Compositions and methods for rapidly reprogramming astrocytes into neurons

Abstract

The present invention relates to the field of biotechnology, and discloses a composition and method for rapidly reprogramming astrocytes into induced neurons, wherein the composition is composed of the small molecule PT109B and one of the PDE family inhibitors. The method comprises the following steps: S1, culturing astrocytes to maturity, so that the cell fusion of the astrocytes is 60%-80%; S2, replacing the culture medium of the astrocytes with a culture medium containing the composition, and continuing to culture for at least 3 days. The present invention combines the small molecule PT109B with the PDE inhibitor to rapidly reprogram astrocytes into induced neurons within 3 days, which not only solves the problems of the large number of small molecule compounds used in the chemical reprogramming technology in the prior art, the complicated steps, and the long cycle, but also completely avoids the risks associated with genetic manipulation, providing a breakthrough solution for the radical treatment of NDDs.

Description

Technical Field

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

[0002] Neurodegenerative diseases (NDDs) are a major disease cluster characterized by progressive neuronal loss in the central and peripheral nervous systems. The resulting cognitive impairment and motor impairment have become a major challenge to global public health. Due to the inherent non-regenerative nature of mature neurons, the structural neural damage caused by NDDs often presents irreversible pathological features. Their core pathogenesis involves the progressive degeneration of neurons and their myelin sheaths, ultimately leading to neural circuit dysfunction.

[0003] Current clinical treatment strategies are primarily based on symptomatic interventions such as dopaminergic replacement therapy. While these can temporarily alleviate some symptoms and slow disease progression, they are unable to achieve substantial regeneration of damaged neurons. More importantly, long-term use of existing drugs can induce serious side effects such as gastrointestinal reactions and motor complications, and they are unable to prevent neuropathological processes such as β-amyloid deposition. Therefore, developing novel treatment modalities that combine neuroregeneration with pathological regulation has become a core scientific proposition in the field of NDDs research.

[0004] With breakthroughs in regenerative medicine theory, in situ neural regeneration technology based on astrocyte transdifferentiation has shown significant therapeutic potential. Since Chen Gong's team first demonstrated that NeuroD1 gene therapy can achieve glial-neuron transdifferentiation, a series of research advances have been made in this field. However, existing technologies generally rely on viral vector-mediated gene editing operations, and their potential risks of genomic integration, tumorigenic transformation, and immune rejection reactions seriously restrict the prospects for clinical translation. 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 has shown 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 control 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 neuroregenerative therapy. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention provides a composition and method for rapidly reprogramming astrocytes into induced neurons. This innovatively designed and developed streamlined dual-molecule induction composition system specifically modulates key signaling pathways, enabling rapid transdifferentiation of astrocytes into induced neurons within three days while completely avoiding the risks associated with genetic manipulation. This provides a breakthrough solution for the radical treatment of NDDs.

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

[0012] A composition for rapidly reprogramming astrocytes into neurons, consisting 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 mass 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. The number of small molecule compounds used is small and the steps are simple, solving the problems of the existing chemical reprogramming technology, which requires a large number of small molecule compounds, complicated steps and a long cycle.

[0017] In some embodiments, 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.

[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 mature and reach a cell confluence of 60%-80%;

[0021] S2. Replace the culture medium of astrocytes with a 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 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.

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

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

[0026] In some embodiments, step S2 specifically comprises: discarding the culture medium in the culture medium of the astrocytes obtained in S1, adding a culture medium containing the composition, and continuing to culture 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. Immunofluorescence assay showed that more than 98% of the cells expressed the astrocyte marker GFAP or S100β, indicating that the cells were mature. The culture was continued until the confluence of the astrocytes on the cell plate reached 60%-80%.

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

[0030] The culture medium in the astrocyte culture medium obtained in S1 was discarded, and the above-mentioned neuronal induction medium was added to the culture dish in which the astrocytes were cultured, replacing 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. This system can achieve rapid transdifferentiation of astrocytes into induced neurons within 3 days by specifically regulating key signaling pathways, and completely avoids the 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 rapidly (within 3 days) reprogram astrocytes into induced neurons. The number of small molecule compounds used is small and the steps are simple. This solves the problems of the existing chemical reprogramming technology, which uses a large number of small molecule compounds, complicated steps, and long cycles, 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 an embodiment of the present invention for rapidly reprogramming astrocytes into induced neurons;

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

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

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

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

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

[0040] The present invention provides a composition for rapidly reprogramming astrocytes into neurons, comprising the small molecule PT109B and one of the PDE family inhibitors, hereinafter collectively referred to as a dual-small molecule induction composition.

