Uses of cell surface adhesion molecule CD22

Abstract

The present invention discloses the use of cell surface adhesion molecule CD22, specifically the use of cell surface adhesion molecule CD22 in screening non-addictive drugs for analgesia. The present invention knocks out CD22 by genetic means, and finds that mice knocked out of CD22 have mechanical hyperalgesia; by intrathecal injection of siRNA targeting CD22, it is found that inhibiting CD22 expression will lead to mechanical hyperalgesia in mice; by tail vein injection of targeted CD22 antibodies, it is found that inhibiting CD22 will also lead to mechanical hyperalgesia in mice; by tail vein injection of CD22 overexpression virus, and then performing SNL surgery, neuropathic pain caused by SNL can be alleviated. The present invention helps to better understand the important role of cell surface adhesion molecules in the development of pain, and provides new possibilities for the development of clinical analgesic drugs as well as prevention and treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the use of cell surface adhesion molecule CD22. Background Art

[0002] Pain, as a kind of somatic sensation, can serve as a warning when the body is hurt, causing a series of defensive protective reactions in the body to avoid further harm. But when pain loses its normal protective function and persists for months or years, it becomes pathological pain (chronic pain) that harms physical and mental health. According to the World Health Organization, chronic pain is the most common disease with the heaviest disease burden. According to statistics, about 30% of adults suffer from chronic pain. Chronic pain is a common symptom in clinical practice. Inflammation, surgery, nerve trauma, viral infection (such as herpes zoster), cancer, metabolic diseases (such as diabetes), immune diseases (such as multiple sclerosis, AIDS), etc. can all cause chronic pain. Patients will have hyperalgesia with increased sensitivity to noxious stimuli and lowered reaction thresholds, as well as tactile allodynia caused by non-painful stimuli (such as touch), which seriously affects the patient's physical and mental health and quality of life.

[0003] In addition, analgesia is also a clinical difficulty. Most of the existing analgesics are opioid analgesics and non-opioid auxiliary analgesics (non-steroidal anti-inflammatory drugs, antidepressants, anticonvulsants). Opioid analgesics such as morphine, methadone, fentanyl, oxycodone, etc. are often accompanied by respiratory depression and circulatory inhibition side effects, which are common as constipation, dizziness, nausea, vomiting, symptoms such as pruritus and dry mouth. Celecoxib, a cyclooxygenase-2 inhibitor commonly used in non-steroidal anti-inflammatory drugs, is accompanied by digestive tract side effects, including symptoms such as abdominal pain, indigestion and nausea. The auxiliary analgesics based on anticonvulsants and antipsychotics, such as carbamazepine, clonidine, ketamine, etc., all have different types of adverse reactions, such as symptoms such as fatigue, headache, thrombocytopenia, skin itching, congestive rash, insomnia, constipation. Therefore, further studying the mechanism of development of chronic pain, finding effective drug action targets from a new perspective has important academic value and practical significance.

[0004] CD22, also known as Siglec-2, is a member of the sialic acid-binding immunoglobulin-type lectin family (Siglecs). It is a type I transmembrane protein that can specifically bind to sialic acid (Sia)-containing polysaccharides and inhibit B cell receptor (BCR) signal transduction through its immunoreceptor tyrosine inhibitory motif (ITIM), playing a role in maintaining humoral immune homeostasis. It is commonly found in normal B cells and B cell malignancies. CD22 is mainly expressed in mature B cells. It is a cell surface adhesion molecule that regulates B cell activation and helps control the sensitivity of B cells to antigen responses. It is currently believed that CD22 expression is specific in B cells and is developmentally regulated in both mice and humans. CD22 can be expressed in pro-B cells and pre-B cells and gradually transferred to the cell surface, but the expression level is low; in IgM + and IgD + It is highly expressed in mature B cells, follicular B cells, mantle cells, and marginal zone B cells, but ultimately in CD27 + In memory B cells, especially plasma cells, the expression is downregulated. In addition, recent studies have found that CD22 expression is also observed in mouse intestinal acid granulocytes, which is a new expression pattern, indicating that CD22 may have eosinophil regulatory function.

[0005] CD22 has multiple ligands, and its extracellular domain can specifically bind to α2-6 sialic acid residues present on a variety of cell surface glycoproteins. CD22 can interact with sialic acid-containing cells, including T cells, B cells, neutrophils, monocytes, and erythrocytes. Among them, the sialic acid ligands on the surface of B cells that can interact with the CD22 extracellular ligand binding domain are cis ligands, and other cell surface ligands are trans ligands. CD22 binds to ligands on the surface of B cells through cis action, and to other cell surface ligands, soluble glycoproteins, or cell-bound antigens through trans action.

