Compositions and methods for rapidly reprogramming astrocytes into induced neurons

Abstract

The invention relates to the technical field of biology, and discloses a composition and a method for rapidly reprogramming astrocytes into induced neurons, and the composition is composed of one of micromolecule PT109B and a PDE family inhibitor. The method comprises the following steps: S1, culturing astrocytes until the astrocytes are mature, so that the cell fusion degree of the astrocytes is 60-80%; and S2, replacing a culture medium of astrocytes with a culture medium containing the composition, and continuously culturing for at least 3 days. After the micromolecule PT109B and the PDE inhibitor are used together, astrocytes can be rapidly reprogrammed into induced neurons within 3 days, the problems that in the prior art, the number of small molecule compounds used in a chemical reprogramming technology is large, the steps are complex, and the period is long are solved, and related risks of gene manipulation are completely avoided; and a breakthrough solution is provided for the radical treatment of the NDDs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a composition and a method for rapidly reprogramming astrocytes into induced neurons. Background Art

[0002] Neurodegenerative diseases (NDDs) are a group of major diseases characterized by progressive neuronal loss in the central and peripheral nervous systems. The cognitive dysfunction and motor system damage caused by NDDs have become a major challenge in the field of global public health. Due to the inherent non-regenerative nature of mature neurons, structural nerve damage caused by NDDs often presents irreversible pathological characteristics. Its core pathogenesis involves the progressive degeneration of neurons and their myelin sheaths, which ultimately leads to dysfunction of neural circuits.

[0003] Current clinical treatment strategies are mainly based on symptomatic interventions such as dopaminergic replacement therapy, which can temporarily relieve some symptoms and delay the progression of the disease, but cannot achieve substantial regeneration of damaged neurons. More importantly, long-term use of existing drugs may induce serious side effects such as gastrointestinal reactions and motor complications, and cannot prevent neuropathological processes such as β-amyloid deposition. Therefore, the development of a new treatment model that combines neuroregeneration function with pathological regulation has become a core scientific proposition in the field of NDDs research.

[0004] With the breakthrough in regenerative medicine theory, in situ neural regeneration technology based on astrocyte transdifferentiation has shown great therapeutic potential. Since Chen Gong's team first confirmed that NeuroD1 gene therapy can achieve glial-neuron transdifferentiation, a series of research progress has been made in this field. However, existing technologies generally rely on viral vector-mediated gene editing operations, and their potential genome integration risks, tumorigenic transformation possibilities, and immune rejection reactions seriously restrict the prospects for clinical transformation. The rise of chemical reprogramming technology has provided a new direction for breaking through the limitations of gene therapy, especially the research on inducing astrocyte transdifferentiation through a combination of small molecule compounds, which shows a higher feasibility of clinical application.

[0005] However, the existing chemical reprogramming system has significant technical bottlenecks:

[0006] (1) The need to use more than four small molecules in combination makes it difficult to regulate pharmacokinetic parameters;

[0007] (2) The transdifferentiation cycle generally exceeds 14 days, which is difficult to meet the needs of acute nerve repair;

[0008] (3) Multi-step sequential dosing regimens seriously affect the operability of treatment.

[0009] Therefore, developing a composition and method for rapidly reprogramming astrocytes to induce neurons, a streamlined dual-small molecule combination with a clear structure-activity relationship, and establishing an efficient and rapid in vitro / in vivo transdifferentiation system have become key breakthroughs in achieving clinical-grade neural regeneration therapy. Summary of the invention

[0010] In view of the shortcomings of the prior art, the present invention provides a composition and method for rapidly reprogramming astrocytes into induced neurons. The present invention innovatively designs and develops a streamlined dual-small molecule induction composition system, which can achieve rapid transdifferentiation of astrocytes into induced neurons within 3 days by specifically regulating key signaling pathways, and completely avoids risks associated with genetic manipulation, providing a breakthrough solution for the radical treatment of NDDs.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] A composition for rapidly reprogramming astrocytes into induced neurons, which is composed of the small molecule PT109B and one of the PDE family inhibitors.

[0013] Among them, the small molecule PT109B is one of the configurations of PT109, its chemical name is 5-(1,2-dithiolan-3-yl)-N-((1r,4r)-4-(isoquinolin-5-ylamino)cyclohexyl) ntanamide, and its chemical formula is C 23 H 31 N3OS2, molecular weight is 429.64, and the structural formula is as follows:

[0014] .

