Method for reprogramming rat skin fibroblasts into neuronal cells

Abstract

The invention relates to the technical field of somatic cell reprogramming and cell transplantation therapy, in particular to a method for reprogramming rat skin fibroblasts into neuronal cells, which comprises the following steps: taking in-vitro rat embryo skin fibroblasts, and reprogramming into intermediate state cells under the action of VPA, CHIR99021, Repsox, Forskolin, GO6983, Y-27632, Melatonin and P7C3-A20; and performing continuous induced maturation on the intermediate state cells by using CHIR99021, Forskolin, Dorsomorphin, P7C3-A20, NT3, N2, B27 containing vitamin A, and cAMP, so as to obtain mature neuronal cells. The neuronal cells obtained by the method provided by the invention can survive in a Parkinson rat brain microenvironment for a long time, and have a certain repairing effect on rat dyskinesia.

Description

Technical Field

[0001] The present invention relates to the technical field of somatic cell reprogramming and cell transplantation therapy, and in particular to a method for reprogramming rat skin fibroblasts into neuronal cells using only small molecule compounds. Background Art

[0002] Parkinson's disease (PD) is a neurodegenerative disease that causes motor dysfunction, such as prohibitive tremors and bradykinesia, and its symptoms worsen over time. There are many factors that cause Parkinson's disease, but the main one is age, which is more common in the elderly over 60 years old. Its pathogenesis is mainly the death of a large number of dopaminergic neurons in the substantia nigra-striatum pathway of the midbrain, which causes a significant decrease in the dopamine content of the striatum and leads to damage to the body's motor function. At present, the main clinical treatments for Parkinson's disease include drug therapy such as L-levodopa and surgical treatment such as deep brain stimulation (DBS). Surgical treatment causes greater mechanical damage to brain tissue, and long-term use of drugs may cause relatively serious sequelae, and Parkinson's disease cannot be fundamentally cured. The main reason is that the neuronal regeneration capacity of the central nervous system (CNS) of adult mammals is extremely limited, which leads to a poor prognosis for patients with CNS damage. Therefore, finding new treatment strategies has become a research hotspot.

[0003] Somatic cell reprogramming technology provides a possible solution. Through this technology, researchers can reprogram patients' somatic cells, such as skin cells, into induced pluripotent stem cells (iPSCs), which then differentiate into neurons. These neurons can be used to establish disease models, screen drugs, and use cell replacement therapy. However, traditional somatic cell reprogramming technology relies on transgenic methods to introduce exogenous genes, which poses potential safety risks in clinical applications.

[0004] In this context, small molecule-induced cell reprogramming technology has emerged. Compared with traditional genetic engineering methods, small molecule compounds have many advantages: they can be obtained through simple chemical synthesis, easy to control dosage and duration, and more importantly, they do not involve genetic modification and therefore have higher safety. Pluripotent stem cell (PSC) transplantation is considered to be the most promising method for the treatment of neurodegenerative diseases, but the activation process of PSC pluripotency and the abnormal expression of certain oncogenes and tumor suppressor genes have a high risk of tumorigenicity. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a method for reprogramming rat skin fibroblasts into neuronal cells using only small molecule compounds. The obtained neuronal cells can survive for a long time in the brain microenvironment of Parkinson's rats and have a certain repair effect on the motor dysfunction of PD rats, providing factual basis and theoretical foundation for the treatment of related neurodegenerative diseases.

[0006] The first object of the present invention is to provide a method for reprogramming rat skin fibroblasts into neuronal cells, comprising the following steps:

[0007] Rat embryonic skin fibroblasts were isolated and reprogrammed into intermediate cells under the action of VPA, CHIR99021, Repsox, Forskolin, GO6983, Y-27632, Melatonin, and P7C3-A20;

[0008] CHIR99021, Forskolin, Dorsomorphin, P7C3-A20, NT3, N2, B27, and cAMP were used to continuously induce the maturation of intermediate cells to obtain mature neuronal cells.

