Application of CD22 gene as target spot in preparation of medicine for treating spinal cord injury related diseases

Abstract

The invention discloses application of a cell surface adhesion molecule CD22 as a target spot in preparation of drugs for treating spinal cord injury related diseases. The change of gene expression in the glial scar formation process is represented by space transcriptome sequencing, and the specific expression of CD22 in the glial scar region is up-regulated. According to single cell sequencing, in-situ hybridization and immunohistochemistry, specific high expression CD22 of part of microglial cells in a glial scar area is found. CD22 is knocked out through a genetic means, and it is found that formation of glial scars after spinal cord injury is remarkably reduced through inhibition of CD22. Behavioral detection finds that the error rate of irregular horizontal ladders is remarkably reduced by inhibiting CD22, and fine movement recovery of hind limbs of mice after spinal cord injury is promoted. The siRNA specifically targeting CD22 is injected into the sheath, so that the expression of microglial cells CD22 is inhibited, the error rate of irregular horizontal ladders of hind limbs can be reduced, and the recovery of the fine movement function of the hind limbs of the mouse after spinal cord injury is promoted. The invention provides a new possibility for development of drugs for spinal cord injury and treatment of spinal cord injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of the CD22 gene as a target in the preparation of drugs for treating spinal cord injury-related diseases. Background Art

[0002] Spinal cord injury (SCI) severely damages the central nervous system, often resulting in the loss of sensory and motor abilities below the SCI area, and patients may face decades of permanent disability. SCI is mostly caused by accidents, and frequent wars, natural disasters, industrial injuries, and traffic accidents are very likely to cause spinal cord injury. It is estimated that there are currently more than 27 million SCI patients worldwide, with approximately 930,000 new cases each year, and it has increasingly become a clinical problem faced by countries around the world. Currently, fully restoring the structure and function of the damaged spinal cord is a great challenge. Clinically, the treatment methods for promoting spinal cord injury repair are too limited, and the treatment effect also depends on the injured segment. Currently, the main reasons considered for SCI-induced limb dysfunction are as follows: 1) After the central nervous system of adult mammals is damaged, the intrinsic regenerative ability of neurons is poor, and the vast majority of neurons cannot regenerate axons. 2) A glial scar forms in the spinal cord injury area of adult mammals. The glial scar can protect the surviving nerve tissue after injury in the early stage of injury, but it will also inhibit axon regeneration in the chronic stage. Currently, it is considered that the glial scar is composed of various cells and extracellular components. For example, ependymal cells, NG2 + oligodendrocyte precursor cells, oligodendrocytes, astrocytes, macrophages, fibroblasts, and microglia; the extracellular matrix includes chondroitin sulfate proteoglycan, myelin-associated glycoprotein, fibronectin, oligodendrocyte myelin glycoprotein, matrix glycoprotein tenascin C, and hyaluronic acid fragments. The current findings are just the tip of the iceberg, and the cellular components and specific regulatory mechanisms of the glial scar have not been elucidated yet.

[0003] Some studies have found that inhibiting glial scars promotes functional recovery. The research group led by Professor Stupp SI developed a degradable amphiphilic supramolecular peptide fiber scaffold, which contains peptide sequences that promote nerve regeneration: laminin signal peptide and basic fibroblast growth factor mimetic peptide. Single injection of this scaffold can reverse paralysis after spinal cord injury and repair spinal cord tissue. This fiber scaffold significantly reduces the glial scar tissue that may cause physical barriers to regeneration and repair. Even paralyzed mice regained the ability to walk. The research group led by Professor Zhigang He also found that scarless healing and spontaneous axonal regeneration can be achieved after spinal cord crush injury in neonatal mice of the C57BL / 6 strain. Microglia in neonatal mice are crucial for coordinating the injury response and are the key to achieving scarless healing. In addition, in adult mammals, scarless healing and functional recovery can also be achieved after complete transection of the spinal cord in the spiny mouse (Acomys cahirinus). These all suggest that there are significant differences in the ability of scarless healing after spinal cord injury in mammals among different developmental stages and different strains. Therefore, further studying the causes of glial scar formation after spinal cord injury in adult mammals and finding effective drug action targets from a new perspective have important academic value and practical significance.

[0004] CD22 is a type I transmembrane protein, also known as Siglec-2, and belongs to the members of the sialic acid-binding immunoglobulin-type lectin family (Siglecs). It can specifically bind to sialic acid-containing glycans. CD22 has multiple ligands, and its extracellular domain can specifically bind to α2-6 sialic acid residues present on various cell surface glycoproteins, and inhibit B cell receptor (BCR) signaling through its immunoreceptor tyrosine-based inhibitory motif, playing a role in maintaining the homeostasis of humoral immunity. It is commonly present in normal B cells and B cell malignancies. CD22 can interact with sialic acid-containing cells, including T cells, B cells, neutrophils, monocytes, and red blood cells. CD22 is mainly expressed in mature B cells and is a cell surface adhesion molecule with the function of regulating B cell activation, which helps to control the sensitivity of B cells to antigen responses. Recent studies have found that CD22 expression has also been observed in murine intestinal eosinophils, which is a novel expression pattern, indicating that CD22 may have eosinophil regulatory functions. Summary of the Invention

[0005] In view of the world-class problem of glial scar formation after spinal cord injury, the present invention uses spatial transcriptome sequencing to characterize the changes in gene expression during glial scar formation, and finds that CD22 is specifically upregulated in the glial scar area. Further research shows that inhibiting CD22 significantly reduces the formation of glial scars after spinal cord injury and promotes the recovery of fine motor function of the hind limbs in mice after spinal cord injury.