[0041] Among them, the structural formula of the small molecule PT109B is shown below:

[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) and degassed with nitrogen bubbling for 1 hour. Pd2(dba)3 (1.32 g, 1.44 mmol, 0.025 eq.) was added. Under nitrogen protection, the mixture was heated to reflux for 8 hours. The reaction progress was monitored by TLC and the reaction was quenched with saturated aqueous NH4Cl. The DME was evaporated under reduced pressure and the mixture was extracted three times with ethyl acetate (100 mL x 3). 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 desired 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 from step (1) (12.8 g, 374.9 mmol, 1.0 eq.) was dissolved in dioxane (500 mL) and a 4M HCl solution in dioxane (500 mL) was added. The reaction mixture was heated to reflux until the conversion of the starting material was complete. 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 carried to the next step.

[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). The mixture was cooled to 0°C. The product from step (2) (374.9 nmol, 1.0 eq.) and iPr2NEt (14.54 mg, 1.12 nmol, 3.0 eq.) were added sequentially. The reaction mixture was allowed to warm to room temperature and stirred until the conversion of the starting materials was complete. The DMF was evaporated under reduced pressure. Ethyl acetate (1 L) was added, washed with water, dried, and concentrated. The residue was purified by column chromatography (DCM / MeOH = 100:1 to 50:1) to obtain 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 combines the small molecule PT109B with a PDE inhibitor to achieve rapid reprogramming of astrocytes 1 into induced neurons 2. The number of small molecule compounds used is small and the steps are simple, which solves the problems of the existing chemical reprogramming technology that uses a large number of small molecule compounds, complicated steps and a long cycle.

[0050] In some embodiments, the dual-molecule induction 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. For example, the concentration of the small molecule PT109B in the neuronal induction culture medium containing the dual-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] An 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 mature and reach a cell confluence of 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 astrocytes with neuronal morphology changes.

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

[0057] In some embodiments, the astrocyte culture medium is selected from 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 culture medium specially designed for cell culture. It can provide appropriate 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 multiplying 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 specifically comprises: extracting astrocytes from the brain of a newborn SD rat and culturing them for 5-7 days. Immunofluorescence analysis indicates that more than 98% of the cells express the astrocyte marker GFAP or S100β, indicating that mature astrocytes have been cultured. Culturing is continued until the astrocyte confluence on the cell plate reaches 60%-80%. Cell confluence in cell culture refers to the proportion of the culture surface occupied by cells adherently growing and interconnected in a culture vessel, such as a cell culture dish or flask, and is typically expressed as a percentage. For example, the confluence of astrocytes on the cell plate may be 60%, 65%, 70%, 75%, or 80%.

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

[0061] In some embodiments, step S2 specifically comprises: discarding the culture medium in the astrocyte culture medium 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 dual-molecule induction 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. For example, the concentration of the small molecule PT109B in the neuronal induction culture medium containing the dual-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 will be further described below with reference to 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 containing the astrocytes, replacing the original culture medium. Culture is continued for 3 days. During this period, the cell status is observed and recorded daily, and the culture medium is replaced every 1-2 days according to the cell status.

[0069] Among them, the preparation method of 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 in Example 1, except that the culture medium containing the dual-molecule induction composition is prepared 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 of DMEM culture medium according to the required concentrations to prepare a neuronal induction culture medium containing the dual-molecule induction composition.

[0073] Example 3 (PT109B+Roflumilast)

[0074] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, except that the culture medium containing the dual-molecule induction composition is prepared 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 of DMEM culture medium according to the required concentration to prepare a neuronal induction medium containing the dual-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, except that the culture medium containing the dual-molecule induction composition is prepared 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 of DMEM culture medium according to the required concentration to prepare a neuronal induction medium containing the dual-molecule induction composition.

[0077] Example 5 (PT109B+Sildenafil)

[0078] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, except that the culture medium containing the dual-small molecule induction composition is prepared 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 of DMEM culture medium according to the required concentration to prepare a neuronal induction 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 in Example 1, except that the culture medium containing the dual-molecule induction composition is prepared 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 of DMEM culture medium according to the required concentration to prepare a neuronal induction medium containing the dual-molecule induction composition.

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

[0082] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, except that the culture medium containing the dual-molecule induction composition is prepared 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 of DMEM culture medium according to the required concentrations to prepare a neuronal induction medium containing the dual-molecule induction composition.

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

[0084] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, except that the culture medium containing the dual-molecule induction composition is prepared 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 of DMEM culture medium according to the required concentration to prepare a neuronal induction culture medium containing the dual-molecule induction composition.