[0009] Furthermore, the specific reprogramming process of the intermediate cells is: after the rat embryonic skin fibroblasts in vitro grow to 80-90%, they are induced and cultured in a small molecule induction medium to obtain the intermediate cells;

[0010] The induction medium is prepared by mixing DMEM / F-12 medium and Neurobasal medium in a volume ratio of 1:1, and the induction medium includes the following small molecule substances: 0.5mmol / L VPA, 3μmol / L CHIR99021, 1μmol / L Repsox, 30μmol / L Forskolin, 10μmol / L Melatonin, 5μmol / L GO6983, 5μmol / LY-27632, 0.5% N2, 1% B27, 10μmol / L cAMP, 20ng / mL bFGF, 3μmol / LP7C3-A20, and 1% penicillin-streptomycin solution.

[0011] Furthermore, the intermediate cells are obtained by inducing the culture medium for 5 to 7 days, and the medium is replaced by half the amount every 3 days during the induction culture.

[0012] Furthermore, the maturation induction is carried out in a maturation medium for continuous induction, during which fresh medium is replaced every two days;

[0013] The maturation medium is prepared by mixing DMEM / F-12 medium and Neurobasal medium in a volume ratio of 1:1, and the maturation medium includes the following substances: 3 μmol / L CHIR99021, 30 μmol / L Forskolin, 3 μmol / L P7C3-A20, 1 μmol / L Dorsomorphin, 0.5% N2, 1% B27, 10 μmol / L cAMP, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, and 1% penicillin-streptomycin solution.

[0014] Furthermore, the continuous induction is induced culture in the maturation medium for 10 to 14 days.

[0015] The second object of the present invention is to provide a method for obtaining neuronal cells according to the above method.

[0016] The third object of the present invention is to provide a use of the neuronal cells in preparing a drug for treating or preventing neurodegenerative diseases.

[0017] Furthermore, the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

[0018] The fourth object of the present invention is to provide a drug for improving neurodegenerative diseases, which comprises the chemically induced neuronal cells and a pharmaceutically acceptable excipient or carrier.

[0019] Furthermore, the drug is an injection solution comprising the neuronal cells, and the number of neuronal cells in the injection solution is at least 1×10 5 indivual.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention reprograms fibroblasts into neuronal cells in stages. In the first stage, intermediate cells are produced by induction for one week by VPA, CHIR99021, Repsox, Forskolin, GO6983, Y-27632, Melatonin, and P7C3-A20, thereby reducing the risk of tumorigenicity. In the second stage, the intermediate cells are induced for another 10 days to obtain mature induced neuronal cells.

[0022] 2. The present invention adds small molecules Melatonin (melatonin) and P7C3-A20 during the induction process, which effectively reduces the cell death during the induction process and improves the efficiency of induction. Melatonin is a lipid-water soluble neurohormone produced by the pineal gland, which participates in a variety of physiological functions, can scavenge hydroxyl radicals and peroxyl radicals, and reduce the number of cells that die due to oxidative stress during the induction process. P7C3-A20 is a neurogenic protective factor, and its main mechanism of action is to activate the PI3K / protein kinase B / glycogen synthase kinase 3β signaling pathway to exert a neuroprotective effect.

[0023] 3. In the present invention, only small molecule compounds are used to reprogram fibroblasts into neuronal cells. Compared with the reprogramming method using proteins or transcription factors, the possibility of mutation or tumor occurrence due to genomic instability is reduced. In addition, small molecule compounds are easier to introduce and operate, and the cost is also lower.

[0024] 4. The present invention uses HE staining of brain sections and immunohistochemistry to detect tyrosine hydroxylase expression, effectively verifying the survival, migration and differentiation of transplanted induced neuronal cells in the host brain; using apomorphine-induced rotation test, open field test and fatigue rotarod test, it is effectively shown that the transplanted neuronal cells have obvious therapeutic effects on Parkinson's rats. The neuronal cells obtained by the method provided by the present invention can survive for a long time in the brain microenvironment of Parkinson's rats, and have obvious repair effects on rat motor dysfunction, laying a foundation for the treatment of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease with cell transplantation.

