CN119405806A

CN119405806A - Application of dystrophin domain 2 inhibitor in the preparation of drugs for promoting nerve growth

Info

Publication number: CN119405806A
Application number: CN202411086541.0A
Authority: CN
Inventors: 易晟; 赵莉莉; 张岚; 钱雨悦; 龚蕾蕾
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2024-10-25
Filing date: 2024-10-25
Publication date: 2025-02-11

Abstract

The invention provides application of a small muscular dystrophin domain 2 (dysbindin domain containing 2, dbdd 2) inhibitor in preparing a medicament for promoting nerve growth. The invention discloses that the gene expression of the knockdown Dbndd < 2 > can promote the growth of the neurites and the regeneration of the damaged nerves for the first time, which shows that Dbndd < 2 > can be used as a drug design target point, and the Dbndd < 2 > inhibitor can promote the growth of the neurites and is used for treating traumatic nerve injury and nerve injury related neurodegenerative diseases.

Description

Application of small muscular dystrophin domain 2-containing inhibitor in preparation of nerve growth promoting medicine

Technical Field

The invention relates to the field of biological medicine, in particular to application of a small myotrophic protein domain 2 (dysbindin domain containing, dbdd 2) inhibitor in preparation of a medicament for promoting nerve growth.

Background

Traumatic nerve injury can lead to injury of neuron axons, cause interruption of nerve conduction, cause serious sensory and motor dysfunction, cause pain, burning sensation, abnormal sensation, fibrosis, muscular atrophy and other complications, and seriously impair the life quality of patients. In the pathological process of neurodegenerative diseases such as amyotrophic lateral sclerosis and glaucoma, the neurites of neurons are also obviously changed in structure and morphology. The molecular mechanism of nerve axon growth is analyzed, molecules which have an effect of blocking axon regeneration are excavated, and the use of the molecular inhibitor can play a role of promoting damaged axon regeneration, thereby being beneficial to promoting the recovery of traumatic nerve injury or neurodegenerative diseases related to axon injury.

Dorsal root ganglion (dorsal root ganglion, DRG) neurons are pseudounipolar neurons with pericytes that project to the peripheral nerve and central processes that project to the spinal cord. After the peripheral processes of the DRG neurons are damaged, the damaged axons can realize a certain degree of functional recovery. However, DRG neurons are difficult to regenerate after CNS injury, resulting in severe sensory dysfunction. Genes that exhibit a down-regulation trend within the DRG following peripheral process injury and genes that exhibit an opposite up-regulation trend within the DRG following central process injury may act as a barrier to axon regeneration of neurons.

The small dystrophin domain contains 2 (dysbindin domain containing, dbndd 2), also known as casein kinase-1 binding protein, which is involved in the negative regulation of protein kinase activity. It was found that e.g. the small dystrophin domain 2 can reduce the phosphorylation and oligomerization of the alpha-synuclein Ser129 by binding to casein kinase-1 and reduce the level of insoluble alpha-synuclein, an important potential target for delaying the progression of alpha-synucleinopathies. At present, no study on the nerve regeneration process of the small muscular dystrophin domain 2 is reported.

Disclosure of Invention

According to the invention, after the perineurite and the central process injury of the DRG neuron are analyzed, the gene with the expression change is shown in the DRG, and the gene Dbndd which codes for the small muscular dystrophin domain 2 in the DRG is down-regulated after the perineurite injury and up-regulated after the central process injury. By knocking down Dbndd, the gene expression of Dbndd is reduced, so that the growth of neuron axons and the regeneration of damaged nerves can be promoted, and Dbndd inhibitor can be used for preparing medicines for promoting the growth of the nerves.

The specific technical scheme of the invention is as follows:

use of an inhibitor comprising 2 (dysbindin domain containing, dbndd 2) in the small dystrophin domain for the preparation of a medicament for promoting nerve growth.

The invention relates to Dbndd 2. 2mRNANCBl Reference Sequence:NM 001047111.2 (SEQ ID NO: 1).