[0015] PDE family inhibitors include PDE 1 inhibitor Vinpocetine, PDE 3 inhibitor Milrinone, PDE 4 inhibitor Roflumilast, PDE 4 inhibitor Apremilast, PDE 5 inhibitor Sildenafil, PDE 5 inhibitor Avanafil, PDE 7 inhibitor BRL-50481, PDE 9A inhibitor PF-04447943, and PDEs inhibitor theophylline.

[0016] The present invention combines the small molecule PT109B with a PDE inhibitor to rapidly (within 3 days) reprogram astrocytes into induced neurons, uses a small number of small molecule compounds and has simple steps, thereby solving the problems of a large number of small molecule compounds, complicated steps and a long cycle used in the prior art chemical reprogramming technology.

[0017] In some embodiments, the composition is added to the culture medium at a concentration of 1-20 μM for the small molecule PT109B and 1-50 μM for the PDE family inhibitor.

[0018] In some embodiments, the concentration of the PDE 1 inhibitor Vinpocetine is 30 μM, or the concentration of the PDE 3 inhibitor Milrinone is 10 μM, or the concentration of the PDE 4 inhibitor Roflumilast is 10 μM, or the concentration of the PDE 4 inhibitor Apremilast is 1 μM, or the concentration of the PDE 5 inhibitor Sildenafil is 10 μM, or the concentration of the PDE 5 inhibitor Avanafil is 10 μM, or the concentration of the PDE 7 inhibitor BRL-50481 is 5 μM, or the concentration of the PDE 9A inhibitor PF-04447943 is 1 μM, or the concentration of the PDEs inhibitor theophylline is 50 μM.

[0019] Another object of the present invention is to disclose a method for rapidly reprogramming astrocytes into induced neurons, comprising the following steps:

[0020] S1. Cultivate astrocytes until they are mature and the cell confluence of astrocytes is 60%-80%;

[0021] S2. Replace the culture medium of astrocytes with the culture medium containing the above composition, and continue culturing for at least 3 days.

[0022] In some embodiments, the astrocytes are primary SD rat astrocytes.

[0023] In some embodiments, the culture medium for astrocytes is selected from one of a DMEM-based culture medium, a serum-free induction culture medium, and a PSC neural induction culture medium.

[0024] In some embodiments, the main components of the PSC neural induction medium include 45 mL DMEM medium, 10% fetal bovine serum, and 1% double antibody solution.

[0025] In some embodiments, step S1 is specifically as follows: astrocytes are extracted from the brain of newborn SD rats and cultured for 5-7 days. When more than 98% of the cells express astrocyte markers GFAP or S100β by immunofluorescence detection, it is considered that mature astrocytes have been cultured. The culture is continued until the degree of confluence of astrocytes on the cell plate is 60%-80%.

[0026] In some embodiments, step S2 specifically includes: discarding the culture medium in the culture medium of astrocytes obtained in S1, adding a culture medium containing the composition, and continuing culturing for at least 3 days.

[0027] In some embodiments, a method for rapidly reprogramming astrocytes to induce neurons comprises the following steps:

[0028] S1. Primary SD rat astrocytes were cultured in DMEM high-glucose medium for 5-7 days. When more than 98% of the cells expressed astrocyte markers GFAP or S100β by immunofluorescence detection, mature astrocytes were considered to have been cultured. The culture was continued until the confluence of astrocytes on the cell plate reached 60%-80%.

[0029] S2, adding the composition, 10% fetal bovine serum, and 1% double antibody solution into 45 mL of DMEM culture medium to prepare a neuron induction culture medium containing the composition;

[0030] The culture medium in the culture medium of astrocytes obtained in S1 was discarded, and the above-mentioned neuron induction medium was added to the culture dish in which astrocytes were cultured to replace the original culture medium, and the culture was continued for at least 3 days.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention innovatively designs and develops a streamlined dual-small molecule induction composition system, which can achieve rapid transdifferentiation of astrocytes into induced neurons within 3 days by specifically regulating key signaling pathways, and completely avoids risks associated with genetic manipulation, providing a breakthrough solution for the radical treatment of NDDs.