[0025] 5. The starting cells used in the present invention are fibroblasts, which are widely present in the normal body and will not cause serious damage when obtained, which can effectively reduce the difficulty and cost of obtaining induced starting cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 is the induction process and cell morphology changes; A is the roadmap for reprogramming rat skin fibroblasts into neuronal cells using small molecule compounds; B is the cell morphology changes at various stages in the process of reprogramming rat embryonic skin fibroblasts into neuronal cells; B1 is a fibroblast, B2 is an intermediate cell at the end of the first stage of induction, and B3 is a neuronal cell at the end of the second stage of induction.

[0028] Figure 2 Figure 2 is the effect of small molecule compounds on the morphology and survival of induced cells. A is the removal of forskolin, the cell survival rate decreased during the induction process, only about 44% of the cells survived, and the cell morphology was almost unchanged, still maintaining the REF morphology, and the proportion of neuron-like cells decreased to about 6%; B is the removal of GO6983, the cell survival rate and the number of neuron-like cells decreased; C is the removal of Y-27632, a large number of cells died in the early stage of induction, only about 5% of the cells survived, and no neuron-like cells were produced; D is the effect of removing small molecules alone on the cell survival rate in the first stage of induction; E is the effect of removing small molecules alone on the induction efficiency in the first stage; ns: P>0.05, *P<0.05, ****P<0.0001.

[0029] Figure 3 Immunofluorescence identification of induced Stage2 cell surface markers; A: Immunofluorescence detection of cell surface TUJ1 and PAX6 expression; B: Immunofluorescence detection of cell surface TUJ1 and SOX2 expression; C: Immunofluorescence detection of cell surface TUJ1 and Nestin expression; DAPI: cell nuclei labeled with nuclear dye; TUJ1: Immunofluorescence detection of cell surface neuronal marker TUJ1 expression; PAX6: Immunofluorescence detection of cell surface neural progenitor cell marker PAX6 expression; SOX2: Immunofluorescence detection of cell surface neural progenitor cell marker SOX2 expression; Nestin: Immunofluorescence detection of cell surface neural progenitor cell marker Nestin expression; Merge: combined image.

[0030] Figure 4 Immunofluorescence identification of induced Stage 3 cell surface markers; A: Immunofluorescence detection of cell surface TUJ1 and TH expression; B: Immunofluorescence detection of cell surface MAP2 and VGLUT1 expression; DAPI: cell nuclei labeled with nuclear dye; TUJ1: Immunofluorescence detection of cell surface neuronal marker TUJ1 expression; MAP2: Immunofluorescence detection of cell surface microtubule-associated protein MAP2 expression; TH: Immunofluorescence detection of cell surface dopaminergic neuron marker TH expression; VGLUT1: Immunofluorescence detection of cell surface glutamatergic neuron marker VGLUT1 expression; Merge: combined image.

[0031] Figure 5 Stage2 cells were transplanted to treat Parkinson's rats and their behavior was tested; A: Rats were fixed in a stereotaxic instrument; B: APO-induced rat rotation; C: fatigue rotarod test; D: movement trajectory of rats in the open field test.

[0032] Figure 6Statistical graphs of the APO-induced rotation experiment; A is the rotation data of transplanted cells at 4 weeks; B is the rotation data of transplanted cells at 8 weeks; ns: P>0.05; ***P<0.001.

[0033] Figure 7 Statistical graphs of the open field experiment; A is the open field data at 4 weeks after cell transplantation; B is the open field data at 8 weeks after cell transplantation; *P<0.05; ****P<0.0001.

[0034] Figure 8 The figures are statistical diagrams of the fatigue rotarod test; A is the data of the time of transplanted cells on the rotarod at 4 weeks; B is the data of the time of transplanted cells on the rotarod at 8 weeks; *P<0.05; ***P<0.001.