[0006] However, there is no literature reporting whether there is a correlation between CD22 and pathological pain. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides the use of the cell surface adhesion molecule CD22, which provides new possibilities for the development of analgesic drugs and the prevention and treatment of pain.

[0008] The present invention is achieved through the following technical solutions:

[0009] Application of cell surface adhesion molecule CD22 in screening non-addictive drugs for analgesia, wherein the NCBI number of the full-length sequence of CD22 is NM_001043317.2.

[0010] Preferably, the drug is an agent that promotes the expression or function of CD22.

[0011] Preferably, the reagent is a CD22 overexpression vector or a CD22 protein.

[0012] Preferably, the CD22 overexpression vector is a lentivirus or adeno-associated virus containing the full-length sequence of CD22.

[0013] Preferably, the type of pain induced by the analgesic is neuropathic pain.

[0014] A pharmaceutical composition for treating neuropathic pain comprises the above reagent and a pharmaceutically acceptable carrier.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention uses qRT-PCR, Western blot, immunohistochemistry and fluorescence in situ hybridization to find that CD22 is expressed in mouse DRG neurons and sciatic nerves; knocking out CD22 by genetic means, and finding that mice knocking out CD22 have mechanical hyperalgesia; intrathecal injection of siRNA targeting CD22, finding that inhibition of CD22 expression leads to mechanical hyperalgesia in mice; tail vein injection of CD22-targeted antibodies, finding that inhibition of CD22 also leads to mechanical hyperalgesia in mice; tail vein injection of CD22 overexpression virus, followed by spinal nerve ligation (SNL) surgery, can alleviate neuropathic pain caused by SNL. The present invention helps to better understand the important role of cell surface adhesion molecules in the development of pain, and provides new possibilities for the development of clinical analgesic drugs, as well as prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The expression of CD22 in DRG neurons in Example 1: A is the immunofluorescence FISH detection of CD22 mRNA level in DRG neurons; B is the immunofluorescence double labeling detection of CD22 protein expression level in DRG neurons; C is the Western Blot detection of CD22 expression in DRG neurons and sciatic nerve; D is the immunofluorescence double labeling detection of CD22 expression on DRG neuron axons in the sciatic nerve;

[0018] Figure 2 The results of the CD22 knockout mouse behavior experiment in Example 2 are as follows: *** P < 0.001, T test, n = 8;

[0019] Figure 3The experimental results of inhibiting the level of DRG CD22 mRNA in Example 3: A is DRG neurons cultured in vitro, transfected with three CD22 siRNAs, and PCR was used to detect the level of CD22 mRNA; B is intrathecal annotated siRNA, mice were killed 72 hours later, DRGs were collected, and real-time fluorescence quantitative PCR was used to examine the expression of CD22 mRNA; C is normal C57BL / 6J mice injected intrathecally with CD22 siRNA, and mechanical hyperalgesia behavior was detected 6h, 24h, 48h and 72h after injection;

[0020] Figure 4 CD22 antibody or an equal amount of IgG was injected into the tail vein of normal mice in Example 4, and the plantar mechanical hyperalgesia behavior was detected at 1h, 2h, 3h, 5h and 24h;

[0021] Figure 5 The experimental results of overexpression of CD22 in the SNL mouse model in Example 5: A is the expression of CD22 mRNA in DRG detected by real-time fluorescence quantitative PCR 4 weeks after intravenous injection of AAV-PHP.S-CD22 or AAV-PHP.S-Negative control in mice, *** P<0.001, T test, n=8; B is the mechanical hyperalgesia threshold of AAV-PHP.S-CD22 mice 13 days after SNL compared with AAV-PHP.S-Negative control mice, ** P<0.01, T test, n=8. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art, and the experimental methods without specifying the specific conditions are all conventional methods in the art.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] The experimental animals used in the following examples were C57BL / 6J mice. All animal experiments were performed in accordance with the animal care guidelines and were ethically approved by the Jiangsu Provincial Laboratory Animal Management Committee. The animal experiment license is 20150304-004.