[0033] The present invention combines the small molecule PT109B with a PDE inhibitor to quickly (within 3 days) reprogram astrocytes into induced neurons. The number of small molecule compounds used is small and the steps are simple, which solves the problems of a large number of small molecule compounds, complicated steps and long cycles used in the chemical reprogramming technology in the prior art, and provides a simple and efficient new strategy for reprogramming astrocytes into neurons. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the principle of the dual small molecule induction composition in the embodiment of the present invention to rapidly reprogram astrocytes into induced neurons;

[0035] Figure 2 Schematic diagram of nuclear magnetic resonance of small molecule PT109B in an embodiment of the present invention;

[0036] Figure 3is a picture of astrocyte morphology of Example 1-9 and Comparative Example 1-2 after administration for 72 hours;

[0037] Figure 4 It is an immunofluorescence cytochemical staining image of the astrocyte neuron marker Map2 of Example 1-9 and Comparative Example 1-2 after administration for 72 hours;

[0038] Figure 5 It is a schematic diagram of the statistical results of the positive expression rate of the astrocyte neuron marker Map2 in Example 1-9 and Comparative Example 1-2 72 hours after administration. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. The reagents and instruments used in the embodiments are all conventional products that can be obtained commercially unless otherwise specified. The experimental methods used in the embodiments are all conventional experimental methods or technical means in the art unless otherwise specified.

[0040] The embodiment of the present invention provides a composition for rapidly reprogramming astrocytes to induce neurons, which is composed of a small molecule PT109B and one of the PDE family inhibitors, which are collectively referred to as a dual small molecule induction composition below.

[0041] Among them, the structural formula of the small molecule PT109B is as follows:

[0042] .

[0043] In some embodiments, the small molecule PT109B is prepared by the following steps:

[0044] (1) 5-Bromoisoquinoline (12.0 g, 57.68 mmol, 1.0 eq.), 1-N-Boc-trans-1,4-cyclohexanediamine (13.60 g, 63.44 mmol, 1.1 eq.) and XPhos (1.38 g, 2.88 mmol, 0.05 eq.) were dissolved in DME (240 mL), degassed by nitrogen bubbling for 1 hour, and Pd2(dba)3 (1.32 g, 1.44 mmol, 0.025 eq.) was added. Under N2 protection, the mixture was heated to reflux for 8 hours. The reaction progress was monitored by TLC, and the reaction was quenched with saturated NH4Cl aqueous solution. The DME was evaporated under reduced pressure and concentrated. The mixture was extracted three times with ethyl acetate (100 mLx3). The organic phases were combined, washed with water, dried and concentrated. The residue was purified by column chromatography (DCM / MeOH = 100:1 to 50:1) to obtain the target product as a light yellow solid (10.3 g, 52.3%). 1HNMR (400 MHz, CDCl3): δ 9.12 (s, 1H), 8.43 (d, J= 8 Hz, 1H), 7.50 (m, 1H), 7.42 (s, 2H), 7.28 (s,1H), 6.74 (d, J= 8 Hz, 1H), 4.52 (s, 1H), 4.11 (s, 1H), 3.51 (s, 1H), 3.39 (s, 1H), 2.23 (m, 2H), 2.10 (m, 2H), 1.44 (s, 9H), 1.38-1.20 (m, 4H).

[0045] (2) The product of step (1) (12.8 g, 374.9 mmol, 1.0 eq.) was dissolved in dioxane (500 mL), and a 4M HCl dioxane solution (500 mL) was added thereto. The reaction solution was heated to reflux until the raw material was completely converted. The mixture was cooled to room temperature, filtered, and the filter cake was washed twice with dioxane (200 mL x 2). The filter cake was vacuum dried and directly subjected to the next step of reaction.