[0035] Fig. 9 Immunofluorescence images of transplanted cells surviving in the host brain; A: 8 weeks after transplantation, the cells survived in the rat brain and expressed TUJ1; B: 8 weeks after transplantation, the cells survived in the rat brain and expressed TH; C: 8 weeks after transplantation, the cells survived in the rat brain and expressed FOXA2. DAPI: cell nuclei labeled with nuclear dye; TUJ1: immunofluorescence detection of the expression of cell surface neuron marker TUJ1; TH: immunofluorescence detection of the expression of cell surface dopaminergic neuron marker TH; VGLUT1: immunofluorescence detection of the expression of cell surface glutamatergic neuron marker VGLUT1; CM-DiI: cells labeled with cell dye CM-DiI; Merge: combined image. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0037] Researchers can use somatic cell reprogramming technology to reprogram patients' somatic cells, such as skin cells, into induced pluripotent stem cells (iPSCs), which can then differentiate into neurons. These neurons can be used to establish disease models, screen drugs, and replace cell therapies. However, traditional somatic cell reprogramming technology relies on transgenic methods to introduce exogenous genes, which poses potential safety risks in clinical applications.

[0038] In this context, small molecule-induced cell reprogramming technology has emerged. Compared with traditional genetic engineering methods, small molecule compounds have many advantages: they can be obtained through simple chemical synthesis, easy to control dosage and duration, and more importantly, they do not involve genetic modification and therefore have higher safety. Pluripotent stem cell (PSC) transplantation is considered to be the most promising method for the treatment of neurodegenerative diseases, but the activation process of PSC pluripotency and the abnormal expression of certain oncogenes and tumor suppressor genes have a high risk of tumorigenicity.

[0039] Based on this, the present invention provides a method for reprogramming rat skin fibroblasts into neuronal cells, comprising the following steps:

[0040] Rat embryonic skin fibroblasts were isolated and reprogrammed into intermediate cells under the action of VPA, CHIR99021, Repsox, Forskolin, GO6983, Y-27632, Melatonin, and P7C3-A20;

[0041] CHIR99021, Forskolin, Dorsomorphin, P7C3-A20, NT3, N2, B27, and cAMP were used to continuously induce the maturation of intermediate cells to obtain mature neuronal cells.

[0042] The present invention reprograms fibroblasts into neuronal cells in stages. In the first stage, intermediate cells are generated by induction for one week through VPA, CHIR99021, Repsox, Forskolin, GO6983, Y-27632, Melatonin, and P7C3-A20, thereby reducing the risk of tumorigenicity. In the second stage, the intermediate cells are induced for another 10 days to obtain mature induced neuronal cells.

[0043] Example 1

[0044] This example provides a method for reprogramming rat skin fibroblasts into neuronal cells using only small molecule compounds.

[0045] 1. Experimental Materials

[0046] 1. Reagents

[0047] NEAA (non-essential amino acids), L-Glutamax, B27, DMEM basal medium, DME M / F-12 medium, Neurobasal medium, 0.25% EDTA-containing trypsin, fetal bovine serum (FBS), and N2 were purchased from Gibco; valproic acid (VPA), TritonX-100, and Rb-Olig2 were purchased from Sigma; CHIR99021, Repsox, GO6983, Y-27632, Forskolin, P7C3-A20, Melatonin, NT3, and Dorsomorphin were purchased from Selleckchem; basic fibroblast growth factor (bFGF) was purchased from R&D; BDNF and GDNF were purchased from PEPROTECH; penicillin-streptomycin solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; donkey serum (NDS) and donkey anti-rabbit Cy3 were purchased from Jackson Lab; donkey anti-mouse Alexa Fluor 488 and CellTrackerTM CM-DiIDye, Rb-VGLUT1, and anti-fluorescence quencher were purchased from Invitrog en; Rb-TH and Rb-Vimentin antibodies were purchased from Biosun; m-TUJ1, m-Nestin, Rb-SOX2, Rb-PAX6, and Rb-FOXA2 antibodies were purchased from ABCAM; m-MAP2 antibody was purchased from Santa Crμz, and injectable penicillin was purchased from Shandong Shengwang Pharmaceutical Co., Ltd.

[0048] All culture media and additives used in the embodiments of the present invention are of clinical grade; the additives include VPA, CHIR99021, Repsox, Forskolin, GO6983, Y-27632, Melatonin, P7C3-A20, Dorsomorphin, NT3, bFGF, N2, B27, BDNF, GDNF, cAMP, penicillin-streptomycin solution, NEAA and GlutaMax.