[0026] Example 1 CD22 is expressed in DRG neurons and sciatic nerve

[0027] 1. Experimental methods

[0028] (1) RNA extraction and real-time fluorescence quantitative PCR

[0029] Total RNA was extracted according to the Trizol method, and 1 μg of total RNA was taken after the concentration was determined. Reverse transcription reaction was performed according to the instructions of Vazyme's HiScript IIIRT SuperMix Kit. The obtained cDNA product was diluted 8 times for the template of real-time fluorescence quantitative PCR. 2 μL template was added to each PCR system. The target gene CD22 ID (NM_001043317.2) was searched in NCBI, and all mRNA or CDS sequences were selected. The sense primer (SEQ ID NO.1: 5'-GCCAAGCGTGTGAGACTTTT-3') and antisense primer (SEQ ID NO.2: 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 real-time fluorescence quantitative PCR SYBR Green Master Mix, 10 μL of reaction system was prepared and placed on the Bio-Rad CFX real-time fluorescence quantitative PCR amplification instrument for reaction. ΔΔCT Methods The results were calculated and statistically analyzed, and GAPDH was used as an internal reference to correct the copy number of the PCR template and eliminate the error of adding samples between groups.

[0030] (2) Protein extraction and Western Blot

[0031] After C57BL / 6J mice were perfused with saline through the heart, the dorsal root ganglia and sciatic nerves of the lumbar spinal cord (L4, L5) were removed and placed in a tissue lysis buffer containing protease inhibitors. The samples were homogenized on ice and the supernatant was taken after centrifugation to obtain a tissue protein solution. The total protein concentration of the sample was determined by the BCA protein content detection kit, and the protein loading buffer was added to calibrate the uniform protein concentration. After boiling in boiling water for 5 minutes, the protein was denatured. After SDS-polyacrylamide gel electrophoresis and wet transfer, the membrane was placed in a 5% skim milk powder blocking solution and blocked at room temperature for 2 hours. After blocking, the primary antibody diluted with 5% skim milk powder was incubated at 4°C overnight, and the secondary antibody was incubated after washing the membrane. After washing the membrane, imaging was performed using an imaging system. Image protein band analysis was performed using ImageJ software.

[0032] (3) Immunofluorescence staining

[0033] C57BL / 6J mice were fixed by perfusion with 4% paraformaldehyde, and the dorsal root ganglia of the lumbar segment (L4, L5) were removed and dehydrated with 30% sucrose solution. After sinking to the bottom, frozen sections were made with a thickness of 12 μm. The sections were rinsed with 0.01M PBS solution for 3 times, 10 min / time, and blocked with 1% BSA (containing 0.5% Triton X-100) at room temperature for 1.5 hours. The primary antibodies: CD22 (rabbit, 1:200, Bioss), neuronal marker TUJ1 (mouse, 1:800, Millipore) were incubated at 4°C overnight. The sections were rinsed with 0.01M PBS solution for 3 times, 10 min / time, and the secondary antibodies: Cy3-goat anti-rabbit (1:1000, Jackson), Alexa Fluor 488-donkey anti-goat (1:1000, Jackson), were incubated at room temperature for 2 hours. The sections were rinsed with 0.01M PBS solution for 3 times, 10 min each time, mounted with fluorescent mounting medium, and observed and photographed under a fluorescence microscope.

[0034] (4) FISH

[0035] C57BL / 6J mice were fixed by perfusion with 4% paraformaldehyde, and the dorsal root ganglia of the lumbar segment (L4, L5) were removed and dehydrated with a 30% sucrose solution. After sinking to the bottom, frozen sections were made with a thickness of 12 μm. The tissue sections were encapsulated on SUPERFROST PLUS slides. The slides were baked at 60°C for 30 minutes, and then the slides were immersed in pre-cooled 4% PFA solution at 4°C and incubated for 15 minutes for post-fixation. The sections were dehydrated with 50% EtOH, 200mL 70% EtOH and 400mL 100% EtOH in sequence, repaired with 1× antigen repair solution, and treated with RNAscope hydrogen peroxide and protease III. Use 3-plex Mm-fluorescence positive control probe and RNAscope Probe-Mm-CD22 fluorescent probe according to In situ hybridization and immunofluorescence co-staining were performed using a multi-channel second-generation fluorescent kit (Cat. No. 323100). After mounting, the slides were sealed with fluorescent mounting media and observed and photographed under a fluorescence microscope.

