6,2 ' -O-dibutyryladenosine 3 ', 5 ' -sodium cyclic phosphate salt (sigma, D0627), 20ng/ml Recombinant HumanNT-3(Peprotech, 450-03), 20ng/ml brain-derived neurotrophic factor (Peprotech,450-02), 20ng/ml (Peprotech,450-10) glial-derived neurotrophic factor) to be matured and cultured for 7-14 days.

The experimental procedures were repeated to treat foreskin fibroblasts from 21-week, 8-year and 13-year old people, human 21-week dorsal skin fibroblasts, monkey tail fibroblast, murine fetal fibroblasts and non-proliferating cell mitomycin C-treated murine fetal fibroblasts, and the results of the Tuj1 positive rate and the MAP2 positive rate are shown in Table 1.

TABLE 1

As shown in Table 1, when the conversion of fibroblast to neuron cell was induced by using myostatin inhibitor (-) -Blebbistatin, the conversion of neuron cell was not optimal although the conversion of neuron cell was excellent at a concentration of 15. mu.M relative to the concentration of the neuron inducing medium; whereas, when the concentration is 20. mu.M to 30. mu.M, the neuronal cell conversion rate is highest.

To further illustrate the effect of the myostatin inhibitor, (-) -Blebbistatin, applicants provided FIG. 1A-FIG. 1H.

Fig. 1A shows the cell morphology and Tuj1 immunofluorescence staining pictures of adult foreskin fibroblasts (HFF20y, beijing stem cell bank).

FIG. 1B shows photomicrographs (left) and images of Tuj1 immunofluorescent staining (right) of the fibroblasts of FIG. 1A after 7 days of culture with neural induction medium containing 20. mu.M (-) -Blebbistatin. As shown in the figure, the cultured cells have obvious neuron morphology, large cell bodies, long axons and the like, and are stained with a neuron fate marker Tuj1, and the Tuj1 positive rate (Tuj1 positive cell/cell nucleus ratio) is counted, and the Tuj1 positive rate is close to 100%.

FIG. 1C shows Marker expressing mature neurons after 7 days of culture in neural induction medium containing 20. mu.M (-) -Blebbistatin and 14 days of culture in neuronal maturation medium for the fibroblasts of FIG. 1A: map2(santa cruz biotechnology, sc-20172), Nf200(Abcam, ab4680), and NeuN (chemicon, MAB 377).

FIG. 1D shows the expression of classical presynaptic marker proteins Syn1(millipore, AB1543P) and Syt1(abcam, AB133856) and postsynaptic marker protein PSD95(abcam, AB18258) for mature neurons after 14 days of culture in neuronal maturation medium after 7 days of neural induction medium containing 20 μ M (-) -Blebbistatin for the fibroblasts of FIG. 1A.

FIG. 1E is a graph showing the results of a mature neuronal patch clamp assay on the fibroblasts of FIG. 1A after 7 days of culture in induction medium containing (-) -Blebbistatin and after 14 days of culture in maturation medium. It shows that the cells have the activity of sodium current and potassium current of mature neurons and certain action potential.

FIG. 1F shows type marker protein staining for the fibroblasts of FIG. 1A after 7 days of culture with neural induction medium containing 20. mu.M (-) -Blebbistatin, with the marker proteins GABA (sigma, SAB4501067), GAD65/67(santa cruzbiotechnology, sc-7513) that are nearly 100% gabaergic expressing, followed by 14 days of culture with neuronal maturation medium.

FIG. 1G shows neuronal expression of the mesencephalic dopaminergic marker TH (santa cruzbiotechnology, sc-14007) and the glutamatergic marker vGlut1(santa cruz biotechnology, sc-377425) for the fibroblasts of FIG. 1A after 7 days of culture with induction medium comprising (-) -Blebbistatin followed by 14 days of culture with maturation medium. It was shown that the induced neurons did not substantially express the mesencephalic dopaminergic marker TH (santa cruz biotechnology, sc-14007) and the glutamatergic marker vGlut1(santa cruz biotechnology, sc-377425).

FIG. 1H shows that ultra-high performance liquid-ultra-high resolution mass spectrometry detection indicates that GABA neurons are induced to release gamma aminobutyric acid in response to treatment with high potassium buffer.

FIGS. 1B-1H clearly show that fibroblasts treated with the myostatin inhibitor (-) -Blebbistatin used in the present invention have very high neuronal transformation efficiency.