[0006] The specific technical solution of the present invention is as follows:

[0007] Use of the CD22 gene as a target in the preparation of a medicament for treating spinal cord injury-related diseases. The medicament is a substance that inhibits the expression of the CD22 gene, and is selected from one or more of compounds, proteins, polypeptides, polysaccharides, glycoproteins, glycopeptides, and nucleic acids.

[0008] The medicament can reduce the formation of glial scars after spinal cord injury. Preferably, the substance that inhibits the expression of the CD22 gene is CD22 gene siRNA or sgRNA.

[0009] In a preferred embodiment, the target sequences of the CD22 gene complementary to the siRNA are shown as SEQ ID NO: 1-3.

[0010] Specific examples adopted in the present invention

[0011] si-m-Cd22_001:

[0012] Target sequence (5'->3'): GTCCAAGTGCAACAAACTA (SEQ ID NO: 1).

[0013] Sense strand (5'->3'): GUCCAAGUGCAACAAACUA (SEQ ID NO: 4).

[0014] Antisense strand (5'->3'): UAGUUUGUUGCACUUGGAC (SEQ ID NO: 5).

[0015] si-m-Cd22_002:

[0016] Target sequence (5'->3'): GTAGGTACAACTCCAGCAA (SEQ ID NO: 2).

[0017] Sense strand (5'->3'): GUAGGUACAACUCCAGCAA (SEQ ID NO: 6).

[0018] Antisense strand (5'->3'): UUGCUGGAGUUGUACCUAC (SEQ ID NO: 7).

[0019] si-m-Cd22_003:

[0020] Target sequence (5'->3'): TCTGAGATCACCTCCATCA (SEQ ID NO: 3).

[0021] Sense strand (5'->3'): UCUGAGAUCACCUCCAUCA (SEQ ID NO: 8).

[0022] Antisense strand (5'->3'): UGAUGGAGGUGAUCUCAGA (SEQ ID NO:9).

[0023] In a specific example of the present invention, the drug is an injection preparation. It can be administered by intrathecal injection for treatment. The drug has siRNA as the main active ingredient.

[0024] Furthermore, the siRNA of the present invention can be further modified or optimized without changing the nucleic acid sequence of the siRNA. For example:

[0025] Phosphodiester bond modification: By chemically altering the structure of the phosphodiester bond of siRNA, the stability of siRNA in cells and its ability to resist nuclease degradation can be improved. Common modifications include 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), and phosphodiester choline (PC), etc.

[0026] Nucleobase modification: Modifying the nucleobases of siRNA can improve its pairing efficiency and specificity with mRNA. Common modifications include 2'-O-methyl, 2'-amino, and N-methyl diisulfonium urea, etc.

[0027] siRNA terminal modification: Introducing chemical modification groups at the 3' or 5' end of siRNA can change the structure and hydrophilicity of siRNA, thereby improving the stability of siRNA in cells. Common modifications include hydroxyl group (OH), phosphodiester group (PO4), and cholesterol (Chol), etc.

[0028] Amino acid modification: Introducing amino acid modification into siRNA can improve the specificity of siRNA in cells. Common modifications include N-acetylcysteine (Ac-Cys) and N-acetyllysine (Ac-Lys).

[0029] In a specific example of the present invention, the chemical modification is methoxy modification and / or cholesterol modification.

[0030] Advantages of the present invention:

[0031] The present invention uses the CD22 gene as a molecular intervention target. By inhibiting the expression of CD22, the error rate of the irregular horizontal ladder of the hind limbs of mice after spinal cord injury is reduced, and the recovery of fine motor function of the hind limbs is promoted. The present invention uses spatial transcriptome sequencing to characterize the changes in gene expression during the formation of glial scars, and finds that CD22 is specifically up-regulated in the glial scar area. Single-cell sequencing, in situ hybridization, and immunohistochemistry reveal that some microglia in the glial scar area specifically highly express CD22. By genetically knocking out CD22, it is found that inhibiting CD22 significantly reduces the formation of glial scars after spinal cord injury; behavioral tests show that inhibiting CD22 significantly reduces the error rate of the irregular horizontal ladder and promotes the recovery of fine motor function of the hind limbs of mice after spinal cord injury. Further, by intrathecal injection of siRNA specifically targeting CD22 to inhibit the expression of CD22 in microglia, the results show that it can reduce the error rate of the irregular horizontal ladder of the hind limbs and promote the recovery of fine motor function of the hind limbs of mice after spinal cord injury. The present invention provides new possibilities for the development and treatment of drugs for spinal cord injury. Description of the Drawings

[0032] Figure 1 It is the single-cell sequencing result of the spinal cord scar tissue of mice with spinal cord injury. Single-cell sequencing finds that CD22 is highly expressed in microglia.

[0033] Figure 2 It is the result of reclustering and subgroup analysis of microglia obtained by single-cell sequencing. Figure 2 A shows microglia that appear at different time points after spinal cord injury; Figure 2 B shows different microglia subgroups after spinal cord injury; Figure 2 C is a UMAP map showing the expression of CD22 in microglia; Figure 2 D shows the expression of CD22 in microglia at different time points after injury.