[0046] (3) α-lipoic acid (81.2 mg, 393.6 nmol, 1.05 eq.), EDCI (7.55 mg, 393.6 nmol, 1.05 eq.) and HOBt (5.32 mg, 393.6 nmol, 1.05 eq.) were dissolved in anhydrous DMF (500 ml) solution. The mixture was cooled to 0°C, and the product of step (2) (374.9 nmol, 1.0 eq.) and iPr2NEt (14.54 mg, 1.12 nmol, 3.0 eq.) were added thereto in sequence. The reaction mixture was naturally heated to room temperature and the reaction was continued under stirring until the raw materials were completely converted. DMF was evaporated under reduced pressure. Ethyl acetate (1 L) was added, washed with water, dried, concentrated, and the residue was purified by column chromatography (DCM / MeOH = 100:1 to 50:1) to give the desired product as a slightly yellow solid (120.0 mg, 74.5%). 1H NMR (400MHz, CDCl3): 9.13 (s, 1H), 8.44 (d, J = 6.0 Hz, 1H), 7.54 (d, J = 6.0 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 7.35 – 7.18 (m, 1H), 6.75 (d, J = 7.7 Hz, 1H), 5.47 (d, J = 8.0 Hz, 1H), 4.26 (d, J = 6.8 Hz, 1H), 3.98 – 3.78 (m, 1H), 3.67– 3.50 (m, 1H), 3.42 (s, 1H), 3.28 – 3.03 (m, 2H), 2.45 (dd, J = 12.6, 6.2Hz, 1H), 2.27 (d, J = 12.2 Hz, 2H), 2.23 – 2.03 (m, 3H), 1.91 (dt, J = 13.0,6.4 Hz, 2H), 1.68 (m, 3H), 1.57 – 1.20 (m, 5H).

[0047] The product of step (3) is the small molecule PT109B, see Figure 2 Schematic diagram of nuclear magnetic resonance.

[0048] PDE family inhibitors, including PDE 1 inhibitor Vinpocetine, PDE 3 inhibitor Milrinone, PDE 4 inhibitor Roflumilast, PDE 4 inhibitor Apremilast, PDE 5 inhibitor Sildenafil, PDE 5 inhibitor Avanafil, PDE 7 inhibitor BRL-50481, PDE 9A inhibitor PF-04447943, and PDEs inhibitor theophylline.

[0049] like Figure 1 As shown, the present invention can achieve rapid reprogramming of astrocytes 1 into induced neurons 2 by combining the small molecule PT109B with a PDE inhibitor, and the number of small molecule compounds used is small and the steps are simple, which solves the problems of a large number of small molecule compounds used in chemical reprogramming technology in the prior art, complicated steps and long cycles.

[0050] In some embodiments, the concentration of the dual small molecule induction composition added to the culture medium is: the concentration of the small molecule PT109B is 1-20 μM, and the concentration of the PDE family inhibitor is 1-50 μM. For example, the concentration of the small molecule PT109B in the neuron induction culture medium containing the dual small molecule induction composition is 1 μM, 3 μM, 10 μM, 15 μM or 20 μM, etc.

[0051] In some embodiments, in the neuronal induction medium containing the dual small molecule induction composition, the concentration of the PDE 1 inhibitor Vinpocetine is 30 μM, or the concentration of the PDE 3 inhibitor Milrinone is 10 μM, or the concentration of the PDE 4 inhibitor Roflumilast is 10 μM, or the concentration of the PDE 4 inhibitor Apremilast is 1 μM, or the concentration of the PDE 5 inhibitor Sildenafil is 10 μM, or the concentration of the PDE 5 inhibitor Avanafil is 10 μM, or the concentration of the PDE 7 inhibitor BRL-50481 is 5 μM, or the concentration of the PDE 9A inhibitor PF-04447943 is 1 μM, or the concentration of the PDEs inhibitor theophylline is 50 μM.

[0052] The embodiment of the present invention also provides a method for rapidly reprogramming astrocytes into induced neurons, comprising the following steps:

[0053] S1. Cultivate astrocytes until they are mature and the cell confluence of astrocytes is 60%-80%;

[0054] S2. Replace the culture medium of astrocytes with the culture medium containing the dual small molecule induction composition, and continue culturing for at least 3 days.

[0055] In some embodiments, the induced neurons include at least one of the neuronal marker Map2 and the astrocytes having neuronal morphology changes.

[0056] In some embodiments, the astrocytes are preferably primary SD rat astrocytes. Primary SD rat cells generally refer to cells obtained directly from Sprague-Dawley (SD) rats. SD rats are a commonly used experimental animal strain with the advantages of fast growth, strong fertility, good adaptability to the environment, and relatively docile temperament.

[0057] In some embodiments, the astrocyte culture medium is selected from one of a DMEM-based culture medium, a serum-free induction culture medium, and a PSC neural induction culture medium. Preferably, the main components of the PSC neural induction culture medium include 45 mL of DMEM culture medium, 10% fetal bovine serum, and 1% double antibody solution.