[0085] Example 9 (PT109B+theophylline)

[0086] A method for rapidly reprogramming astrocytes to induce neurons is the same as in Example 1, except that the culture medium containing the dual-molecule induction composition is prepared as follows: PT109B (3 μM), the PDEs inhibitor theophylline (50 μM), 10% fetal bovine serum, and 1% dual-antibody solution are added to 45 mL of DMEM culture medium according to the required concentrations to prepare a neuronal induction culture medium containing the dual-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, except that the culture medium containing the dual small molecule induction composition is replaced with the following blank culture medium. The preparation method is as follows: 10% fetal bovine serum and 1% dual antibody solution are added to 45 mL of 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, except that the culture medium containing the dual-small molecule induction composition is replaced with the following culture medium containing the single small molecule PT109B. The preparation method is as follows: PT109B (3 μM), 10% fetal bovine serum, and 1% dual antibody solution are added to 45 mL of DMEM culture medium according to the required concentration to prepare a culture medium containing the single small molecule PT109B.

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

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

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

[0095] In summary, the present invention provides a method for rapidly reprogramming astrocytes into induced neurons using the small molecule combination PT109B in combination with a PDE inhibitor. This method significantly improves the efficiency and speed of astrocyte-to-neuron conversion by combining PT109B with a PDE family inhibitor (such as Vinpocetine, Milrinone, and Roliumilast). The specific implementation steps include culturing astrocytes to maturity, replacing the culture medium with PT109B and a PDE inhibitor, continuing the culture for three days, and finally verifying the expression of the neuronal marker Map2 by immunofluorescence. The experimental results showed that the combination of PT109B and a PDE inhibitor led to more pronounced neuronal-like changes in astrocytes and significantly enhanced expression of the neuronal marker Map2. This effect was particularly pronounced when PT109B was combined with PDE3, PDE4, and PDE5 inhibitors. This method overcomes the problems of the existing art, which require a large number of small molecule compounds, complex steps, and long processing times, and provides a simple and efficient new strategy for reprogramming astrocytes into neurons.

[0096] The above embodiments provide 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 intended 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 contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for rapidly reprogramming astrocytes into induced neurons, characterized in that: The following steps are involved: S1. Cultivate astrocytes in astrocyte culture medium until they mature and reach a cell confluence of 60%-80%; S2. replacing the astrocyte culture medium with a culture medium containing a composition for rapidly reprogramming astrocytes into neurons, and continuing the culture for at least 3 days; The rapid reprogramming of astrocytes is a composition for inducing neurons, which is composed of the small molecule PT109B and a PDE family inhibitor; The structural formula of the small molecule PT109B is shown below: ； The PDE family inhibitor is 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 9A inhibitor PF-04447943 or PDEs inhibitor theophylline; The concentration of the composition added to the culture medium is: the concentration of the small molecule PT109B is 1-20 μM; 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 PDE9A inhibitor PF-04447943 is 1 μM, or the concentration of the PDEs inhibitor theophylline is 50 μM.

2. The method for rapidly reprogramming astrocytes into neurons according to claim 1, characterized in that: The astrocytes are primary SD rat astrocytes.

3. The method for rapidly reprogramming astrocytes to induce neurons according to claim 1, 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.

4. The method for rapidly reprogramming astrocytes to induce neurons according to claim 3, characterized in that: The PSC neural induction medium includes 45 mL of DMEM medium, 10% fetal bovine serum and 1% double antibody solution.

5. The method for rapidly reprogramming astrocytes to induce neurons according to claim 1, characterized in that: Step S1 specifically comprises: extracting astrocytes from the brain of newborn SD rats and culturing them for 5-7 days. Immunofluorescence detection is performed and if more than 98% of the cells express the astrocyte marker GFAP or S100β, it is considered that mature astrocytes have been cultured. The culture is continued until the confluence of the astrocytes on the cell plate reaches 60%-80%.

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

7. The method for rapidly reprogramming astrocytes to induce neurons according to claim 1, characterized in that: The following steps are involved: S1. Primary SD rat astrocytes were cultured in DMEM high-glucose medium for 5-7 days. Immunofluorescence assay showed that more than 98% of the cells expressed the astrocyte marker GFAP or S100β, indicating that the cells were mature. The culture was continued until the confluence of the astrocytes on the cell plate reached 60%-80%. S2. Add the composition for rapidly reprogramming astrocytes to induce neurons, 10% fetal bovine serum, and 1% double-antibody solution to 45 mL of DMEM medium to prepare a medium containing the composition for rapidly reprogramming astrocytes to induce neurons, i.e., a neuron induction medium; The culture medium of astrocytes in S1 was discarded, and the above-mentioned neuronal induction medium was added to the culture dish of astrocytes, replacing the original culture medium, and the culture was continued for at least 3 days.

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