[0034] Figure 3 It is the expression of CD22 in the scar area at different time points after spinal cord injury characterized by spatial transcriptome sequencing.

[0035] Figure 4 It is the detection of the expression of CD22 in the spinal cord injury area by fluorescence in situ hybridization.

[0036] Figure 5 It is the detection of the expression of CD22 in the spinal cord injury area by immunofluorescence staining.

[0037] Figure 6 It is the situation of scar formation of microglia and astrocytes in the spinal cord injury area of CD22 gene knockout mice. Figure 6A shows the spinal cord tissues of WT mice and CD22 - / - mice at 8 weeks after injury co - labeled with Iba1, GFAP, and CD68. Iba1 (red) represents microglia in the scar area, CD68 (purple) represents activated microglia, and GFAP (green) represents astrocytes. Bar = 100μm; Figure 6 B shows the statistical results of the proportion of activated microglia and the width of the gap in the center of the scar formed by GFAP - positive astrocytes. Mean ± SEM, Students’s t - test, * represents P < 0.05 vs WT, ** represents P < 0.01 vs WT, with statistical significance.

[0038] Figure 7 It shows the formation of fibrous scar in the spinal cord injury area of CD22 gene - knockout mice. Figure 7 A shows the results of immunofluorescence staining of spinal cord tissues. P4HB (red) represents fibroblasts in the scar area, GFAP (green) represents astrocytes, and DAPI (blue) shows the cell nuclei. Bar = 100μm; Figure 7 B shows the measurement results of the fibrous scar area. The average fluorescence intensity of P4HB, with WT as the positive control, Mean ± SEM, Students’s t - test, * represents P < 0.05 vs WT, ** represents P < 0.01 vs WT, with statistical significance.

[0039] Figure 8 It shows the axonal regeneration in the spinal cord injury scar area of CD22 gene - knockout mice. Figure 8 A shows the spinal cord tissues of WT mice and CD22 - / - mice at 8 weeks after injury co - labeled with Tuj1 and DAPI. Tuj1 (red) represents the regenerated axons in the scar area, and DAPI shows the cell nuclei. Bar = 100μm; Figure 8 B shows the statistical results of the number of Tuj1 - positive axons passing through the scar area, with WT as the positive control, Mean ± SEM, Students’s t - test, * represents P < 0.05 vs WT, ** represents P < 0.01 vs WT, with statistical significance.

[0040] Figure 9 It shows the blood vessel distribution in the spinal cord injury scar area of CD22 gene - knockout mice. Figure 9 A shows the spinal cord tissues of WT mice and CD22 - / - mice at 4 weeks and 8 weeks after injury co - labeled with CD31 and DAPI. CD31 (green) represents the blood vessels in the scar area, and DAPI shows the cell nuclei. Bar = 100μm; Figure 9To count the number of CD31-positive blood vessels in the scar area, WT was used as a positive control, Mean±SEM, Students’s t-test, * represents P<0.05 vs WT, ** represents P<0.01 vs WT, with statistical significance.

[0041] Figure 10 It is the test result of the horizontal ladder in the behavioral level of CD22 gene knockout spinal cord injury mice. Figure 10 A is the detection time point of the irregular horizontal ladder; Figure 10 B is the schematic diagram of the irregular horizontal ladder test; Figure 10 C is the statistics of the error rate (%) of the irregular horizontal ladder. The error rate (%) is on the Y-axis, and the time point is on the X-axis, Mean±SEM, Students’s t-test, *, ** are P<0.05, P<0.01 vs WT respectively, with statistical significance.

[0042] Figure 11 It is the evaluation of the recovery of hindlimb fine motor function after spinal cord injury in CD22 knockout mice by the fine motor dynamic evaluation system (TSE MotoRater System). Figure 11 A is the stick figure of the right hindlimb in the TSE test; Figure 11 B is the statistical chart of the knee-right ankle bone-right hindfoot angle. Mean±SEM, Students’s t-test, *, ** are P<0.05, P<0.01 vs WT respectively, with statistical significance.

[0043] Figure 12 It is the inhibitory effect of siRNA on the expression of CD22 in vitro cultured BV2 microglia. Figure 12 A shows that CD22 is localized on the cell membrane of BV2 by immunohistochemistry. CD22 is green, and the microglia marker Iba1 is red; Figure 12 B is the interference efficiency of three siRNAs on the expression of CD22.

[0044] Figure 13 It is the test result of the horizontal ladder in the behavioral level of spinal cord injury mice injected with CD22 siRNA intrathecally. Specific implementation mode

[0045] The following is a further detailed description of the above content of the present invention through specific implementation modes in the form of examples. However, the examples should not be construed as any limitation to the present invention. The protection scope of the present invention is subject to the claimed rights. Except as otherwise specified, conventional existing technologies are used in the following examples.

[0046] Example 1 Specific expression of CD22 in microglia after spinal cord injury

[0047] I. T10 right hemisection model of adult mouse spinal cord

[0048] Eight-week-old female C57BL / 6J mice were anesthetized, and the skin was incised along the midline of the thoracic vertebrae. T10 laminectomy was performed, and the tip of an iris knife (BVI Beaver, Oakville, Canada) was carefully inserted into the posterior median sulcus of the spinal cord to completely transect the right spinal cord. The muscle layer was sutured, and then the skin was fixed with wound clips. The mice were placed at 37 °C for recovery until fully awake, and pain was relieved with painkillers. All animal experiments were conducted in accordance with the animal care guidelines and were ethically approved by the Jiangsu Provincial Laboratory Animal Management Committee. The animal experiment license number was 20150304-004.

