Application of Bcl11a in the preparation of drugs for nerve injury repair
By targeting and overexpressing Bcl11a, astrocytes are reprogrammed into pyramidal neurons, solving the problem of repair after central nervous system injury and achieving significant neuronal repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANTONG UNIV
- Filing Date
- 2026-01-28
- Publication Date
- 2026-05-26
AI Technical Summary
There is a lack of effective drugs for repairing nerve damage in the current technology, especially drugs that cannot effectively promote the repair of damage to the central nervous system.
Targeted overexpression of Bcl11a promotes the reprogramming of cortical astrocytes into pyramidal neurons. The dynamic trajectory of cells is depicted using spatial transcriptome sequencing and single-cell sequencing technologies. Stereoscopic injection of AAV-rBcl11a into the brain significantly improves neuronal loss after central nervous system injury.
It significantly promoted the repair of the central nervous system after injury, and by targeting and overexpressing Bcl11a, it increased the conversion rate of astrocytes to pyramidal neurons and slowed down the loss of neurons.
Smart Images
Figure CN122075671A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Bcl11a in the preparation of drugs for nerve injury repair. Background Technology
[0002] Most tissues and organs in mammals lack strong regenerative capabilities, and the irreversible regeneration of neurons in the central nervous system (CNS) after adulthood remains a challenge in neuroscience research. Astrocytes (ASTs), the most widely distributed cell type in the CNS, number almost five times that of neurons, play crucial roles in providing structural support to neurons, maintaining metabolic homeostasis, and regulating synaptic function. Furthermore, ASTs and neurons originate from the same precursor cells, exhibiting similar lineage specificity and high homology, making astrocytes an ideal cell source for neuronal reprogramming.
[0003] Bcl11a is a zinc finger protein belonging to the transcription factor family. It is a key mediator of the regulatory network responsible for the development of the brain and various cell lineages. Studies have shown that Bcl11a and Bcl11b are necessary conditions for the generation and differentiation of cortical projection neurons.
[0004] The present invention aims to clarify that Bcl11a is a key transcription factor in the connection between pyramidal cells and glial cells. Summary of the Invention
[0005] Technical problem solved: In view of the shortcomings of the prior art, this application provides the application of Bcl11a in the preparation of drugs for nerve injury repair. Targeted overexpression of Bcl11a can significantly promote the reprogramming of astrocytes in the M1 area of the cerebral motor cortex into pyramidal neurons after spinal cord injury, thus promoting the repair of spinal cord injury and solving the technical problems of lack of effective drugs for nerve injury repair in the prior art.
[0006] Technical solution: To achieve the above objectives, this application provides the following technical solution: Application of Bcl11a in the preparation of drugs for nerve injury repair.
[0007] Furthermore, targeted overexpression of Bcl11a promotes the transformation of cortical astrocytes into pyramidal neurons.
[0008] Furthermore, the nerve injury is a central nervous system injury.
[0009] A drug for nerve injury repair process, with BCL11A as the drug design target.
[0010] Furthermore, the medication used in the nerve injury repair process may be an injectable or non-injectable formulation.
[0011] Furthermore, the medication used in the nerve injury repair process is a pharmaceutically permissible oral dosage form, injection dosage form, powder dosage form, ointment dosage form, or transdermal patch dosage form.