[0058] Among them, DMEM medium is a basic medium specially designed for cell culture. It can provide suitable nutrients for cell growth, and its physical and chemical properties such as osmotic pressure and pH are also suitable for cell survival and development. Fetal bovine serum is an additive that can provide some nutrients and growth factors necessary for cell growth. The double-antibody solution is a mixture of penicillin and streptomycin, usually containing 100 U / mL penicillin and 100 μg / mL streptomycin, mainly to prevent bacterial contamination. Penicillin can inhibit the synthesis of bacterial cell walls, while streptomycin mainly interferes with the synthesis of bacterial proteins. The combined use of the two can effectively prevent most common bacteria from growing and reproducing in the culture medium, thereby protecting the purity of the neuronal cell culture system and ensuring that neurons can grow in a relatively sterile environment.

[0059] In some embodiments, step S1 is specifically as follows: astrocytes are extracted from the brain of newborn SD rats and cultured for 5-7 days. When more than 98% of the cells express astrocyte markers GFAP or S100β by immunofluorescence detection, it is considered that mature astrocytes are cultured, and the culture is continued until the fusion degree of astrocytes on the cell plate is 60%-80%. The cell fusion degree in cell culture refers to the proportion of cells that grow adherently and connect to each other in a culture container such as a cell culture dish or culture bottle, occupying the culture surface, usually expressed as a percentage. For example, the fusion degree of astrocytes on the cell plate is 60%, 65%, 70%, 75% or 80%, etc.

[0060] In some embodiments, the medium for culturing astrocytes to maturity can be Dulbecco's Modified Eagle Medium (DMEM high glucose medium), which is a synthetic medium widely used in cell culture.

[0061] In some embodiments, step S2 specifically includes: discarding the culture medium in the culture medium of astrocytes obtained in S1, adding a culture medium containing the dual small molecule induction composition, and continuing to culture for at least 3 days.

[0062] In some embodiments, the concentration of the dual small molecule induction composition added to the culture medium is: the concentration of the small molecule PT109B is 1-20 μM, and the concentration of the PDE family inhibitor is 1-50 μM. For example, the concentration of the small molecule PT109B in the neuron induction culture medium containing the dual small molecule induction composition is 1 μM, 3 μM, 10 μM, 15 μM or 20 μM, etc.

[0063] In some embodiments, in the neuronal induction medium containing the dual small molecule induction composition, the concentration of the PDE 1 inhibitor Vinpocetine is 30 μM, or the concentration of the PDE 3 inhibitor Milrinone is 10 μM, or the concentration of the PDE 4 inhibitor Roflumilast is 10 μM, or the concentration of the PDE 4 inhibitor Apremilast is 1 μM, or the concentration of the PDE 5 inhibitor Sildenafil is 10 μM, or the concentration of the PDE 5 inhibitor Avanafil is 10 μM, or the concentration of the PDE 7 inhibitor BRL-50481 is 5 μM, or the concentration of the PDE 9A inhibitor PF-04447943 is 1 μM, or the concentration of the PDEs inhibitor theophylline is 50 μM.

[0064] The present invention is further described below by way of Examples 1-9 and Comparative Examples 1-2.

[0065] Example 1 (PT109B+Vinpocetine)

[0066] A method for rapidly reprogramming astrocytes into induced neurons, comprising the following steps:

[0067] (1) Primary SD rat astrocytes were cultured in DMEM high-glucose medium for 5-7 days. Immunofluorescence detection showed that more than 98% of the cells expressed astrocyte markers GFAP or S100β, which was considered to be mature astrocytes. The cells were cultured until the cell confluence reached 60-80%.

[0068] (2) The astrocytes obtained in step (1) are cultured continuously. On the first day of culture, the original culture medium is discarded, and the culture medium containing the dual small molecule induction composition is added to the culture dish for culturing astrocytes, replacing the original culture medium, and continuing the culture for 3 days. During this period, the cell status is observed and recorded every day, and the culture medium is replaced every 1-2 days according to the cell status.

[0069] Among them, the method for preparing the culture medium containing the dual small molecule induction composition is as follows: according to the concentration requirements, PT109B (3 μM), PDE 1 inhibitor Vinpocetine (30 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium to prepare a neuronal induction culture medium containing the dual small molecule induction composition.

[0070] (3) On the third day, cell immunofluorescence technology was used to detect the expression of the neuronal marker Map2 in induced neurons.