[0049] II. Single-cell sequencing and spatial transcriptome sequencing of spinal cord scar tissue

[0050] At 12 h, 1 d, 3 d, 7 d, 14 d, 60 d, and 90 d after T10 right hemisection of the spinal cord, segments about 3 mm long in the spinal cord injury area were collected. Single-cell suspensions were obtained according to the spinal cord single-cell dissociation procedure, and 10× single-cell sequencing was performed by Shanghai Biochip Co., Ltd. At 3 d, 7 d, 14 d, and 28 d after T10 right hemisection of the spinal cord, segments about 3 mm long in the spinal cord injury area were collected. The spinal cord was embedded according to the spatial transcriptome sequencing tissue sample processing procedure, and 10× Visum spatial transcriptome sequencing was performed by Shanghai Biochip Co., Ltd. Relevant data analysis was completed by Shanghai Biochip Co., Ltd.

[0051] The results of single-cell sequencing of spinal cord scar tissue in spinal cord injured mice are as Figure 1 shown, showing that CD22 is highly expressed in microglia. The results of reclustering and subgroup analysis of microglial cells obtained by single-cell sequencing are as Figure 2 shown, Figure 2 A shows microglial cells that appear at different time points after spinal cord injury. Figure 2 B shows different microglial cell subgroups after spinal cord injury. Figure 2 C is a UMAP plot showing the expression of CD22 in microglial cells. Figure 2 D shows the expression of CD22 in microglial cells at different time points after injury. Figure 3 shows the expression of CD22 in the scar area at different time points after spinal cord injury characterized by spatial transcriptome sequencing. The results show that CD22 begins to be expressed in a part of microglial cells 7 d after spinal cord injury.

[0052] III. Fluorescence in situ hybridization (FISH)

[0053] C57BL / 6J mice were perfused and fixed with 4% paraformaldehyde. A spinal cord segment about 3 mm long in the injured area was removed, dehydrated in a 30% sucrose solution, and then subjected to cryosectioning at a thickness of 12 μm after sedimentation. The tissue sections were mounted on SUPERFROST PLUS slides. The slides were baked at 60 °C for 30 minutes, and then immersed in pre-cooled 4% PFA solution at 4 °C and incubated for 15 minutes for post-fixation. The sections were dehydrated successively with 50% EtOH, 200 mL of 70% EtOH, and 400 mL of 100% EtOH, repaired with 1× antigen repair solution, and treated with RNAscope hydrogen peroxide and protease III. Using 3-plex Mm-fluorescent positive control probe and RNAscope Probe-Mm-Cd22 fluorescent probe according to the multi-channel second-generation fluorescence kit (product number 323100) for in situ hybridization and immunofluorescence co-staining. After mounting the sections, they were sealed with a fluorescent mounting medium and observed and photographed under a fluorescence microscope. The distribution of the CD22 probe at the injury site was observed, photographed, and its distribution in various cell types was counted. The results are as Figure 4 shown. FISH showed that CD22 was not expressed in the normal spinal cord, and high expression of CD22 appeared in microglia (Iba1+) after injury. The CD22 probe signal was red dots, the Iba1 signal was green (488), DAPI showed the cell nucleus, Bar = 20 μm.

[0054] IV. Immunofluorescence staining

[0055] C57BL / 6J mice were perfused and fixed with 4% paraformaldehyde. The lumbar dorsal root ganglia (L4, L5) were removed, dehydrated in a 30% sucrose solution, and then subjected to cryosectioning at a thickness of 12 μm after sedimentation. The sections were rinsed 3 times with 0.01 M PBS solution for 10 minutes each time, incubated with 1% BSA (containing 0.5% Triton X-100) at room temperature for 1.5 hours for blocking, and incubated with the primary antibody: CD22 (rabbit, 1:100, Invitrogen) at 4 °C overnight. The sections were rinsed 3 times with 0.01 M PBS solution for 10 minutes each time, and incubated with the secondary antibody: Alexa Fluor488-donkey anti-rabbit (1:1000, Jackson) at room temperature for 2 hours. The sections were rinsed 3 times with 0.01 M PBS solution for 10 minutes each time. After mounting the sections, they were sealed with a fluorescent mounting medium and observed and photographed under a fluorescence microscope. The results are as Figure 5 shown. The results showed that the CD22 signal (green) appeared in the scar area at different time points after injury, and DAPI showed the cell nucleus.

[0056] Bar = 100 μm. (Since the CD22 antibody has the same origin as the commonly used Iba1 antibody for labeling microglia, double-labeling co-localization cannot be performed).