[0012] This application provides the application of Bcl11a in the preparation of drugs for nerve injury repair, which has the following advantages compared with the prior art: 1. This invention uses Bcl11a as a molecular intervention target, targeting the M1 motor area of the cerebral cortex, overexpressing Bcl11a in astrocyte subset 2 (i.e., intermediate cells), promoting the reprogramming of astrocytes into pyramidal neurons, and promoting the repair of the central nervous system after injury. 2. This invention utilizes spatial transcriptome sequencing and single-cell sequencing technologies to depict the dynamic trajectory between astrocytes and pyramidal cells, supporting the hypothesis that intermediate cells may transform into pyramidal cell subclusters 3 after spinal cord injury, and demonstrating that Bcl11a and Zmart4 are key transcription factors in the connection between pyramidal cells and glial cells. 3. This invention, through stereotactic injection of AAV-rBcl11a into the brain, can significantly improve neuronal loss following central nervous system injury. Attached Figure Description
[0013] Figure 1 UMAP diagram of different astrocyte subsets after spinal cord injury in Example 1; Figure 2 This is a graph showing the percentage of each astrocyte subset at different time points after injury, as in Example 1. Figure 3 This is a diagram illustrating the differentiation trajectory of astrocytes after spinal cord injury using CellRank analysis, as shown in Example 1. Figure 4 The diagram shows a significant connection between intermediate-state astrocytes (subpopulation 2) and pyramidal cells (subpopulation 3) in the pseudo-temporal trajectory of Example 1. Figure 5 The partition-based graph abstraction method of Example 1 reveals a significant connection between intermediate-state astrocytes (subpopulation 2) and pyramidal cell subpopulation 3. Figure 6 CellRank analysis for Example 1 depicts the dynamic trajectory between astrocytes and pyramidal cells; Figure 7 This is a differential gene map of intermediate-state astrocytes (subpopulation 2) and pyramidal cell subpopulation 3, used for analysis and screening in Example 1. Figure 8A transcription factor tree diagram was constructed for the differentially expressed genes selected in Example 1; Figure 9 This is a graph showing the analysis of single-cell nuclear sequencing and spatial transcriptome sequencing data from Example 1; Figure 10 This is a Venn diagram of upstream regulatory transcription factors of neuronal classical marker genes in Example 1; Figure 11 This is a gene expression heatmap from Example 1; Figure 12 Protein interaction network analysis in Example 1 confirmed Bcl11a as a potential hub gene. Figure 13 The diagram shows that Foxp2, Bcl11a, and Zmart4 were highly enriched and significantly expressed in intermediate cells, as shown in Example 1. Figure 14 The ST spectrum of Example 2; Figure 15 This is an image showing the immunofluorescence staining results of Example 2; Figure 16 This is a quantitative analysis diagram of the immunofluorescence staining results from Example 2; Figure 17 This is an immunofluorescence staining image of Bcl11a overexpression in vivo in Example 3; Figure 18 Immunofluorescence staining image of Bcl11a overexpression in astrocytes in Example 3, which slows down neuronal loss in the SCI model. Figure 19 This is an immunofluorescence colocalization map of Bcl11a, NeuN, and mcherry in the in vivo astrocyte overexpression of Bcl11a to slow down SCI model of Example 3. Detailed Implementation
[0014] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and equivalent alterations or modifications also fall within the scope defined by the claims of this application.
[0015] Example 1: Application of Bcl11a in the preparation of drugs for nerve injury repair, analysis of single-cell nuclear sequencing and spatial transcriptome sequencing data: Tissue acquisition and sequencing: Motor cortex tissues were collected from 18 rats at six time points (0 hours, 3 hours, 12 hours, 1 day, 3 days, and 7 days) following spinal cord injury. Cell nuclei were isolated using the 10x Genomics platform for sNuc-seq and ST analysis. The relationship between astrocytes and pyramidal neurons was analyzed using CellRank analysis, pseudo-temporal trajectory analysis, and PAGA assay.