[0049] 2. Culture medium

[0050] DMEM high-glucose medium was purchased from Hyclone; DMEM / F12 medium and Neurobasal medium were purchased from Gibco. Culture media used at different stages: (1) Normal culture of rat embryonic skin fibroblasts: DMEM high-glucose medium supplemented with 10% serum, 1% NEAA, 1% GlutaMax, and 1% penicillin-streptomycin solution.

[0051] (2) Transdifferentiation of rat embryonic skin fibroblasts to intermediate cells: DMEM / F-12:Neurobasal (1:1), 0.5mmol / L VPA, 3μmol / L CHIR99021, 1μmol / L Repsox, 30μmol / L Forskolin, 10μmol / L Melatonin, 5μmol / L GO6983, 5μmol / LY-27632, 0.5% N2, 1% B27, 10μmol / L cAMP, 20ng / mL bFGF, 3μmol / L P7C3-A20, and 1% penicillin-streptomycin solution.

[0052] (2) Inducing differentiation of intermediate cells into neuronal cells: DMEM / F-12:Neurobasal (1:1), 3μmol / L CHIR99021, 30μmol / L Forskolin, 3μmol / L P7C3-A20, 1μmol / L Dorsomorphin, 0.5% N-2, 1% B27, 10μmol / L cAMP, 20ng / mL bFGF, 20ng / mL BDNF, 20ng / mL GDNF, 20ng / mL NT3, and 1% penicillin-streptomycin solution.

[0053] 3. Cells used for reprogramming: The somatic cell type used for transdifferentiation induction is rat embryonic skin fibroblasts.

[0054] 2. Experimental Methods

[0055] The roadmap provided by the present invention for reprogramming rat skin fibroblasts into neuronal cells using only small molecule compounds is as follows Figure 1 As shown in A.

[0056] 1. Primary Isolation and Culture of Rat Embryonic Skin Fibroblasts

[0057] SD rats at 13 to 15 days of pregnancy were selected, and the pregnant rats were killed by cervical dislocation. The fetuses were taken out, the heads were cut off with scissors, and the limbs, internal organs, gonads and other tissues were removed with forceps. The skin of the trunk was peeled off and placed in a culture dish filled with PBS. The blood and fat tissue on the skin surface were carefully removed with forceps. After rinsing, the skin was placed in a new culture dish and the skin tissue was cut into 1 mm with scissors. 2 For tissue blocks of different sizes, evenly stick them to the bottom of the culture bottle, add 3 mL of fibroblast culture medium to each bottle, invert and place them in 37°C, CO2 culture medium for 6 hours, then turn the culture bottle over and continue culturing. When the cells grow out from around the tissue blocks and reach a certain size, digest the cells and re-lay them in the bottle. After that, change the medium every three days. When the cells grow to 80-90%, freeze some of the cells for later use and continue culturing the rest.

[0058] It should be noted that the rat embryonic skin fibroblasts used in the present invention can also be obtained from rat embryos in vitro.

[0059] 2. Reprogramming of rat embryonic skin fibroblasts into neurons

[0060] (1) Induction of rat embryonic dermal fibroblasts (REF) into intermediate cells:

[0061] Rat embryonic skin fibroblasts with good growth status were selected and inoculated on the well plate pre-coated with matrix gel. After the cells grew to 80-90%, they were induced in the small molecule induction medium for 5-7 days, during which the medium was replaced by half every 2 days to obtain intermediate cells.

[0062] Among them, the induction medium is prepared by mixing DMEM / F-12 and Neurobasal in a volume ratio of 1:1, and the induction medium also contains the following substances: 0.5mmol / LVPA, 3μmol / L CHIR99021, 1μmol / L Repsox, 30μmol / L Forskolin, 10μmol / L Melatonin, 5μmol / L GO6983, 5μmol / L Y-27632, 0.5% N2, 1% B27, 10μmol / L cAMP, 20ng / mL bFGF, 3μmol / L P7C3-A20, and 1% penicillin-streptomycin solution.

[0063] (2) Induction of intermediate cells into neurons:

[0064] The old culture medium was discarded, and new maturation culture medium containing small molecules was added to the intermediate cells obtained by induction, and the induction was continued for about 10 days, during which time the fresh culture medium was replaced every 2 days.