The application of the invention is that the inhibitor is a negative regulator of the small muscular dystrophin domain containing 2 protein or the encoding gene thereof, including a protein inhibitor targeting the small muscular dystrophin domain containing 2 protein, a nucleic acid aptamer and/or an inhibitor of the small muscular dystrophin domain containing 2 interference RNA, gRNA, microRNA of the encoding gene, a small molecular compound inhibitor and the combination thereof.

In a specific example of the invention, the negative modulator is an siRNA targeting the dystrophin domain 2 gene and/or a vector-based shRNA.

The sequence of the siRNA targeting the small dystrophin domain containing the 2 gene is as follows:

5'GAAGUUCUUCGAGGACAUU 3' of si-r-Dbndd2 001 sense strand, SEQ ID NO. 6;

3'CUUCAAGAAGCUCCUGUAA 5' of the si-r-Dbndd2 001 antisense strand, SEQ ID NO. 7;

5'GGUGGAAUUUAUUGACCUU 3' of si-r-Dbndd2 002 sense strand, 8 of SEQ ID NO;

3'CCACCUUAAAUAACUGGAA 5' of si-r-Dbndd2 002 antisense strand, SEQ ID NO 9;

5'GCAGUCCAAAUCCAAGUGA 3' of si-r-Dbndd2 sense strand and 10 of SEQ ID NO;

The si-r-Dbndd2 antisense strand 3'CGUCAGGUUUAGGUUCACU 5' with SEQ ID NO. 11.

The shRNA comprises a sense strand segment and an antisense strand segment, and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and the sequence is shown as SEQ ID No. 14.

The application of the invention, the nerve is peripheral nerve, preferably spinal nerve.

In a specific example of the present invention, the nerve is an sciatic nerve.

The medicine disclosed by the invention can promote the growth of axons, and can be used for treating traumatic nerve injury or neurodegenerative diseases related to the axon injury.

Another object of the present invention is to provide a2 inhibitor in the small dystrophin domain, which is a small interfering RNA or shRNA AAV virus in which the small dystrophin domain contains 2 genes.

According to the invention, after the peri-and central-process injury of the rat DRG neuron is found through transcriptome sequencing, the gene Dbndd which is differentially expressed in the DRG is verified through a tissue immunofluorescence staining experiment, dbndd is found to be down-regulated after the peri-process injury of the DRG neuron, and the expression after the central-process injury is up-regulated (figure 1), so that different changes of the expression of Dbndd after the peri-and central-process injury can be one of reasons for the difference of the regeneration capacity of the axon after the peri-and central-process injury of the DRG neuron. The change in Dbndd2 in neurons in DRGs of neonatal and adult rats was examined by single cell sequencing and Dbndd2 was found to be expressed less with development (fig. 2), suggesting that Dbndd2 may have a neurological growth promoting effect during development. Culturing primary neonatal rat DRG neurons, transfecting siRNA fragments against Dbndd2, found that Dbndd siRNA transfection significantly reduced the expression of Dbndd2, indicating Dbndd siRNA is a potent inhibitor of Dbndd2 (fig. 3A). Dbndd2 expression decreased, significantly increasing the axon length of the DRG neurons of the neonatal rats (fig. 3B and 3C). Transfection of the siRNA fragment against Dbndd2 in primary adult rat DRG neurons also reduced Dbndd2 expression and significantly increased the axon length of adult rat DRG neurons (fig. 4). Adult rat DRG neurons knocked down Dbndd still had longer axon length on cell culture dishes coated with the axon growth inhibitory factor myelin compared to transfected control siRNA, indicating that reduced Dbndd2 expression still favors axon growth in an inhibitory microenvironment (fig. 5). The expression of knock down Dbndd was found to significantly accelerate the regeneration process after the DRG neuronal axon injury in adult rats using microfluidic cells to construct an in vitro DRG neuronal axon injury model (fig. 6). Ischial nerve injury was performed after intrathecal injection of AAV virus expressed by knock-down Dbndd in adult rats, and it was found that expression of knock-down Dbndd2 in rats was beneficial for growth of injured nerves (fig. 7-8).

The Dbndd2 can be directly used as a target point of a medicine to design a medicine (inhibitor) for inhibiting the expression of the medicine, and the medicine can be interacted with the medicine to inhibit the expression of Dbndd2 in neurons so as to play a role in regulating the growth of nerves.