[0016] like Figures 1-8 The single-cell nuclear sequencing and spatial transcriptome sequencing data analysis described in Example 1 are shown below. Figure 1 The UMAP diagram for different astrocyte subsets after spinal cord injury in Example 1 shows six different astrocyte subsets. Figure 2 The percentage of each astrocyte subset at different time points after injury is shown in Example 1, indicating that the proportion of astrocyte subsets 1 and 2 increased significantly three days after injury. Figure 3 Example 1 uses CellRank analysis to infer the differentiation trajectory of astrocytes after spinal cord injury, showing that there is connectivity between pyramidal cells and astrocytes, while there is no such phenomenon between OPCs, indicating that some astrocytes may have been transformed into pyramidal cells. Figure 4 The pseudo-timeline trajectory analysis diagram of Example 1 shows that there is a significant connection between intermediate-state astrocytes (subpopulation 2) and pyramidal cell subpopulation 3; Figure 5 The partition-based graph abstraction method in Example 1 revealed a significant connection between intermediate-state astrocytes (subpopulation 2) and pyramidal cell subpopulation 3. Figure 6 CellRank analysis for Example 1 depicted a dynamic trajectory between astrocytes and pyramidal cells, supporting the hypothesis that intermediate-state astrocytes (subset 2) may transform into pyramidal cell subset 3 after spinal cord injury. Figure 7 To analyze and screen differential gene maps of intermediate cells and pyramidal cell subpopulation 3 in Example 1, differentially expressed genes (DEGs) between the two subpopulations were screened. Figure 8 A transcription factor tree diagram was constructed based on the differentially expressed genes screened in Example 1; Figure 9 The image shows the data analysis of single-cell nuclear sequencing and spatial transcriptome sequencing in Example 1. The heatmap visualization shows that there are multiple transcription factors with statistically significant differences between the two cell subpopulations. Figure 10 The Venn diagram of upstream regulatory transcription factors of neuronal classical marker genes in Example 1 shows that Foxp2, Bcl11a, and Zmart4 are highly enriched and significantly expressed in intermediate-state astrocytes (subgroup 2). Figure 11 The gene expression heatmap for Example 1 shows that Foxp2, Bcl11a, and Zmart4 are highly enriched and significantly expressed in intermediate-state astrocytes (subgroup 2). Figure 12The protein interaction network analysis diagram of Example 1 confirms that Bcl11a is a potential hub gene with the highest interaction score; Figure 13 The map shown in Example 1 illustrates that Foxp2, Bcl11a, and Zmart4 are highly enriched and significantly expressed in intermediate-state astrocytes (subgroup 2), suggesting that Bcl11a and Zmart4 may be key transcription factors in the connection between pyramidal cells and glial cells.
[0017] Example 2: Application of Bcl11a in the preparation of a drug for nerve injury repair; changes in Bcl11a expression after spinal cord injury: 1. SD rat spinal cord T10 right hemisection model: Rats were anesthetized by intraperitoneal injection of 0.35 mL / 100 g to ensure complete sedation and painlessness throughout the procedure. The surgical area around the T10 vertebra was shaved and disinfected. The rats were placed prone on a sterile surgical platform. Under a surgical microscope, a midline skin incision was made above the thoracic vertebrae to expose the anterior margin of the T10 vertebra. The right hemisection of the spinal cord was carefully performed using a fine scalpel. The blade was inserted vertically into the dorsal side of the spinal cord at T10, with the cut being as precise as possible to ensure complete damage to only the right hemisection of the spinal cord while preserving the left hemisection. Bleeding was controlled by gently pressing the surgical site with a cotton swab. The muscle and skin layers at the injured site were carefully sutured. The rats were monitored postoperatively for any signs of discomfort, infection, or complications. Analgesics were administered according to the approved protocol for pain management. The rats were ensured to have access to water and food.
[0018] 2. Tissue dehydration and embedding: The perfused tissue was dehydrated in a 30% sucrose solution; the dehydrated tissue was then placed in OCT tissue embedding medium and frozen at -80°C. Sections were then prepared using a CM3050S cryostat.
[0019] 3. Immunofluorescence staining of tissue sections: Sections were dried in a 55°C oven for 30 min; 0.5% PBST was used to incubate the sections for 15 min, followed by membrane rupture at room temperature; the sections were wiped dry, and a histochemical pen was used to draw circles around the tissue; approximately 200 μL of blocking solution was added to the tissue, and the sections were incubated at room temperature for 2 h; the blocking solution was wiped dry, and the prepared primary antibody was added, and the sections were incubated overnight at 4°C; the primary antibody was washed with PBS for 10 min each time, for 3 washes; after wiping the PBS clean, the secondary antibody was added, and the sections were incubated at room temperature for 2 h; the secondary antibody was washed with 0.1% PBST for 10 min each time, for 3 washes; the sections were mounted with anti-fluorescence quenching mounting solution (containing DAPI).
[0020] like Figure 14 and Figure 15 As shown, this illustrates the changes in Bcl11a expression after injury observed using the spinal cord hemisection model established in Example 2 of this paper; where... Figure 14 The ST spectrum of Example 2; Figure 15 and Figure 16 The image shows the results of immunofluorescence staining in Example 2. On day 3 after spinal cord injury, the number of triple-labeled (Bcl11a+, NueN+, and GFAP+) cells increased significantly.