[0036] 2. Experimental results

[0037] After C57BL / 6J mice were perfused transcardially with saline, the dorsal root ganglia and sciatic nerves of the lumbar spinal cord (L4, L5) were removed for testing. Figure 1 shown.

[0038] Immunofluorescence FISH was used to detect the level of CD22 mRNA in DRG neurons. Figure 1 As shown in A, immunofluorescence double labeling was used to detect the expression level of CD22 protein in DRG neurons. Figure 1 As shown in B, CD22 mRNA and protein expression are present in DRG neurons; Figure 1 As shown in C, Western Blot confirmed the expression of CD22 in DRG neurons and sciatic nerves; Figure 1 As shown in middle D, double immunofluorescence labeling experiments confirmed that CD22 was expressed on the axons of DRG neurons in the sciatic nerve.

[0039] Example 2 Knockout of CD22 gene induces mechanical hyperalgesia in mice

[0040] 1. Experimental methods

[0041] (1) Experimental animals: 86 8-week-old male wild-type C57BL / 6J mice and 8-week-old male CD22- / - mice. The mechanical hyperalgesia behavior of the mice was assessed using von Frey filaments.

[0042] (2) Mechanical hyperalgesia detection method

[0043] Before the behavioral test, mice were placed in a plexiglass box on a mechanical pain test frame to adapt to the environment until it was quiet. The room temperature was 23±1℃ for 3 consecutive days. According to the "up and down" method reported by Dixon et al., the mechanical touch-induced pain threshold of the bottom of the left hind paw of mice was detected using von Frey filament. The measurement started with 0.16g von Frey filament, and the bottom of the left hind paw of the mouse was stimulated. The slight bending of the filament was used as the standard for full force. The stimulation lasted for 2s. If the mouse showed signs of paw retraction, paw flicking, and paw licking, it was recorded as pain. Then a filament with a smaller gram was used for stimulation. If it was not painful, a filament with a higher gram was used for stimulation. The stimulation interval was 3min, and 6 stimulation results were recorded. Finally, the paw retraction threshold (threshold) was found by comparing it with the threshold table. The unit is "g". The lower the paw retraction threshold, the more severe the mechanical hyperalgesia.

[0044] 2. Experimental results

[0045] like Figure 2 As shown in Figure 2, compared with wild-type (WT) mice, the mechanical hyperalgesia threshold of CD22- / - mice to von Frey filament stimulation was significantly reduced, and the difference was statistically significant ( **** P<0.0001). This indicates that CD22 gene knockout induces mechanical hyperalgesia in mice.

[0046] Example 3 Inhibition of CD22 expression in DRG induces mechanical hyperalgesia

[0047] 1. Experimental methods

[0048] (1) Isolation and culture of DRG neurons

[0049] Prepare 2-3 mL of dissecting solution and place it in a medium dish on ice; take newborn P1 mice, disinfect them and kill them; open the medullary cavity along the back, take out the DRG, and place them in a culture dish on ice; add 3 mg / mL collagenase and digest at 37°C for 30 min; digest at 37°C for 20 min with 0.25% trypsin; then add complete culture medium (DMEM+10% FBS) to terminate digestion, make a single cell suspension, pass through a sieve, and centrifuge at 1000 rpm / min for 5 min; discard the supernatant, add 15% BSA, resuspend the precipitate, and centrifuge at 900 rpm / min for 5 min; resuspend the cells in complete culture medium and inoculate them in a medium dish; after 4-6 hours, add neuronal culture medium (Neuronbasal Medium+B27+Glutamine+Pen-Strep double antibody), and add Arac to remove non-neuronal cells.

[0050] (2) Design and synthesis of mouse CD22 siRNA

[0051] siRNA design and synthesis was completed by Guangzhou Ruibo Biotechnology Co., Ltd.: The full-length sequence of mouse CD22 mRNA (NM_001043317.2) was obtained from the National Center for Biotechnology Information (NCBI) database of the United States. According to the RNAi principle, combined with the design software, three candidate siRNAs targeting the mouse CD22 mRNA gene transcript were designed. After BLAST comparison to ensure that there was no homology with other genes, chemical synthesis was performed. The target sequence information of the three candidate siRNAs is shown in Table 1 below.