The invention has the advantages that the research result shows that the expression of Dbndd can be reduced by Dbndd2 inhibitor, thereby improving the ability of neurite outgrowth and axon regeneration. Dbndd2 inhibitors can be used in the treatment of traumatic nerve injury and neurodegenerative diseases associated with axonal injury by promoting nerve growth.

Drawings

FIG. 1 shows changes in the DRG of Dbndd following peri-and central-process injury to the neurons of the adult rat. (FIG. 1A shows the expression trend of Dbndd gene in DRG after injury of peri-neuronal and central-neuronal processes of adult rat, and FIG. 1B shows the expression of DBNDD protein in DRG after injury of peri-neuronal and central-neuronal processes of adult rat.

FIG. 2 shows the change in Dbndd2 in the DRG neurons of neonatal and adult rats. the tSNE plot shows the distribution of Dbndd2 within neuronal cells in the cytograms of single cell sequencing detection of DRG tissues in neonatal and adult rats. FIG. 1B shows the average expression level of Dbndd gene in DRG tissues of neonatal red skin SD rats and adult SD rats by single cell sequencing.

FIG. 3 is a graph showing the effect of knock down Dbndd2 on the neurite outgrowth in a neonatal rat DRG neuron. (FIG. 3A shows the expression of Dbndd gene after transfection of control siRNA or Dbndd siRNA into primary cultured dorsal root ganglion neurons of newborn rat. FIG. 3B shows the Tuj1 staining result of axons of DRG neurons of newborn rat after transfection of control siRNA or Dbndd siRNA, with a scale of 50 μm. FIG. 3C shows statistics of the longest length and total length of axons of DRG neurons of newborn rat).

FIG. 4 is a graph showing the effect of knock down Dbndd2 on adult rat DRG neuron axon growth. (FIG. 4A shows the expression of Dbndd gene after transfection of control siRNA or Dbndd siRNA in primary cultured adult rat dorsal root ganglion neurons. FIG. 4B shows the Tuj1 staining result of adult rat DRG neuron axons after transfection of control siRNA or Dbndd siRNA, scale bar is 50 μm. FIG. 4C shows statistics of the longest length and total length of adult rat DRG neuron axons).

FIG. 5 is a graph showing the effect of knock down Dbndd on adult rat DRG neuron axons grown on axon growth inhibitory factor myelin coated dishes. (FIG. 5A is Tuj1 staining results of adult rat DRG neuron axons on a culture dish coated with myelin after transfection of control siRNA or Dbndd siRNA, scale bar 50 μm. FIG. 5B is a maximum length and total length statistic of adult rat DRG neuron axons).

FIG. 6 is an effect of knock down Dbndd2 on regeneration after axonal injury of adult rat DRG neurons. (FIG. 6A shows Tuj1 staining results of regenerated axons after cross-sectional injury on the axon side of adult rat DRG neurons transfected with control siRNA or Dbndd siRNA in a microfluidic cell culture system, scale bar 50 μm. FIG. 6B shows regenerated axon length statistics).

FIG. 7 shows the effect of interference on Dbndd gene and the effect of infection in neuronal cells in DRG tissue after intrathecal injection of control shRNA or Dbndd shRNA AAV in adult rats. (FIG. 7A shows the expression of Dbndd gene in DRG tissue after intrathecal injection of control shRNA or Dbndd shRNA AAV in adult rats. FIG. 7B shows the expression of EGFP fluorescent tag protein in DRG tissue after intrathecal injection of control shRNA AAV in adult rats. FIG. 7C shows the expression of DBNDD protein in DRG tissue after intrathecal injection of Dbndd shRNA AAV in adult rats.

FIG. 8 is the effect on axon regeneration following injury to sciatic nerve following intrathecal injection Dbndd of a shRNA AAV in adult rats. FIG. 8A shows the result of SCG10 staining, a regenerated sensory nerve marker at the sciatic nerve of a rat after sciatic nerve injury, with a scale of 1000. Mu.m. Fig. 8B is a graph showing nerve regeneration length statistics at sciatic nerve 3 days after sciatic nerve injury).

Detailed Description

The following examples illustrate the specific steps of the present invention, but are not limited thereto.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.