[0065] Among them, the maturation medium is prepared by mixing DMEM / F-12 and Neurobasal in a volume ratio of 1:1, and the maturation medium also contains the following substances: 3μmol / L CHIR99021, 30μmol / L Forskolin, 3μmol / L P7C3-A20, 1μmol / L Dorsomorphin, 0.5% N2, 1% B27, 10μmol / L cAMP, 20ng / mL bFGF, 20ng / mL BDNF, 20ng / mL GDNF, 20ng / mL NT3, and 1% penicillin-streptomycin solution.

[0066] 3. Experimental Results

[0067] 1. Reprogramming of rat embryonic skin fibroblasts into intermediate cells

[0068] The first stage is the chemical induction of the production of intermediate cells. Rat embryonic skin fibroblasts are induced to produce intermediate cells on the 6th day under the combined action of 0.5mmol / LVPA, 3μmol / L CHIR99021, 1μmol / L Repsox, 30μmol / L Forskolin, 10μmol / L Melatonin, 5μmol / L GO6983, 5μmol / LY-27632, 0.5% N2, 1% B27, 10μmol / L cAMP, 20ng / mLbFGF, and 3μmol / LP7C3-A20. The cells show a bipolar morphology.

[0069] 2. Induce intermediate cells to differentiate into neurons

[0070] The intermediate cells obtained in the second stage were replaced with a maturation medium containing CHIR99021, Forskolin, Dorsomorphin, P7C3-A20, NT3, N2, B27 (containing vitamin A), and cAMP to continue induction until the 17th day, and neuronal cells ( Figure 1 (B).

[0071] 3. Effects of small molecule combinations on cells during reprogramming

[0072] Previous studies by the present invention team have demonstrated that small molecules VPA, CHIR99021, and Repsox can promote neuronal transformation of fibroblasts during reprogramming. The present invention illustrates the effect of the combination of several small molecules on cells during reprogramming by removing any small molecule in the small molecule combination.

[0073] The results are as follows Figure 2 As shown. A is the removal of Forskolin, the cell survival rate decreased during the induction process, only about 44% of the cells survived, and the cell morphology was almost unchanged, still maintaining the REF morphology, and the proportion of neuron-like cells decreased to about 6%; B is the removal of GO6983, the cell survival rate and the number of neuron-like cells decreased; C is the removal of Y-27632, a large number of cells died in the early stage of induction, only about 5% of the cells survived, and no neuron-like cells were produced; D is the effect of small molecule removal on cell survival rate, the removal of Forskolin, the cell survival rate dropped from about 65% to about 44%, the removal of GO6983, the cell survival rate dropped to about 55%, the removal of Y-27632, the cell survival rate dropped to about 5%; E is the effect of each small molecule removal on the cell induction efficiency, the removal of Forskolin, the induction efficiency dropped from about 56% to about 6%, the removal of GO6983, the induction efficiency dropped to about 31%, the removal of Y-27632, almost no neuron-like cells were produced.

[0074] Example 2

[0075] Functional identification of the induced neuronal cells obtained in Example 1

[0076] 1. Analysis of protein marker staining for intermediate cells

[0077] The intermediate cells were stained with Nestin, SOX2, and PAX6 by immunofluorescence staining. Nestin, SOX2, and PAX6 are all markers of neural progenitor cells. The results of immunofluorescence staining showed that the induced intermediate cells expressed Nestin, SOX2, and PAX6 at the same time, and had obvious characteristics of neural progenitor cells. Figure 3 .

[0078] 2. Protein marker staining analysis of induced mature neurons

[0079] Immunofluorescence staining of TUJ1, MAP2, TH and VGLUT1 was performed on the induced mature neuronal cells. TH is a marker of dopamine neurons, VGLUT1 is a marker of glutamatergic neurons, TUJ1 is a marker of neural microtubules, and MAP2 is a marker of mature neurons. Figure 4 It was demonstrated that the induced neurons highly expressed TH, TUJ1 and MAP2, and some cells expressed VGLUT1.