[0021] Example 3: In vivo targeted overexpression of Bcl11a slows down neuronal loss in an SCI model: 1. Stereotactic brain injection AAV5- GfaABC1D- rBcl11a: Rats were anesthetized by intraperitoneal injection of 0.35 mL / 100 g to ensure complete sedation and painlessness throughout the procedure. The rats were placed in a stereotaxic apparatus, and their heads were stabilized using ear rods and dental rods to prevent movement. The skull was kept horizontal between the anterior and posterior fontanelles for accurate localization. Under a surgical microscope, an incision was made along the midline of the scalp to expose the skull. The anterior fontanelle or another anatomical landmark was identified as a reference point for calculating the injection coordinates. The skull in the stereotaxic region was drilled open with a craniotomy burr to expose the left cerebral cortex of the rat, taking care to avoid damaging the underlying brain tissue.
[0022] The glass electrode was attached to the stereotactic arm, and the needle was lowered to the desired depth according to the coordinates of the stereotactic atlas (six injection sites were performed in the M1 region of the left motor cortex for each rat: AP = -1.5 mm, ML = 2.0 mm, DV = -1.5 mm; AP = -1.5 mm, ML = 2.5 mm, DV = -1.5 mm; AP = -2.0 mm, ML = 2.0 mm, DV = -1.5 mm; AP = -2.0 mm, ML = 2.5 mm, DV = -1.5 mm; AP = -1.5 mm, ML = 3.0 mm, DV = -1.5 mm; AP = -2.5 mm, ML = 2.0 mm, DV = -1.5 mm). mm. Wait a few minutes to minimize tissue damage and allow pressure to equalize. After determining the injection site, aspirate the virus using a capillary glass electrode and slowly inject the substance over several minutes to ensure diffusion into the brain tissue. Adjust the injection volume and speed according to experimental requirements and the size of the target area. After injection, leave the needle in place for 5 minutes to allow the substance to diffuse and prevent backflow along the injection path. Then slowly withdraw the needle, minimizing interference with the brain tissue. After gently cleaning the area around the surgical site with a cotton swab and applying hemostatic agents, carefully suture the muscle and skin layers of the injured area. Monitor the rats postoperatively for any signs of discomfort, infection, or complications. Administer analgesics for pain management according to the approved protocol. Ensure the rats have access to water and food.
[0023] 2. SD rat spinal cord T10 right hemisection model, same as in Example 2; 3. Tissue dehydration and embedding, same as in Example 2; 4. Immunofluorescence staining of tissue sections, same as in Example 2.
[0024] like Figure 17 As shown, the in vivo injection of the virus into the motor M1 region of the cerebral cortex led to overexpression of Bcl11a in the M1 region; Figure 18 The results indicate that overexpression of Bcl11a in the M1 motor region increases Neu expression, demonstrating that targeted overexpression of Bcl11a in vivo slows down neuron loss in the SCI model. Figure 19 This indicates that after overexpression of Bcl11a, the number of triple-labeled (Bcl11a+, Nuen+, and GFAP+) cells significantly increased on day 3 after spinal cord injury, further validating the conclusion that targeted overexpression of Bcl11a in vivo slows down neuronal loss in the SCI model.
[0025] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.
Claims
1. The application of Bcl11a in the preparation of drugs for nerve injury repair.
2. The application of Bcl11a in the preparation of a drug for nerve injury repair according to claim 1, characterized in that: Targeted overexpression of Bcl11a promotes the transformation of astrocytes in the cerebral cortex into pyramidal neurons.
3. The application of Bcl11a in the preparation of a drug for nerve injury repair according to claim 1, characterized in that: The nerve injury refers to damage to the central nervous system.
4. A drug for nerve injury repair, characterized in that: BCL11A as a drug design target.
5. The drug according to claim 4, characterized in that: The medication used in the nerve injury repair process can be either injectable or non-injectable.
6. The drug according to claim 5, characterized in that: The medications used in the nerve injury repair process are pharmaceutically permissible oral dosage forms, injection dosage forms, powder dosage forms, ointment dosage forms, or transdermal patch dosage forms.