[0057] Case 2: Scar formation of microglia and astrocytes in the spinal cord injury area of CD22 gene knockout mice

[0058] I. Right hemi-section model of the spinal cord of adult mice at T10

[0059] Eight 8-week-old female C57BL / 6J mice and eight 8-week-old female CD22 - / - mice (Jiangsu Jicui Yakang Biotechnology Co., Ltd., product number: T028402). After anesthesia, the skin was incised along the midline of the thoracic vertebrae, a T10 laminectomy was performed, and the tip of an iris knife (BVI Beaver, Oakville, Canada) was carefully inserted into the posterior median sulcus of the spinal cord to completely transect the right spinal cord. The muscle layer was sutured, and then the skin was fixed with wound clips. They were placed at 37 °C for resuscitation until fully awake, and pain was relieved with a pain-relieving injection. All animal experiments were conducted in accordance with animal care guidelines and were ethically approved by the Jiangsu Provincial Laboratory Animal Management Committee. The animal experiment license was 20150304-004.

[0060] II. Immunofluorescence staining of spinal cord tissue and measurement of inflammatory cells in the scar area

[0061] After 8 weeks, perfusion was performed with 4% paraformaldehyde. Then, post-fixation was carried out with 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, the samples were washed three times with PBS for 10 minutes each time. The laminae were stripped to expose the spinal cord, and the integrity of the spinal cord was maintained as much as possible. The spinal cord tissue was removed, and the spinal cord at a distance of 3 mm before and after the injury site was collected. After dehydration with 30% sucrose, it was embedded in OCT, and the frozen section thickness was 10 μm. Immunohistochemical blocking solution was added and blocked at room temperature for 1 hour. The primary antibodies anti-Iba-1 antibody (Wako 019-19741, Rabbit 1:100) and GFAP (abcam ab4674, Chicken 1:500) were diluted with immunohistochemical primary antibody diluent. After adding the primary antibodies, they were incubated overnight at 4 °C. The primary antibodies were discarded, and the samples were washed 3 times with PBS for 5 minutes each time. The fluorescent secondary antibodies Alexa 647 AffiniPure Sheep Anti-rabbit IgG (invitrogen A21244, 1:500), Alexa Fluor TM 488 Goat anti-Chicken IgY (H+L) (invitrogen, A11039, 1:500) were diluted with immunohistochemical secondary antibody diluent. After adding the secondary antibodies, they were incubated for 2 hours at room temperature in the dark. The secondary antibodies were discarded, and the samples were washed 3 times with PBS for 5 minutes each time. An appropriate amount of fluorescent mounting medium was added, and observations and photographs were taken under a ZEISS upright fluorescence microscope.

[0062] Observe the morphology of activated microglia and astrocytes at the injury site, and count the number and boundaries of activated microglia and astrocytes in each group. The results are as Figure 6 shown Figure 6 A shows the spinal cord tissues of WT mice and CD22- / - mice at 8 weeks after injury co-labeled with Iba1, GFAP, and CD68. Iba1 (red) represents microglia in the scar area, CD68 (purple) represents activated microglia, and GFAP (green) represents astrocytes. Bar = 100 μm. Figure 6 B shows the statistical analysis of the proportion of activated microglia and the fluorescence density of GFAP-positive astrocytes. Mean±SEM, Students’s t-test, * represents P<0.05 vs WT, ** represents P<0.01 vs WT, with statistical significance. The results show that at 8 weeks after injury, the activated CD68 - / - microglia in the spinal cord scar area of the CD22 + group were significantly reduced, the gap width in the scar center area formed by GFAP-positive astrocytes was significantly narrowed, and some GFAP-positive astrocytes were interconnected to form a "bridge".

[0063] Example 3 Fibrous scar formation in the spinal cord injury area of CD22 gene knockout mice

[0064] I. Refer to Example 2 to construct a right-sided T10 hemisection model of the spinal cord in adult mice.

[0065] II. Immunofluorescence staining of spinal cord tissue and measurement of fibrous scar area

[0066] After 8 weeks, perfuse with 4% paraformaldehyde. Then post-fix with 4% paraformaldehyde for 12 hours. After discarding the paraformaldehyde, wash three times with PBS, 10 minutes each time. Dissect the lamina to expose the spinal cord, and try to keep the spinal cord intact. For the removed spinal cord tissue, collect the spinal cord at a distance of 3 mm before and after the injury site. After dehydration with 30% sucrose, embed in OCT, and the frozen section thickness is 10 μm. Add immunohistochemical blocking solution and block at room temperature for 1 h. Dilute the primary antibody anti-P4HB antibody (abcam ab137110, Mouse 1:100) with immunohistochemical primary antibody diluent. After adding the primary antibody, incubate overnight at 4°C. Discard the primary antibody and wash 3 times with PBS, 5 minutes each time. Dilute the fluorescent secondary antibody Alexa 647 AffiniPure Sheep Anti-mouse IgG (invitrogen A31571, 1:500). After adding the secondary antibody, incubate at room temperature for 2 h in the dark. Discard the secondary antibody and wash three times with PBS for 5 min each time. Add an appropriate amount of fluorescent mounting medium and observe and photograph under a ZEISS upright fluorescence microscope. Observe the fibroblast distribution at the injury site, photograph, and measure the size of the fibrotic scar area in each group. The results are as Figure 7 shown. Figure 7 Panel A shows the results of immunofluorescence staining of spinal cord tissue. P4HB (red) indicates fibroblasts in the scar area, GFAP (green) indicates astrocytes, and DAPI (blue) shows cell nuclei. Bar = 100 μm; Figure 7 Panel B shows the results of measuring the fibrotic scar area. The mean fluorescence intensity of P4HB, with WT as the positive control, Mean ± SEM, Students’s t-test, * represents P < 0.05 vs WT, ** represents P < 0.01 vs WT, with statistical significance. The results show that the number of fibroblasts and the area of the fibrotic scar in the spinal cord scar area of the CD22 - / - group were significantly reduced.