[0052] Table 1 Target sequence information of candidate siRNA

[0053] Candidate siRNA number SEQ ID Target sequence si-mouse-CD22_001(siRNA-1) 3 GTCTACTACAGTCCAGAGA si-mouse-CD22_002(siRNA-2) 4 GCAATGGATGACACCGTTA si-mouse-CD22_003(siRNA-3) 5 CTTACTCGGTGATACAGAA

[0054] (3) CD22 siRNA intervention experiment

[0055] Prepare transfection reagent, take a 1.5mL EP tube, add Lipofectamine RNAiMAX, miRNA mimics and Opti-MEM TM Serum-free culture medium was placed on ice and incubated for 10-15 minutes. The cultured DRG neurons were transfected with different siRNAs using RNAMAX. After 48 hours, the cells were lysed to extract RNA, and the transfection efficiency was detected by real-time fluorescence quantitative PCR after reversal.

[0056] (4) Total RNA extraction and real-time fluorescence quantitative PCR analysis

[0057] The total RNA of DRG neurons in the four groups was extracted by Trizol method, and the absorbance (A260 / 280nm) was detected by nucleic acid protein analyzer to calculate RNA concentration and purity. The total RNA was reverse transcribed with reference to the third-generation high-efficiency cDNA single-strand synthesis kit of Novazon, and the product cDNA can be directly used for PCR reaction or stored at -80℃. The target gene CD22 ID (NM_001043317.2) was searched in NCBI, all mRNA or CDS sequences were selected, and the sense primer (SEQ ID NO.1) and antisense primer (SEQ ID NO.2) were designed by Primer Premier 5.0 software, and the primer sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. According to the instructions of AceQ real-time fluorescence quantitative PCR SYBR Green Master Mix of Vazyme, 10μL reaction system was prepared and placed on Bio-Rad CFX real-time fluorescence quantitative PCR amplification instrument for reaction. 2- ΔΔCT Methods The results were calculated and statistically analyzed, and GAPDH was used as an internal reference to correct the copy number of the PCR template and eliminate the error of adding samples between groups.

[0058] (5) Intrathecal injection of drugs

[0059] In this experiment, after screening out the siRNA with high efficiency against the mouse CD22 gene from three candidate siRNAs, Guangzhou Ruibo Biotechnology Co., Ltd. performed methoxy modification on this siRNA to improve its stability in vivo, and also performed cholesterol modification. The modified siRNA directly enters the cell to interfere with gene expression without the aid of a transfection vector complex, and is used for behavioral testing after intrathecal injection.

[0060] After the hair on the back of the mouse was shaved by electric clippers, the mouse was anesthetized by isoflurane inhalation, and the skin was disinfected with alcohol. A 30-gauge (BD) injection needle was used to inject 10 μL of the drug into the subarachnoid space of the mouse between the fourth and fifth lumbar spinous processes. The tail trembled or suddenly swung sideways as a sign of successful puncture. After the inhalation anesthesia was stopped, the mouse could wake up quickly, which had no effect on the subsequent pain behavior detection and other studies, ensuring the reliability of the experimental results.

[0061] 2. Experimental results

[0062] (1) Screening for highly effective siRNA targeting the mouse CD22 gene

[0063] The experiment was conducted using DRG neurons cultured in vitro to screen out the most effective siRNA that interferes with CD22 expression. Figure 3 As shown in A, all three candidate siRNAs were effective, among which siRNA-3 had the most significant interference effect ( ***P < 0.001 compared with the siRNA-NC group, n = 4, one-way ANOVA and Bonferroni correction test). Therefore, this siRNA-3 was selected for methoxy modification and cholesterol modification to make the siRNA more stable in vivo and to exert interference effects by direct injection in vivo without the aid of transfection reagents, and was used for behavioral experiments.

[0064] (2) siRNA inhibition of CD22 mRNA expression can enhance the sensitivity of mechanical hyperalgesia

[0065] Methoxy-modified and cholesterol-modified siRNA-3 targeting the CD22 gene were injected intrathecally to specifically inhibit the expression of CD22, and the mechanical touch-induced pain threshold of the left hind paw of mice was detected by von Frey filament.

[0066] like Figure 3 As shown in B, after intrathecal injection of high-dose siRNA-3-2OMe+5Chol (5 μg), real-time fluorescence quantitative PCR was used to examine the interference effect 72 hours later, and it was found that high-dose siRNA-3-2OMe+5Chol (5 μg) significantly inhibited CD22 mRNA in DRG.

[0067] like Figure 3 As shown in C, after intrathecal injection of siRNA-003-2OMe+5Chol (5 μg), the CD22 mRNA level in mouse DRG was significantly decreased ( ** P<0.01 compared with siRNA-NC-2OMe+5Chol group, n=4-5, Student's t-test).