[0080] Example 3

[0081] In vivo survival and differentiation ability of induced mature neurons

[0082] 1. Construction of PD model

[0083] Select SPF SD rats weighing about 180-220 g and inject 3% pentobarbital intraperitoneally for anesthesia. Figure 5 As shown in A, the SD rats were fixed on the stereotaxic platform, the brain skin was routinely disinfected, the skin of the surgical area was incised, the periosteum was scraped, and the brain stereotaxic instrument was used for positioning. The bregma was used as the standard reference point, and the coordinates were selected for positioning: ①AP-4.4mm, ML-1.2mm, DV-7.8mm; ②AP-4.0mm, ML-0.8mm, DV-8.0mm. Hamilton microsyringe was used to slowly inject 4μL of 6-OHDA at each point. The injection time was 8min, the needle was stopped for 8min, and then the needle was slowly withdrawn. The brain skin was sutured, and penicillin was injected intraperitoneally for 3 consecutive days after surgery to prevent postoperative infection, and the postoperative recovery was observed every day.

[0084] 2. Behavioral testing

[0085] (1) APO-induced rotation experiment: Two weeks after stereotaxic modeling, apomorphine (0.5 mg / kg) was intraperitoneally injected. The rats with successful modeling showed sniffing, tail pressing, rigidity, and bending the body toward the healthy side and rotating head-to-tail with the healthy hind limb as the support point ( Figure 5 In B), the standard for successful modeling is 210r / 30min.

[0086] (2) Fatigue Rotating Rod Test

[0087] Set the rod speed to 10r / min and the time to 30min to test the rat's time on the rod. When the rat falls, the system will automatically pause and record the time on the rod. To ensure the accuracy of the experimental data, each rat needs to be tested 3 times. Figure 5 Middle C.

[0088] (3) Open field experiment: The open field box is 100×100×40 (length×width×height, cm), and a camera is placed above the center grid. Start the computer and turn on the open field experiment tracking and recording system. The camera will automatically capture and record the activities of the rats in the open field box. Figure 5 Middle D.

[0089] 3. Stereotactic transplantation of induced neurons for the treatment of Parkinson's disease model rats

[0090] Staining before transplantation: Collect cells in good growth state into centrifuge tubes, digest with trypsin, and count them after digestion into single cells to ensure that the number of cells injected into each rat is about 1×10 5 Then prepare the cell staining solution: 5μL CM-DiI dye + 1mL Neurobasal culture medium, add the prepared dye to the cell pellet, mix well and put it in a carbon dioxide incubator for staining for 30-40 minutes. Take it out and blow and mix every 10 minutes. Protect from light throughout the process.

[0091] Treatment of Parkinson's model rats with induced neuronal cell stereotaxic transplantation: A successfully established PD rat model was selected, and the cells were transplanted into the right medial forebrain bundle of the PD rats using the same method as the modeling method.

[0092] 4. Organize the collection of materials

[0093] Four weeks after transplantation, 3% pentobarbital was injected intraperitoneally. After being fully anesthetized, the rat was fixed on a rat dissection board, the abdominal cavity was opened in turn, the mediastinum and all organs below the chest that play a role in fluid circulation were clamped with hemostats, the chest cavity was opened, the heart was completely exposed, and a perfusion needle was inserted from the left ventricle into the aortic arch. The right atrial appendage was quickly cut open, and about 200 mL of normal saline was perfused. When the blood flowing out became clear and the liver turned white, about 200 mL of 4% paraformaldehyde was perfused and fixed. After fixation, the brain was dissected in time, the brain tissue was removed and fixed in 4% paraformaldehyde for about 4 hours, and then dehydrated with 25% sucrose and frozen in a -80°C refrigerator. When slicing, the rat brain tissue was taken out of the -80°C refrigerator, embedded and frozen with OCT embedding agent, and then continuous coronal sections with a thickness of 12 to 20 μm were sliced ​​and stored at -20°C for use.

[0094] 5. Immunofluorescence

[0095] The slices were taken out from -20°C and placed at room temperature for 10 minutes, and then immunofluorescence staining (cells stained with red dye CM-DiI) was performed to observe the survival and differentiation ability of the transplanted cells in vivo.