[0067] Example 4 Axonal regeneration in CD22 gene knockout mice with spinal cord injury

[0068] I. Refer to Example 2 to construct a right T10 hemisection model of the spinal cord in adult mice.

[0069] II. Immunofluorescence staining of spinal cord tissue and measurement of fibrotic scar area

[0070] After 8 weeks, perfuse with 4% paraformaldehyde. Then post-fix in 4% paraformaldehyde for 12 h. Discard the paraformaldehyde and wash three times with PBS for 10 min each time. Dissect the vertebral lamina to expose the spinal cord, and try to keep the spinal cord intact. Take out the spinal cord tissue, collect the spinal cord at a distance of 3 mm before and after the injury site, dehydrate with 30% sucrose, embed in OCT, and cut frozen sections with a thickness of 10 μm. Add immunohistochemical blocking solution and block at room temperature for 1 h. Dilute the primary antibody anti-Tuj1 antibody (cell signaling, D71G9, rabbit, 1:500) with immunohistochemical primary antibody diluent. After adding the primary antibody, incubate overnight at 4°C. Discard the primary antibody and wash three times with PBS for 5 min each time. Dilute the fluorescent secondary antibody Alexa 647 AffiniPure Sheep Anti-rabbit IgG (invitrogen A21244, 1:500). After adding the secondary antibody, incubate in the dark at room temperature for 2 h. Discard the secondary antibody and wash 3 times with PBS for 5 min each time. Add an appropriate amount of fluorescence mounting medium and observe and photograph under a ZEISS upright fluorescence microscope. Observe the distribution of regenerated axons at the injury site, photograph and count the number of regenerated axons in each group. The results are as Figure 8 shown. Figure 8 A shows the spinal cord tissues of WT mice and CD22 - / - mice at 8 weeks after injury co-labeled with Tuj1 and DAPI. Tuj1 (red) represents the regenerated axons in the scar area, and DAPI shows the cell nuclei. Bar = 100 μm; Figure 8 B shows the statistical results of the number of Tuj1-positive axons passing through the scar area. WT is used as a positive control. Mean ± SEM, Students’s t-test, * represents P < 0.05 vs WT, ** represents P < 0.01 vs WT, which is statistically significant. The results show that the number of regenerated axons passing through the spinal cord scar area in CD22 gene knockout mice is significantly increased.

[0071] Example 5 Vascular distribution in the spinal cord scar area of CD22 gene knockout mice with spinal cord injury

[0072] I. Refer to Example 2 to construct a right T10 hemisection model of the spinal cord in adult mice.

[0073] II. Immunofluorescence staining of spinal cord tissue and measurement of fibrous scar area

[0074] At 4 and 8 weeks after injury, perfuse with 4% paraformaldehyde. Then post-fix with 4% paraformaldehyde for 12 h. Discard the paraformaldehyde and wash 3 times with PBS for 10 min each time. Dissect the vertebral lamina to expose the spinal cord, and try to keep the spinal cord intact. Take out the spinal cord tissue, collect the spinal cord at a distance of 3 mm before and after the injury site, dehydrate with 30% sucrose, embed in OCT, and the thickness of the frozen section is 10 μm. Add immunohistochemical blocking solution and block at room temperature for 1 h. Dilute the primary antibody anti-CD31 antibody (R&D, AF3628, goat, 1:100) with immunohistochemical primary antibody diluent. After adding the primary antibody, incubate at 4 °C overnight. Discard the primary antibody and wash 3 times with PBS for 5 min each time. Dilute the fluorescent secondary antibody Alexa Fluor with immunohistochemical secondary antibody diluent TM488Donkey anti-Goat IgG(H+L)Cross-Adsorbed Secondary Antibody (invitrogen, A-11055, 1:500). After adding the secondary antibody, incubate for 2 h at room temperature in the dark. Discard the secondary antibody and wash 3 times with PBS for 5 min each time. Add an appropriate amount of fluorescent mounting medium and observe and photograph under a ZEISS upright fluorescence microscope. Observe the vascular distribution at the injury site, photograph and count the number and length of blood vessels in the scar area of each group. The results are as Figure 9 shown. Figure 9 A shows the spinal cord tissues of WT mice and CD22- / - mice at 4 and 8 weeks after injury co-labeled with CD31 and DAPI. CD31 (green) indicates blood vessels in the scar area, and DAPI shows cell nuclei. Bar = 100 μm; Figure 9 B shows the statistical results of the number of CD31-positive blood vessels in the scar area. WT is used as a positive control. Mean ± SEM, Students’s t-test, * represents P < 0.05 vs WT, ** represents P < 0.01 vs WT, which is statistically significant. The results show that the vascularization in the spinal cord scar area of CD22 knockout mice is significantly increased.