The invention is described in further detail below in connection with specific embodiments and with reference to the data. It should be understood that this example is merely illustrative of the invention and is not intended to limit the scope of the invention in any way.

In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art.

The invention will be further illustrated with reference to specific examples.

Example 1 examination of the relationship between adult rat DRG neuronal injury and Dbndd2

1. Adult rat DRG neuron sciatic nerve injury

Adult Sprague Dawley rats were given a cross-sectional or pinch injury to the hip-mitered exposed nerve at the 1cm sciatic nerve on the bifurcation of the tibial and fibular total nerves, and a model of rat DRG neuron periprocess injury was constructed. Rats with only sciatic nerve exposed and without injury were taken as a peri-process injury sham surgery group.

Adult rat DRG neuron dorsal root injury

Adult Sprague Dawley rat DRG neurites enter spinal cord, back roots are exposed, and transverse or pinching injury is carried out, so that a rat DRG neuron central process injury model is constructed. Rats with only the dorsal root exposed and without injury were taken as the peri-synaptic injury sham.

Drawing and sequencing of adult rat DRG tissue

1 Day after the adult rat is subjected to the peri-or central-process injury or the corresponding pseudo-operation group, taking DRG tissues, extracting tissue RNA, separating and fragmenting mRNA, synthesizing cDNA by taking the mRNA as a template, carrying out gene expression quantification by transcriptome sequencing, and analyzing the change of gene expression after the peri-or central-process injury of the DRG neurons.

FIG. 1A shows the change of Dbndd genes in DRG tissues after analysis of peri-or central-process injury of DRG neurons by transcriptome sequencing. Transcriptome sequencing results show that Dbndd gene expression in DRG tissues is significantly reduced after 1 day of peri-neuronal process injury compared to the DRG-neuronal process sham surgery group. In contrast to the change in Dbndd2 following a DRG neuronal peri-process injury, dbndd gene expression in DRG tissue showed an up-regulation trend after 1 day of DRG neuronal peri-process injury compared to the DRG neuronal central process prosthesis surgery group.

Taking the DRG tissue obtained in the above, embedding, slicing and staining after tissue fixation, incubating with DBNDD antibody, and then incubating with fluorescence-labeled secondary antibody to detect the expression and localization of DBNDD protein in the DRG tissue.

FIG. 1B shows the expression of DBNDD protein in DRG tissue after immunofluorescence staining for detecting peri-or central-process injury of DRG neurons. The results showed that DBNDD protein in DRG tissue had reduced expression following peri-DRG neuronal process injury and increased expression following DRG neuronal process injury.

Example 2 conditions of neuronal intracellular gene expression in dorsal root ganglion tissues of neonatal and adult rats dorsal root ganglion tissues of neonatal red skin and adult Sprague Dawley rats were digested with collagenase type I and trypsin, and after removal of dead cells, cell suspensions were obtained, washed and resuspended in PBS solution containing 0.04% BSA. Cell suspensions were loaded onto 10 XChromium, barcode-labeled microbeads were added, and the droplets were isolated to form GEM and transferred, after which cells were lysed and single cell sequencing was performed using Illumina NovaSeq. The cell map is visualized by using t-SNE nonlinear clustering, and the FINDALLMARKERS function is used for determining the characteristic expression genes of each cell group, so as to analyze the expression conditions of the genes in the neuron cells in the dorsal root ganglion tissues of the neonatal and adult rats.

FIG. 2 shows the nonlinear clustering of t-SNE to detect Dbndd gene expression in neuronal cells in DRG of neonatal and adult rats. The results showed that Dbndd gene expression was lower in neurons of the dorsal root ganglion of the neonatal rat than Dbndd gene expression in neurons of the adult rat, indicating increased Dbndd gene expression during development, suggesting that Dbndd2 may have a blocking effect on the growth of neuronal axons.

Example 3 Effect of Dbdd 2 siRNA on rat neuronal axon growth

1. Drawing and culturing of primary neonatal rat DRG neurons

The method comprises the steps of taking the DRG tissue of a newborn Sprague Dawley rat, cutting into small sections, digesting with collagenase, centrifuging, re-suspending the precipitate, placing the cell suspension in 15% BSA, and centrifuging to obtain the precipitate containing dorsal root ganglion neurons. The neonatal rat DRG neurons were cultured using a Neurobasal medium containing B27 and L-glutamine.