[0096] 6. Results

[0097] The transplanted induced neuronal cells improved the cognitive and motor behavior disorders of Parkinson's model rats. At 4 and 8 weeks after the transplantation of induced neurons, the results of APO-induced rotation behavior test showed that the circling behavior of the transplanted rats was significantly reduced. Figure 6 The total distance of open field movement of the transplanted rats was significantly increased compared with that of the model group rats, and the rats were more active and excited. Figure 7 In addition, compared with the model group, in the fatigue rotarod test, the transplanted group rats significantly increased their time on the rod, and the behavioral improvement effect at week 8 was more obvious than that at week 4. Figure 8 .

[0098] Immunofluorescence staining revealed that the transplanted induced neuronal cells could differentiate into a variety of functional neurons in the host brain, expressing the neuronal surface marker TUJ1 positively and having neuronal characteristics. Fig. 9 Middle A: At the same time, the transplanted cells expressed dopamine neuron-specific markers TH and FOXA2 positively on their surfaces, and the induction of neuronal cell differentiation in vivo was identified. Fig. 9 B, C. The above results indicate that the transplanted induced neurons can differentiate into functional neurons in the host brain and increase the number of positive TH neurons in vivo, thus playing a role in injury repair.

[0099] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0100] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for reprogramming rat skin fibroblasts into neuronal cells, characterized in that: The following steps are involved: Rat embryonic skin fibroblasts were isolated and reprogrammed into intermediate cells under the action of VPA, CHIR99021, Repsox, Forskolin, GO6983, Y-27632, Melatonin, and P7C3-A20; CHIR99021, Forskolin, Dorsomorphin, P7C3-A20, NT3, N2, B27, and cAMP were used to continuously induce the maturation of intermediate cells to obtain mature neuronal cells.

2. The method according to claim 1, characterized in that The specific reprogramming process of the intermediate state cells is: after the rat embryonic skin fibroblasts in vitro grow to 80-90%, they are induced and cultured in a small molecule induction medium to obtain the intermediate state cells; The induction medium is prepared by mixing DMEM / F-12 medium and Neurobasal medium in a volume ratio of 1:1, and the induction medium contains the following small molecules: 0.5mmol / LVPA, 3μmol / L CHIR99021, 1μmol / L Repsox, 30μmol / L Forskolin, 10μmol / LMelatonin, 5μmol / L GO6983, 5μmol / LY-27632, 0.5% N2, 1% B27, 10μmol / L cAMP, 20ng / mL bFGF, 3μmol / L P7C3-A20, and 1% penicillin-streptomycin solution.

3. The method according to claim 2, characterized in that The intermediate cells are obtained by inducing the induction medium for 5 to 7 days, and the medium is replaced by half the amount every 3 days during the induction culture.

4. The method according to claim 1, characterized in that The maturation induction is carried out in a maturation medium for continuous induction, during which fresh medium is replaced every two days; The maturation medium is prepared by mixing DMEM / F-12 medium and Neurobasal medium in a volume ratio of 1:1, and the maturation medium contains the following substances: 3 μmol / L CHIR99021, 30 μmol / L Forskolin, 3 μmol / L P7C3-A20, 1 μmol / L Dorsomorphin, 0.5% N2, 1% B27, 10 μmol / L cAMP, 20 ng / mL bFGF, 20 ng / mL BDNF, 20 ng / mL GDNF, 20 ng / mL NT3, and 1% penicillin-streptomycin solution.

5. The method according to claim 4, characterized in that The continuous induction is induced culture in the maturation medium for 10 to 14 days.

6. A neuronal cell obtained according to the method according to any one of claims 1 to 5.

7. Use of the neuronal cell according to claim 6 in preparing a drug for treating or preventing neurodegenerative diseases.

8. The use according to claim 7, characterized in that The neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

9. A drug for improving neurodegenerative diseases, characterized in that: The method comprises the neuronal cell according to claim 6, and a pharmaceutically acceptable auxiliary material or carrier.

10. The drug according to claim 9, characterized in that The drug is an injection solution comprising the neuronal cells, and the number of the neuronal cells in the injection solution is at least 1×10 5 indivual.

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