[0071] Example 2 (PT109B+Milrinone)

[0072] A method for rapidly reprogramming astrocytes to induce neurons is the same as Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDE 3 inhibitor Milrinone (10 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the required concentration to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0073] Example 3 (PT109B+Roflumilast)

[0074] A method for rapidly reprogramming astrocytes to induce neurons is the same as Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDE 4 inhibitor Roflumilast (10 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the required concentration to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0075] Example 4 (PT109B+Apremilast)

[0076] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDE 4 inhibitor Apremilast (1 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the required concentration to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0077] Example 5 (PT109B+Sildenafil)

[0078] A method for rapidly reprogramming astrocytes to induce neurons is the same as Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDE 5 inhibitor Sildenafil (10 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the required concentration to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0079] Example 6 (PT109B+Avanafil)

[0080] A method for rapidly reprogramming astrocytes to induce neurons is the same as Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDE 5 inhibitor Avanafil (10 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the required concentration to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0081] Example 7 (PT109B + BRL-50481)

[0082] A method for rapidly reprogramming astrocytes to induce neurons is the same as Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDE 7 inhibitor BRL-50481 (5 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the concentration requirements to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0083] Example 8 (PT109B + PF-04447943)

[0084] A method for rapidly reprogramming astrocytes to induce neurons is the same as Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDE 9A inhibitor PF-04447943 (1 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the concentration requirements to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0085] Example 9 (PT109B+theophylline)

[0086] A method for rapidly reprogramming astrocytes to induce neurons is the same as Example 1, but the method for preparing the culture medium containing the dual small molecule induction composition is as follows: PT109B (3 μM), PDEs inhibitor theophylline (50 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the required concentration to prepare a neuron induction culture medium containing the dual small molecule induction composition.

[0087] Comparative Example 1 (blank example)

[0088] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, but the culture medium containing the dual small molecule induction composition is replaced with the following blank culture medium, and the preparation method is as follows: 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the required concentration to prepare a blank culture medium.

[0089] Comparative Example 2 (PT109B)

[0090] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, but the culture medium containing the dual small molecule induction composition is replaced with the following culture medium containing the single small molecule PT109B, and the preparation method is as follows: PT109B (3 μM), 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL DMEM culture medium according to the concentration requirements to prepare a culture medium containing the single small molecule PT109B.

[0091] Figure 3 It is the astrocyte morphology picture of Example 1-9 and Comparative Example 1-2 after administration for 72 hours.

[0092] Figure 4 It is the immunofluorescence cytochemical staining image of the astrocyte neuron marker Map2 after 72 hours of administration of Example 1-9 and Comparative Example 1-2.

[0093] Figure 5It is a schematic diagram of the statistical results of the positive expression rate of the astrocyte neuron marker Map2 in Example 1-9 and Comparative Example 1-2 72 hours after administration.

[0094] from Figure 4 As can be seen in the figure, after three days of single administration of PT109B (3μM), the cells showed neuron-like changes but not obvious. However, after the combination of PT109B (3μM) and PDE inhibitors to form a dual small molecule induction combination, the morphology showed obvious neuron-like changes. Except for the PDE 7 inhibitor BRL-50481, the expression of neuron marker Map2 in other small molecule combinations was statistically different from that in PT109B alone, as shown in Figure 2. Figure 5 As shown. This indicates that the combination of PT109B and PDE inhibitors can quickly reprogram astrocytes into induced neurons. Moreover, the difference in the expression of the neuronal marker Map2 is more significant when PT109B is used in combination with PDE3, PDE4, and PDE5 inhibitors compared to the use of PT109B alone.

[0095] In summary, the embodiment of the present invention provides a method for rapidly reprogramming astrocytes into induced neurons by using a small molecule combination PT109B and a PDE inhibitor. The method significantly improves the efficiency and speed of astrocyte conversion into induced neurons by combining PT109B with a PDE family inhibitor (such as Vinpocetine, Milrinone, Roliumilast, etc.). The specific implementation steps include culturing astrocytes to maturity, replacing them with a culture medium containing PT109B and a PDE inhibitor, continuing to culture for 3 days, and finally verifying the expression of the neuronal marker Map2 by immunofluorescence detection. The experimental results show that after PT109B is used in combination with a PDE inhibitor, the neuronal-like changes of astrocytes are more obvious, and the expression of the neuronal marker Map2 is significantly enhanced, especially when PT109B is used in combination with PDE3, PDE4, and PDE5 inhibitors. The effect is more significant. This method solves the problems of a large number of small molecule compounds, complicated steps, and long cycles in the prior art, and provides a simple and efficient new strategy for reprogramming astrocytes into neurons.