[0075] Example 6 Behavioral test of horizontal ladder in CD22 knockout mice with spinal cord injury

[0076] Start horizontal ladder training for each group of experimental mice three days before modeling, and train each mouse three times a day ( Figure 10 A). Refer to Example 2 to construct a right hemisection model of the spinal cord at T10 in adult mice. After modeling, perform irregular horizontal ladder tests weekly. Randomly arrange the spacing of the ladder at each test time point and record with a camera. Each mouse is photographed 3 times ( Figure 10 B). The total experimental duration is 8 weeks. Two personnel who are unaware of the experimental situation count the error rate of the right hind paw of each group. Figure 10 C shows the statistical results of the error rate (%) of the irregular horizontal ladder. The error rate (%) is on the Y-axis, and the time point is on the X-axis. Mean ± SEM, Students’s t-test, *, ** are P < 0.05, P < 0.01 vs WT respectively, which is statistically significant. The results show that CD22 knockout reduces the error rate of the horizontal ladder after spinal cord injury.

[0077] Example 7 Evaluation of the recovery of hindlimb fine motor function in CD22 knockout mice with spinal cord injury using a fine motor dynamic assessment system (TSE MotoRater System)

[0078] I. Refer to Example 2 to construct a right hemisection model of the spinal cord at T10 in adult mice.

[0079] II. Tests and statistics of TSE

[0080] Eight weeks after modeling, TSE tests were performed on two groups of mice, and TSE tests were also performed on uninjured mice of the same age (16 weeks). The hair on the right side of the mice was shaved, and a luminescent sticker was used and attached to the right pelvis, right hip, knee, right ankle bone, and right hind foot of the mice. TSE Motion was used to test and analyze the foot trajectories and the statistical analysis of the knee-right ankle bone-right hind foot angle ( Figure 11 A). It was found that the step frequency, step distance, and the knee-right ankle bone-right hind foot angle of CD22 knockout mice were closer to those of uninjured normal mice ( Figure 11 B).

[0081] Example 8 Inhibitory effect of siRNA on CD22 expression in in vitro cultured BV2 microglial cells

[0082] I. Culture of BV2 microglial cell line

[0083] The semi-adherent BV2 microglial cell line was purchased from Tongpai (Shanghai) Biotechnology Co., Ltd., and the STR identification was completed by Suzhou Jingda. The culture medium used was: DMEM supplemented with 10% FBS, 1.5 g / L NaHCO3, 4.5 g / L Glucose, 4 mM L-Glutamine, and 1.0 mM. The semi-adherent microglial cell line was passaged at a ratio of 1:2. When passaging the cells, the suspended cells were collected in a culture tube. The adherent cells were washed with PBS and digested with 0.25% trypsin at 37 °C for 5 min. The adherent cells and the suspended cells were centrifuged together at 1000 rpm for 5 min and collected. Then, these cells were added to a new culture dish.

[0084] II. Design and synthesis of mouse CD22 siRNA

[0085] The design and synthesis of siRNA were completed by Guangzhou Ribobio Co., Ltd.: The full-length sequence of mouse CD22 mRNA (NM_001043317.2) was obtained from the National Center for Biotechnology Information (NCBI) database. According to the RNAi principle and combined with design software, 3 candidate siRNAs targeting the mouse CD22 mRNA gene transcript were designed. After BLAST alignment check to ensure no homology with other genes, chemical synthesis was then carried out.

[0086] The information of the 3 candidate siRNAs is as follows:

[0087] si-m-Cd22_001:

[0088] Target sequence (5'->3'): GTCCAAGTGCAACAAACTA.

[0089] Sense strand (5'->3'): GUCCAAGUGCAACAAACUAdTdT。

[0090] Antisense strand (5'->3'): UAGUUUGUUGCACUUGGAC dTdT。

[0091] si-m-Cd22_002:

[0092] Target sequence (5'->3'): GTAGGTACAACTCCAGCAA。

[0093] Sense strand (5'->3'): GUAGGUACAACUCCAGCAA dTdT。

[0094] Antisense strand (5'->3'): UUGCUGGAGUUGUACCUAC dTdT。

[0095] si-m-Cd22_003:

[0096] Target sequence (5'->3'): TCTGAGATCACCTCCATCA。

[0097] Sense strand (5'->3'): UCUGAGAUCACCUCCAUCA dTdT。

[0098] Antisense strand (5'->3'): UGAUGGAGGUGAUCUCAGA dTdT。

[0099] (3) CD22 siRNA interference experiment

[0100] The cultured BV2 microglial cells were transfected with different siRNAs using RNAMAX. After 48 hours, the cells were collected and RNA was extracted for real-time fluorescence quantitative PCR detection.

[0101] Seed 2.5×10 5 BV2 cells into 6-well plates. When the cell confluence reached 30 - 50%, transfection was carried out according to the transfection reagent instruction manual of Guangzhou Ribobio Co., Ltd.: ① Dilute siRNA and siRNA nontarget control: Dilute 5 μl of siRNA or negative control stock solution (20 μM) with 120 μl of 1×riboFECTTM CP Buffer, and mix gently. ② Prepare the mixture: Add 12 μl of riboFECTTM CP Reagent, gently pipette and mix, and incubate at room temperature for 10 min to prepare the transfection complex. ③ Add the transfection complex to the BV2 culture medium and mix gently. ④ Incubate in the incubator for 12 h, observe the status of BV2 cells and replace with fresh complete medium, and continue to culture for another 48 h.