Drawing and culturing of primary adult rat DRG neurons

Adult Sprague Dawley rat DRG tissue was cut into small pieces, digested with collagenase for 2 hours, digested with pancreatin for 30 minutes, centrifuged, the pellet resuspended in Neurobasal A medium containing B27 and L-glutamine, filtered using a cell filter, and adult rat DRG neurons inoculated into poly-L-lysine coated dishes for culture.

Transfection of primary neonatal and adult rat DRG neurons

Dbndd2 siRNA was transfected into cultured primary neonatal and adult DRG neurons using Lipofectamine ^TM RNAiMAX transfection reagent instructions to reduce expression of Dbndd in the neurons.

Dbndd2 inhibitors and inhibitor controls were synthesized by Sharp Biotechnology Inc. of Guangzhou, inc. The Dbndd siRNA sequences used were:

5'GAAGUUCUUCGAGGACAUU 3' of si-r-Dbndd2 001 sense strand, SEQ ID NO. 6;

3'CUUCAAGAAGCUCCUGUAA 5' of the si-r-Dbndd2 001 antisense strand, SEQ ID NO. 7;

5'GGUGGAAUUUAUUGACCUU 3' of si-r-Dbndd2 002 sense strand, 8 of SEQ ID NO;

3'CCACCUUAAAUAACUGGAA 5' of si-r-Dbndd2 002 antisense strand, SEQ ID NO 9;

The sense strand of si-r-Dbndd2 003 5'GCAGUCCAAAUCCAAGUGA 3', SEQ ID NO 10, the antisense strand of si-r-Dbndd2 003 3'CGUCAGGUUUAGGUUCACU 5', SEQ ID NO 11.

The inhibitor control was nonsensical random sequences, the sense strand was 5'GGCUCUAGAAAAGCCUAUGC 3' (SEQ ID NO: 12), and the antisense strand was 3'CCGAGAUCUUUUCGGAUACG 5' (SEQ ID NO: 13).

The transfection reagent Lipofectamine ^TM RNAiMAX was produced by Invitrogen (Invitrogen, carlsbad, calif., USA) and cell transfection was performed according to the instructions.

RNA from the primary DRG neuron cells cultured as described above was reverse transcribed using Oligo dT primer, and RT-PCR was performed using Applied Biosystems Stepone real-TIME PCR SYSTEM instrument and SYBR Green Premix Ex Taq. Pre-denaturation at 95℃for 2min, followed by 40 PCR cycles of 5 sec 95℃plus 10 sec 60 ℃. The Ct values of the target gene Dbndd and the reference Gapdh were detected using Gapdh as an internal reference, and the relative expression amount of Dbndd was calculated by the ΔΔct method.

The primer sequence of the target gene Dbndd is as follows:

Dbndd2(forward):5’CGTCAGACAGGACCACATCC 3’(SEQ ID NO:2)。

Dbndd2(reverse):5’TGTCTCCTCCCCCATCACTT 3’(SEQ ID NO:3)。

the primer sequences of the internal reference gene Gapdh are as follows:

Gapdh(forward):5’ACAGCAACAGGGTGGTGGAC 3’(SEQ ID NO:4)。

Gapdh(reverse):5’TTTGAGGTGCAGCGAACTT 3’(SEQ ID NO:5)。

FIG. 3A shows the expression of Dbndd2 after RT-PCR detection of the control siRNA or Dbndd siRNA transfected with the DRG neurons of the neonates. The results show that after Dbndd siRNA is transfected into the DRG neuron of the newborn rat, the gene expression of Dbndd2 in the neuron is obviously reduced, which shows that Dbndd siRNA can inhibit the gene expression of Dbndd2 in the DRG neuron of the newborn rat and is an effective inhibitor of Dbndd 2.

FIG. 4A shows the expression of Dbndd2 after RT-PCR detection of adult rat DRG neurons transfected with control siRNA or Dbndd siRNA. The results showed that Dbndd gene expression in neurons was significantly reduced after transfection Dbndd of the siRNA into adult rat DRG neurons, indicating that Dbndd siRNA can inhibit Dbndd gene expression in adult rat DRG neurons.