Examples 1 to 2

The experimental procedure of example 1-1 was used except that the myostatin inhibitor used was the myosin light chain kinase (M L CK) inhibitor M L-7.

Foreskin fibroblasts from 20-year-old HFF (human foreskin cells) P15, 21-week-old/8-year-old/13-year-old people, human 21-week-old dorsal skin fibroblasts, monkey caudate fibroblasts, murine fetal fibroblasts, and non-proliferating cell mitomycin C-treated murine fetal fibroblasts were treated, and the results of Tuj1 positive rate and MAP2 positive rate were shown in Table 2.

TABLE 2

As shown in Table 2, the conversion of fibroblasts into neuronal cells induced by the myostatin inhibitor myosin light chain kinase (M L CK) inhibitor M L-7 was not optimal when the concentration of the inhibitor relative to the neuronal induction medium was 15. mu.M, whereas the neuronal cell conversion rate was highest when the concentration was 20. mu.M-30. mu.M.

Examples 1 to 3

The experimental procedure of example 1-1 was used except that mouse tail tip fibroblasts were treated.

FIG. 2A shows the staining images of the neuron marker proteins Tuj1 and Map2 before the fibroblasts at the tip of the mouse are not treated. FIG. 2B shows Marker expression of neurons after 7 days of culture for mouse tip fibroblasts with neural induction medium containing 20. mu.M (-) -Blebbistatin: tuj1, Map2, NF200, and NeuN.

After the mouse tip fibroblasts are cultured for 7 days by using a nerve induction culture medium containing 20 mu M (-) -Blebbistatin and then cultured for 14 days by using a maturation culture medium, the positive rates of expressing Tuj1, Tuj1/Map2 and Map2/NeuN are respectively 96%, 96% and 97%.

FIG. 2C shows the activity of the cells in examples 1-3 in which the mouse tail fibroblasts were cultured in a neural induction medium containing 20. mu.M (-) -Blebbistatin for 7 days and then cultured in a maturation medium for 14 days to have sodium current and potassium current exhibited by neuronal maturation and certain action potentials.

This example shows that mouse tail tip fibroblasts treated with the myostatin inhibitor (-) -Blebbistatin used in the present invention can be transformed into neuronal cells at high transformation rates.

Example 2

The experimental procedure of example 1-1 was used except that foreskin fibroblasts from human 21, 8 and 13 years old, human 21-week dorsal skin fibroblasts, monkey tail fibroblast, murine fetal fibroblasts and non-proliferating cell mitomycin C-treated murine fetal fibroblasts were induced by replacing the myostatin inhibitors with 2. mu.M cytochalasin B (CB, sigma, C6762) and 0.2. mu.M L atrunculin B (BioVision, 2182-1), respectively, as actin assembly inhibitors, and the results of the Tuj1 positive rate and the Map2 positive rate obtained at the end are shown in tables 3 and 4.

TABLE 3

As shown in table 3, when the actin assembly inhibitor Cytochalasin B was used to induce the transformation of fibroblasts into neuronal cells, and the concentration of the inhibitor to the neuronal induction medium was 1.5 μ M, the neuronal cell transformation effect was excellent but not optimal; and when the concentration is 2-3 mu M, the neuron cell conversion rate reaches the highest.

TABLE 4

As shown in Table 4, when actin assembly inhibitor L atrunculin B was used as a medium for inducing the conversion of fibroblasts into neuronal cells, the neuronal cell conversion efficiency was not optimal although it was excellent at a concentration of 0.15. mu.M relative to the neuronal induction medium, and the neuronal cell conversion efficiency was highest at concentrations of 0.2. mu.M and 0.3. mu.M.

To further illustrate the effects of inhibitors of actin assembly, applicants provide FIGS. 3A-3E, FIGS. 3B-3E show the cell morphology of human adult foreskin fibroblasts (HFF20y, Beijing Stem cell Bank) that was neural induced using Cytochaisin B and L atroncin B, FIG. 3A shows the cell morphology after 7 days of culture using DMSO medium without any inhibitor, showing that the treated fibroblasts do not produce any neuronal morphology, FIG. 3B shows the cell morphology after 7 days of culture using medium containing 0.4 μ M Cytochaisin B, showing that the treated fibroblasts have slight changes in neuronal morphology, FIG. 3C shows the cell morphology after 3 days of culture using medium containing 2 μ M Cytochaisin B, showing that the treated fibroblasts have significant changes in neuronal morphology, FIG. 3D shows the cell morphology after 7 days of culture using medium containing 0.2 μ M L atroncin B, showing that the treated neurons have changed in morphology after 2 days of culture using medium, showing that the treated neurons have changed in morphology after 7 days of culture using medium, showing that the treated neurons have reached a state of maturation of neurons, showing that the cells have changed in the cell morphology after 7 days of culture using medium containing 0.2 μ M and showing that the treated neurons have changed in the cell morphology after 7 days.