[0102] Total RNA of BV2 cells in each group was extracted using the Trizol method. The absorbance (A260 / 280nm) was detected with a nucleic acid protein detector, and the RNA concentration and purity were calculated. Reverse transcription of the total RNA was performed with reference to the third-generation high-efficiency cDNA first-strand synthesis kit of Novoprotein. The resulting cDNA could be directly used for PCR reactions or stored at -80°C. The target gene CD22 ID was searched in NCBI, and the full mRNA or CDS sequences were selected. The sense primer (5’-GCCAAGCGTGTGAGACTTTT-3’) and antisense primer (5’-CCTCAACCCCAGATTCCCAC-3’) were designed using Primer Premier 5.0 software, and the primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. According to the instructions of Vazyme's AceQ qPCR SYBR Green MasterMix, a 10 μl reaction system was prepared and reacted on a Bio-Rad CFX real-time fluorescence quantitative PCR amplifier. The 2 -ΔΔCT method was used to calculate and statistically analyze the results. GAPDH was used as an internal reference to correct the copy number of the PCR template and eliminate the pipetting error between groups. The transfection efficiency was detected.

[0103] The immunohistochemical results were as Figure 12 shown. Figure 12 A was immunohistochemical staining, showing that CD22 was localized on the BV2 cell membrane, CD22 was green, and the microglial marker Iba1 was red; Figure 12 B was the interference efficiency of three siRNAs on CD22 expression. The experimental results showed that CD22 was expressed in BV2 microglia, and all three candidate siRNAs could inhibit CD22 expression. Among them, siRNA-3 had the most significant interference effect (***P < 0.001 compared with the siRNA-NC group). Therefore, siRNA-003 was selected as the one with the best interference effect on the target gene. This siRNA was methoxylated and cholesterol-modified to make the siRNA more stable in vivo and directly injected in vivo without the aid of transfection reagents to play an interfering role for further behavioral function verification.

[0104] Example 9 Effect of intrathecal injection of CD22 siRNA on the recovery of fine motor function of the hindlimbs in spinal cord injury mice

[0105] Sangon Biotech (Guangzhou) Co., Ltd. was commissioned to methoxylate siRNA-3 to improve its stability in vivo, and at the same time, cholesterol modification was performed. The modified siRNA could directly enter the cells to play an interfering gene expression role without the aid of a transfection vector complex and was used for behavioral detection after intrathecal injection in vivo.

[0106] Three days before modeling, horizontal ladder training was started for each group of experimental mice, and each mouse was trained three times a day. An adult mouse spinal cord right T10 hemisection model was constructed with reference to Example 2. After modeling, intrathecal injection of cholesterol and methoxy-modified siRNA (siRNA-003-2OMe + 5Chol) was performed twice a week. This continued until 8 weeks after surgery. The specific operation was as follows: After shaving the hair on the back of the mouse's waist with an electric clipper, the mouse was anesthetized by inhalation of isoflurane, the skin was disinfected with alcohol, and a 30-gauge (BD) injection needle was used to inject 10 μl of cholesterol and methoxy-modified siRNA into the subarachnoid space of the mouse between the spinous processes of the fourth and fifth lumbar vertebrae. The successful puncture was marked by the appearance of tail tremors or sudden lateral flicks. After the inhalation anesthesia was stopped, the mouse could wake up quickly, which had no effect on the subsequent horizontal ladder behavioral tests and other studies, ensuring the reliability of the experimental results. Irregular horizontal ladder tests were performed every week.

[0107] Figure 13 It was the statistical result of the irregular horizontal ladder error rate (%). The error rate (%) was on the Y-axis, the time point was on the X-axis, Mean±SEM, Students’s t-test, *, ** were P<0.05, P<0.01 vs WT respectively, which was statistically significant. The results showed that intrathecal injection of high-dose siRNA-003-2OMe + 5Chol (5 μg) could significantly reduce the horizontal ladder error rate from 5 to 8 weeks after spinal cord injury (*P<0.05 compared with the siRNA-NC-2OMe + 5Chol group (synthesized by Guangzhou Ribobio Co., Ltd.), n = 6-8, two-way repeated measures ANOVA, Bonferroni correction test for pairwise comparison). The results indicated that the recovery of fine motor function of the hindlimbs after spinal cord injury could be promoted by inhibiting CD22 expression.

Claims

1. Use of the CD22 gene as a target in the preparation of a medicament for treating diseases related to spinal cord injury.

2. The application according to claim 1, wherein The medicament reduces the formation of glial scars after spinal cord injury.

3. The application according to claim 1, characterized in that The medicament is a substance that inhibits the expression of the CD22 gene, and is selected from one or more of compounds, proteins, polypeptides, polysaccharides, glycoproteins, glycopeptides, and nucleic acids.

4. The application according to claim 3, characterized in that The substance that inhibits the expression of the CD22 gene is CD22 gene siRNA or sgRNA.

5. The application according to claim 4, wherein The target sequences of the CD22 gene complementary to the siRNA are as shown in SEQ ID NO: 1-3.

6. The application according to claim 5, characterized in that The sense strand nucleic acid sequences of the siRNA are as shown in SEQ ID NO: 4, 6, 8.

7. The application according to claim 4, wherein The medicament is an injection preparation.

8. The application according to claim 7, characterized in that The medicament takes siRNA as the main active ingredient.

9. The application according to any one of claims 4-8, characterized in that The siRNA is chemically modified without changing the siRNA nucleic acid sequence.

10. The application according to claim 9, characterized in that The chemical modification is methoxy modification and / or cholesterol modification.

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