4. Neuronal axon growth detection

Taking the new-born or adult rat DRG neurons transfected with the control siRNA or Dbndd siRNA, inoculating the DRG neurons to a cell culture dish, fixing the cell climbing sheet, staining Tuj1 by using cell immunofluorescence, observing the length of the neuronal axons marked by Tuj1, and detecting the influence of the Dbndd2 inhibitor on the growth of the neuronal axons.

FIG. 3B is a representative Tuj1 staining image (scale bar 50 μm) of neonatal rat DRG neurons transfected with control siRNA or Dbndd siRNA. FIG. 3C shows the longest length and total length of the axons of the neonatal rat DRG neurons transfected with control siRNA or Dbndd siRNA. The results show that after Dbndd siRNA transfection, the longest length and the total length of the axon of the DRG neuron of the newborn rat are obviously increased, which indicates that the Dbndd2 inhibitor can promote the growth of the axon of the DRG neuron of the newborn rat.

FIG. 4B is a representative Tuj1 staining image (scale bar 50 μm) of adult rat DRG neurons transfected with control siRNA or Dbndd siRNA. FIG. 4C shows the longest length and total length of adult rat DRG neuron axons transfected with control siRNA or Dbndd siRNA. The results show that the longest length and the total length of the axons of the adult rat DRG neurons are obviously increased after Dbndd siRNA is transfected, which indicates that Dbndd2 inhibitor can promote the growth of the axons of the adult rat DRG neurons.

5. Neurite growth assay of neurons on myelin coated cell culture dishes

The axon growth inhibitory factor myelin was extracted from adult rat nerve tissue and cell culture dishes were coated with myelin. Taking adult rat DRG neurons transfected with control siRNA or Dbndd siRNA, inoculating the DRG neurons to a myelin-coated cell culture dish, fixing the cell climbing slices, performing Tuj1 staining through cell immunofluorescence, observing the length of a Tuj1 marked neuron axon, and detecting the influence of a Dbndd2 inhibitor on the neuron axon growing on the myelin-coated cell culture dish.

FIG. 5A is a representative Tuj1 staining image (scale bar 50 μm) of adult rat DRG neurons transfected with control siRNA or Dbndd siRNA on myelin-coated cell culture dishes. FIG. 5B shows the longest length and total length of adult rat DRG neuron axons transfected with control siRNA or Dbndd siRNA on myelin-coated cell culture dishes. The results show that the maximum length and total length of axons of adult rat DRG neurons are obviously increased after Dbndd siRNA transfection on myelin-coated cell culture dishes, indicating that Dbndd inhibitor can promote the growth of adult rat DRG neuron axons in the inhibitory microenvironment where myelin exists.

6. Neuronal damaged axon regeneration detection

And (3) inoculating the adult rat neurons into a microfluidic cell culture system, and after the dorsal root ganglion neurons axon grows to the axon side, cutting off the axon by vacuum suction to prepare an in-vitro dorsal root ganglion neurons axon dissociation model. Tuj1 staining is performed by cell immunohistochemistry, the length of regeneration after the damage of the axon of the neuron marked by Tuj1 is observed at the axon side of a microfluidic cell culture system, and the axon regeneration condition of the neuron is detected.

FIG. 6A is a representative Tuj1 staining image (scale bar 50 μm) of regenerated axons after axon injury of adult rat DRG neurons transfected with control siRNA or Dbndd siRNA in a microfluidic cell culture system. FIG. 6B is a graph showing the length of regenerated axons after axonal injury from adult rat DRG neurons transfected with control siRNA or Dbndd siRNA. The results show that after Dbndd siRNA transfection, the regeneration length of the damaged axon of the DRG neuron of the adult rat becomes longer, which shows that the Dbndd2 inhibitor can promote the regeneration of the damaged axon of the DRG neuron of the adult rat.