[0096] The above embodiments have provided a detailed introduction to the technical solutions provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A composition for rapidly reprogramming astrocytes into neurons, characterized in that: It is composed of the small molecule PT109B and one of the PDE family inhibitors; The structural formula of the small molecule PT109B is shown below: ； The PDE family inhibitors include PDE 1 inhibitor Vinpocetine, PDE 3 inhibitor Milrinone, PDE 4 inhibitor Roflumilast, PDE 4 inhibitor Apremilast, PDE 5 inhibitor Sildenafil, PDE 5 inhibitor Avanafil, PDE 7 inhibitor BRL-50481, PDE 9A inhibitor PF-04447943, and PDEs inhibitor theophylline.

2. The composition for rapidly reprogramming astrocytes to induce neurons according to claim 1, characterized in that: The concentration of the composition added to the culture medium is: the concentration of the small molecule PT109B is 1-20 μM, and the concentration of the PDE family inhibitor is 1-50 μM.

3. The composition for rapidly reprogramming astrocytes into neurons according to claim 2, characterized in that: The concentration of the PDE 1 inhibitor Vinpocetine is 30 μM, or the concentration of the PDE 3 inhibitor Milrinone is 10 μM, or the concentration of the PDE 4 inhibitor Roflumilast is 10 μM, or the concentration of the PDE 4 inhibitor Apremilast is 1 μM, or the concentration of the PDE 5 inhibitor Sildenafil is 10 μM, or the concentration of the PDE 5 inhibitor Avanafil is 10 μM, or the concentration of the PDE 7 inhibitor BRL-50481 is 5 μM, or the concentration of the PDE 9A inhibitor PF-04447943 is 1 μM, or the concentration of the PDEs inhibitor theophylline is 50 μM.

4. A method for rapidly reprogramming astrocytes into induced neurons, characterized in that: The following steps are involved: S1. Cultivate astrocytes until they are mature and the cell confluence of astrocytes is 60%-80%; S2. Replace the culture medium of astrocytes with a culture medium containing the composition according to any one of claims 1 to 3, and continue culturing for at least 3 days.

5. The method for rapidly reprogramming astrocytes to induce neurons according to claim 4, characterized in that: The astrocytes are primary SD rat astrocytes.

6. The method of rapidly reprogramming astrocytes to induce neurons according to claim 4, characterized in that: The astrocyte culture medium is selected from one of a DMEM-based culture medium, a serum-free induction culture medium, and a PSC neural induction culture medium.

7. A method for rapidly reprogramming astrocytes to induce neurons according to claim 6, characterized in that: The main components of the PSC neural induction medium include 45 mL DMEM medium, 10% fetal bovine serum, and 1% double antibody solution.

8. The method of rapidly reprogramming astrocytes to induce neurons according to claim 4, characterized in that: Step S1 is specifically as follows: astrocytes are extracted from the brain of newborn SD rats and cultured for 5-7 days. When more than 98% of the cells express astrocyte markers GFAP or S100β by immunofluorescence detection, it is considered that mature astrocytes have been cultured. The culture is continued until the degree of confluence of astrocytes on the cell plate is 60%-80%.

9. The method of rapidly reprogramming astrocytes to induce neurons according to claim 4, characterized in that: Step S2 specifically comprises: discarding the culture medium in the culture medium of astrocytes obtained in S1, adding a culture medium containing the composition according to any one of claims 1 to 3, and continuing the culture for at least 3 days.

10. The method of rapidly reprogramming astrocytes to induce neurons according to claim 4, characterized in that: The following steps are involved: S1. Primary SD rat astrocytes were cultured in DMEM high-glucose medium for 5-7 days. When more than 98% of the cells expressed astrocyte markers GFAP or S100β by immunofluorescence detection, mature astrocytes were considered to have been cultured. The culture was continued until the confluence of astrocytes on the cell plate reached 60%-80%. S2. Add the composition according to any one of claims 1 to 3, 10% fetal bovine serum, and 1% double antibody solution into 45 mL of DMEM culture medium to prepare a neuron induction culture medium containing the composition according to any one of claims 1 to 3; The culture medium in the culture medium of astrocytes obtained in S1 was discarded, and the above-mentioned neuron induction medium was added to the culture dish in which astrocytes were cultured to replace the original culture medium, and the culture was continued for at least 3 days.

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