Example 3

This example provides a specific embodiment of the use of cytoskeletal protein inhibitors for the preparation of an anti-cancer drug capable of transdifferentiating glioma cells into neuronal cells.

Antitumor drug I was formulated using DMEM/F12(Gibco, 10565018) mixed with Neurobasal (Gibco,21103-049) 1 to 1, N2 additive (100 ×, Gibco, 17502048), B27 additive (50 ×, Gibco, 17504044), 2% bovine serum albumin (1000 ×, sigma, A8022), β -mercaptoethanol (1000 ×, Gibco, 21985023), Glutamax (200 ×, Gibco, 35050-jar 061), 1. mu.g/ml insulin (Roche, 11376497001), diabodies), and (-) -Blebbistatin (100 mM concentrate of dimethyl sulfoxide dissolved (sigma, D2650), stored at-20 degrees Celsius for 1 month), adjusting the concentration of (-) -Blebbistatin to 20. mu.M relative to the entire drug I.

The following reagents are adopted to prepare the antitumor drug II: based on the antitumor drug I, 100 mu of MN6,2 ' -O-dibutyryladenosine 3 ', 5 ' -cyclic sodium phosphate salt (sigma, D0627), 20ng/ml of Recombinant Human NT-3(Peprotech, 450-03), 20ng/ml of brain-derived neurotrophic factor (Peprotech,450-02), and 20ng/ml of glial-derived neurotrophic factor (Peprotech,450-10) were formulated.

The antitumor drugs I and II are applied in a matching way in sequence, and the antitumor drug II is applied 3-7 days after the antitumor drug I is applied.

The anti-tumor drugs I and II of the present example were used in combination with human glioblastoma cells (U87), astrocyte cells (U251), human glioblastoma cells (L N229) and human glioblastoma cells (T98G), respectively, the morphology of the final glioma cells, the growth curves and transformation results were shown in FIGS. 4A and 4B compared to the control group (i.e., no (-) -Blebbistatin contained in the drug), wherein the amounts of the initial cells of the experimental groups were the same when human glioblastoma cells (U87), astrocyte cells (U251), human glioblastoma cells (L N229) and human glioblastoma cells (T98G) were treated with 20 μ M (-) -Blebbistatin and DMSO of the control group, respectively.

FIG. 4A shows that there was a significant change in neuronal morphology at day 3 with the formulation containing 20. mu.M (-) -Blebbistatin compared to control DMSO, and that cells were significantly rarer than the control.

Optionally, two strains of glioma cells U87 and U251 are respectively planted in a 48-well plate, 5000 cells are counted in each well, the cells are respectively treated by (-) -Blebbistatin containing 20 mu M and DMSO, 3 parallel wells are respectively adopted, Cell viability (Cell proliferation viability) is detected by using a Cell Counting Kit (CCK-8) CCK-8 Kit (sigma, 96992) every 24 hours, and the Cell proliferation viability is detected for 5 days in total, wherein the specific steps comprise (1) inoculating Cell suspension (100 mu L/well) in the 48-well plate, (2) placing the culture plate in an incubator for preculture for a period of time (37 ℃, 5% CO)₂) (ii) a (3) Add 10. mu.l CCK solution to each well; (4) absorbance at 450nm was measured with a microplate reader.

Fig. 4B shows that the antitumor agent of the present example significantly inhibited the proliferation of glioma cells, and the inhibition efficiency on optional glioma cells U87 and U251 was 50% and 99%, respectively, in the first 5 days.

Fig. 4C shows that the antitumor agent of this example transdifferentiates the optional tachyphylaxis glioma cell U87 into a differentiated neuronal cell expressing the neuronal marker protein Tuj1 with a positive rate of approximately 95%.