Example 4 Effect of Dbdd 2 shRNA on sciatic nerve regeneration in adult rats

Adult Sprague Dawley rats were subjected to intrathecal injection of control shRNA virus (pAAV-U6-shRNA (NC) -CMV-EGFP-WPRE) or Dbndd shRNA virus (pAAV-U6-shRNA (Dbndd) -CMV-EGFP-WPRE) using surgical scissors to cut the skin of the L4-L6 segment along the dorsal midline, with ophthalmic scissors to cut the muscles of the lumbar spinous processes on both sides, exposing the intervertebral space to exposure, using a microinjection pump. The viruses used were packaged by Shanghai and Meta-Biol. The shRNA sequence selected for packaging AAV virus is shown as SEQ ID NO. 14.

FIG. 7A shows the detection of Dbndd gene and protein expression in DRG tissues by RT-PCR and tissue immunofluorescence staining experiments after 21 days of intrathecal injection of control shRNA virus or Dbndd shRNA AAV virus in adult rats. The results showed that Dbndd gene and protein expression in DRG tissues was reduced following intrathecal injection of Dbndd shRNA AAV virus in adult rats, indicating that Dbndd shRNA AAV virus is a potent inhibitor of Dbndd 2.

FIG. 8A shows the results of SCG10 staining for sciatic nerve regeneration markers at sciatic nerve sites (scale bar 1000 μm) after 21 days of intrathecal injection of control shRNA virus or Dbndd shRNA AAV virus in adult rats, followed by 3 days of perfusion. Fig. 8B is the length of damaged nerve regeneration at the sciatic nerve of adult rats injected intrathecally with control shRNA virus or Dbndd shRNA AAV virus 3 days after sciatic nerve injury. The results show that after the gene expression of Dbndd is knocked down after the intrathecal injection Dbndd shRNA AAV virus of the adult rat, the regeneration length of damaged sciatic nerve becomes longer, which indicates that Dbndd inhibitor can promote the regeneration of damaged nerve in the adult rat.

Claims

1. Application of dystrophin domain 2 inhibitors in the preparation of drugs that promote nerve growth.

2. The use according to claim 1, characterized in that the inhibitor is a negative regulator of dystrophin domain-containing 2 protein or its encoding gene.

3. The use according to claim 2, characterized in that the negative regulator comprises a protein inhibitor targeting the dystrophin domain-containing 2 protein, a nucleic acid aptamer and/or an interfering RNA, gRNA, microRNA, a small molecule compound inhibitor of the dystrophin domain-containing 2 encoding gene, and an inhibitor of the above combination.

4. The use according to claim 3, characterized in that the negative regulator is siRNA targeting the dystrophin domain-containing 2 gene and/or vector-based shRNA.

5. The use according to claim 4, characterized in that the sequence of the siRNA targeting the dystrophin domain 2 gene is as follows:

si-r-Dbndd2 001 positive strand: 5’GAAGUUCUUCGAGGACAUU 3’, SEQ ID NO: 6;

si-r-Dbndd2 001 antisense strand: 3’CUUCAAGAAGCUCCUGUAA 5’, SEQ ID NO: 7;

si-r-Dbndd2 002 positive strand: 5′GGUGGAAUUUAUUGACCUU 3′, SEQ ID NO: 8;

si-r-Dbndd2 002 antisense strand: 3’CCACCUUAAAUAACUGGAA 5’, SEQ ID NO: 9;

si-r-Dbndd2 003 positive strand: 5’GCAGUCCAAAUCCAAGUGA 3’, SEQ ID NO: 10;

si-r-Dbndd2 003 antisense strand: 3’CGUCAGGUUUAGGUUCACU 5’, SEQ ID NO: 11;

The shRNA comprises a sense strand segment, an antisense strand segment and a stem-loop structure connecting the sense strand segment and the antisense strand segment, and the sequence is shown in SEQ ID No: 14.

6. The use according to claim 1, characterized in that the nerve is a peripheral nerve.

7. The use according to claim 6, characterized in that the nerve is a spinal nerve.

8. The use according to claim 7, characterized in that the nerve is the sciatic nerve.

9. The use according to claim 1, characterized in that the drug is a drug for treating nerve damage or neurodegenerative diseases related to nerve damage.

10. A dystrophin domain-containing 2 inhibitor, characterized by being a small interfering RNA or shRNA AAV virus of the dystrophin domain-containing 2 gene.