Example 4

(siRNA knockdown cytoskeleton system protein component transformation experiment and effect)

Human foreskin fibroblast cell line 1 × 10⁵Was seeded in 12-well plates and cultured with 1ml of fibroblast culture medium (DMEM + 10% FBS). When the cells reach 50-70% confluence

RNAiMAX (13778150, invitrogen) kit transfects targets rock1, rock2, mrlc1, mrlc2, mrlc3, myh9, myh10, mrck α, mrck β, lamna/c, lmnb1, lbr, sun1, sun2, cbx1, cbx3, cbx5, banf1, syne1, syne2 and β -actin respectively, and the specific steps are as follows (1) diluting the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and S.sub.3, respectivelyEQ 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 siRNAs in RNase-free EP tubes 12pmol siRNA duplexes (2.5ul) diluted with 100 μ L/well of serum-free Opti-MEM medium and gently mixed, (2) dilution of transfection reagent

RNAiMAXReagent, gently mix; (3) diluted

After 2min incubation, the RNAimax Reagent was gently mixed with the diluted siRNA of (2) above, and incubated at room temperature for 20min to form siRNA-

The RNAiMAX Reagent mixture, the solution may be cloudy, but not affected. (4) Adding the mixed solution of the step (3) into a cell culture plate containing cells and a culture solution, and slightly shaking to mix the cells and the culture solution; (5) place the plates in 37 ℃ CO₂Culturing in an incubator, and replacing neuron culture solution after transfection for 6-8 hours. (6) Transfection was carried out for 48-72 hours.

Table 5 shows the correspondence of siRNA sequences to their knocked down gene expression.

TABLE 5

There was a clear change in neural morphology at day 7 after knockdown of any target, with the cell morphology change after partial knockdown of the target shown in fig. 5A. Figure 5B immunofluorescence staining results show induction of day 7 expression of the specific protein Tuj1 of neuronal fate.

Neuronal medium (N2B27 broth: DMEM/F12(Gibco, 10565018) was mixed 1:1 with Neurobasal (Gibco, 21103-.

Example 5

(transformation experiment and Effect of interfering with extracellular matrix treatment and Low adhesion)

Firstly, a 1% agarose solution is prepared by sterile double distilled water, the solution is heated to boiling by a microwave oven, then an equal volume of 2 × DMEM (Gibco,12800-017) is added to prepare a 0.5% agarose solution dissolved in the DMEM, and then the agarose solution is poured into a culture dish of 6cm and is cooled and solidified for standby.

Human foreskin fibroblasts were seeded at a density of 1 × 105 in the above described petri dishes and cultured in suspension with neural stem cell culture medium for 7 days, half the way between days, photo micrographs after 7 days of suspension culture are shown in fig. 6a, where neuronal morphology has been shown.

Human fibroblasts cultured on day 7 of suspension culture were centrifuged, digested for 3min in 37 ℃ incubator with Tryple (Gibco, a1285901), diluted with PBS, centrifuged, discarded supernatant resuspended in neural stem cell culture medium, plated in four well plates at 5 × per well, the next day the medium was removed and immunofluorescent stained as follows:

the cells were fixed with PBS solution containing 4% paraformaldehyde (Sigma,158127) for 20min, washed 3 times with PBS, 5min each, then permeabilized 1H in PBS solution containing 0.3% TritonX-100(Solarbio, T8200) and 0.2% BSA (Sigma, A3803) followed by incubation with 2% BSA and one anti-mouse anti-Nestin (Millipore, MAB353,1:100), goat anti-Sox 2(Santa, sc-17320,1:100), mouse anti-Nkx2.2 (abcam, ab 187375), rabbit anti-En 1(abcam, 70993,1:50), rabbit anti-N-cadherin (abhscam, ab12221,1:100), rabbit anti-Pax 6(abcam, 5790,1:50), PBS solution containing 1% paraformaldehyde, PBS, 1:50), then washed with a counterbuff, stained with a PBS solution containing 1H-Invitrogen, then incubated with a counterstained DNA, stained with a PBS solution at room temperature for three times with PBS, 5min, then incubated with a counterstained DNA, and observing the results with a counterstained DNA, stained with a fluorescent microscope, stained with 2, a PBS solution containing 2, a counterstained mouse anti-Nestin (Invitrogen, 1 H.10, 1H, 1H, and then stained with a confocal microscope, and then stained with a fluorescent microscope at 4 ℃ for three times.

The results in FIG. 6B show that human foreskin fibroblasts cultured in low adhesion suspension can be transformed into neural stem cells that are both Nestin and Sox2 positive. Further, these double positive cells were also positive for Nkx2,2, En1, N-cad, and Pax 6.

The neural stem cells identified above were inoculated into four-well plates on day 7 in the same manner as described above, the randomly differentiated group was cultured in the randomly differentiated medium, the directionally differentiated group was cultured in the directionally differentiated medium, the medium was changed half every other day, and immunofluorescence staining was performed on day 14. Wherein the primary antibody is rabbit anti-Tuj 1(abcam, ab18207,1:2000), mouse anti-GFAP (sigma, G3893,1:100), and chicken anti-NF 2000(abcam, ab4680,1: 1000). The detection results are shown in 6C and 6D respectively.

The results in FIG. 6C show that the neural stem cells obtained from the low adhesion treatment were randomly differentiated to obtain GFAP-positive astrocytes, and further, these astrocytes were negative for Tuj 1.

The results in FIG. 6D show that neural stem cells obtained from low adhesion treatment can obtain neurons with Tuj1 and NF2000 double positive after directed differentiation.

Basic culture solution

Prepared from DMEM/F12(Gibco,12400-024) and Neurobasal (Gibco,21103-049) in a volume ratio of 1:1, and 100 × N2(Gibco,17502-048), 50 × B27(Gibco,17504-044), 100 × GlutaMAX (Gibco,35050-079, 1000 ×β -mercaptethanol (Gibco,21985), 1000 × 2% BSA (sigma, A7906-100G), 1000 × Insulin (Roche applied science,11376497001,10mg/m L), 100 × SP (Gibco,15140-122) were added.

Neural stem cell culture solution

Prepared by adding 20ng/ml of bFGF (epidermal growth factor, R & D,233-FB-001MG/CF) to the basal medium.

Random differentiation culture solution

Prepared from the basal medium supplemented with 1% FBS (fetal bovine serum, Gibco, 16000-.

Directional differentiation culture solution

The basal medium was prepared by adding 20ng/ml GDNF (peprotech,450-10), 20ng/ml BDNF (peprotech,450-02), 20ng/ml NT3(peprotech,450-03) and 4ng/ml Forskolin (stemgent, 04-0025).

Example 6

(Primary astrocytes transdifferentiate into neurons)

Adult primary astrocytes (ScienCell, 1800) or primary glial cells isolated from mouse cerebral cortex were plated in each well uniformly with the wash solution removed, and cultured with basal medium (high-glucose DMEM (Gibco, C12430500BT), 1 × sodium pyruvate (100 ×, Gibco, 11360-.

The glial cells treated as above are added into neuron induction culture medium (N2B27 culture medium: DMEM/F12(Gibco, 10565018) and Neurobasal (Gibco,21103-049) 1:1, N2 additive (100 ×, Gibco, 17502048), B27 additive (50 ×, Gibco, 17504044), 2% bovine serum protein (1000 ×, sigma, A8022), β -mercaptoethanol (1000 ×, Gibco, 21985023), Glutamax (200 ×, Gibco, 35050-061),1 μ g/ml insulin (Roche, 11376497001), double antibody). 100mM concentrated storage of myostatin inhibitor (-) -Blebbistatin (dimethyl sulfoxide, D2650) is added, and the neuron induction culture medium is cultured at a final concentration of Blebbistatin culture medium of 15-30 μ g-7 days, and has a significant neuron induction rate of neuron induction, neuron induction culture medium is marked by a neuron growth rate of 587, and the neuron growth rate is large, and the neuron growth rate is counted in the neuron induction culture medium is marked by the neuron growth rate of the neuron growth in the neuron induction medium (Tubco, Tubc.

FIG. 7A shows primary astrocyte morphology (left panel) and the astrocyte-expressing marker protein GFAP (right panel). As shown in FIG. 7B, (-) -Blebbistatin induced astrocytes for 13 days exhibited a classical neuronal morphology. As shown in FIG. 7C, (-) -Blebbistatin induced astrocytes for 20 days, nearly 100% expressed the classical neuronal marker protein Tuj 1.

Sequence listing

<110> institute of animal research of Chinese academy of sciences

<120> a method for converting non-neuronal cells into neuronal cells

<130>PDK03143D2

<141>2017-03-01

<160>21

<170>PatentIn version 3.5

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Claims

2. The method of claim 1, wherein the interference handling is: the extracellular matrix is subjected to a low adhesion treatment.

3. The method of claim 1 or 2, wherein the non-neuronal cells are fibroblasts and/or glial cells.

4. The method of claim 2, wherein the low adhesion treatment comprises culturing non-neuronal cells in suspension with agarose DMEM;

preferably, the suspension culture time is 6 days, 7 days or 8 days;