[0068] The above results indicate that inhibiting CD22 mRNA expression can enhance the sensitivity of mechanical hyperalgesia.

[0069] Example 4 Inhibition of CD22 function induces mechanical hyperalgesia

[0070] 1. Experimental methods

[0071] 8-week-old male C57BL / 6J mice were injected with CD22 antibody (10 μg) or an equivalent amount of IgG via tail vein, and the mechanical touch-induced pain threshold of the left hind paw of the mice was detected by von Frey filament at 1h, 2h, 3h, 5h and 24h. The CD22 antibody used in this example is from BioXCell (Cy34). The antibody specifically targets the extracellular domain of CD22 and inhibits its binding to the ligand.

[0072] 2. Experimental results

[0073] like Figure 4As shown in the figure, compared with mice injected with IgG, mice injected with CD22 antibody by tail vein had significantly lower paw withdrawal threshold at 1 h, which lasted until 24 h, indicating that CD22 antibody induced mechanical hyperalgesia in mice ( * P<0.05, ** P<0.01, *** P < 0.001, compared with the IgG group, n = 8-10, two-way repeated measures ANOVA, Bonferroni correction test for pairwise comparisons).

[0074] The above experimental results indicate that inhibiting the function of CD22 induces mechanical hyperalgesia.

[0075] Example 5 Overexpression of CD22 inhibits mechanical hyperalgesia in SNL mice

[0076] 1. Experimental methods

[0077] (1) Establishment of the mouse spinal nerve ligation (SNL) neuropathic pain model

[0078] After intraperitoneal injection of compound anesthetics into 8-week-old male C57BL / 6J mice, the hair on the back of the waist was shaved with a shaver. After disinfection with iodine and alcohol, a longitudinal incision of about 1 cm long was made about 5 mm to the left and lateral of the posterior midline, and 2 / 3 of the incision was above the iliac crest. Then the thoracolumbar fascia was incised, the erector spinae muscles were bluntly separated and pulled outward, the transverse process of the 6th lumbar vertebra was exposed and bitten off under the operating microscope, the L5 spinal nerve was gently separated with a glass needle, the L5 spinal nerve was tightly ligated with 6-0 silk thread, and then the incision was sutured layer by layer.

[0079] (2) Tail vein injection of virus and detection of mechanical hyperalgesia

[0080] Eight-week-old male C57BL / 6J mice were divided into two groups, with 8 mice in each group. The mechanical hyperalgesia behavior of mice was evaluated using von Frey filament. Before the behavioral test, mice were placed individually in a plexiglass box on a mechanical pain test frame to adapt to the environment until it was quiet. The room temperature was 23±1℃ for 3 consecutive days. According to the "up and down" method reported by Dixon et al., the mechanical touch-evoked pain threshold of the bottom of the left hind paw of mice was detected using von Frey filament. The measurement started with a 0.16g vonFrey filament, and the bottom of the left hind paw of the mouse was stimulated. The slight bending of the filament was used as the standard for full force. The stimulation lasted for 2s. If the mouse showed signs of paw retraction, paw flicking, licking, etc., it was recorded as pain. Then a filament with a smaller gram was used for stimulation. If it was not painful, a filament with a higher gram was used for stimulation. The stimulation interval was 3min, and 6 stimulation results were recorded. Finally, the paw retraction threshold (threshold) was found by comparing it with the threshold table. The unit is "g". The lower the paw retraction threshold, the more severe the mechanical hyperalgesia. The paw withdrawal threshold of normal mice was tested before surgery, which was used as the baseline threshold (BL).

[0081] Next, the two groups of mice were injected with 200 μL of AAV-PHP.S-CD22 virus (adeno-associated virus containing the full-length sequence of CD22, packaged by Guangzhou Yunzhou Biotechnology Co., Ltd.) or control virus (AAV-PHP.S-Negative control, provided by Guangzhou Yunzhou Biotechnology Co., Ltd.) through the tail vein, with a virus titer of 3×10 13 Three weeks later, both groups of mice underwent SNL surgery to induce hyperalgesia. Thirteen days after surgery, the mechanical hyperalgesia behavior of the mice was assessed using von Frey filaments and the paw withdrawal threshold was recorded.

[0082] 2. Experimental results

[0083] The virus-injected mice were killed 4 weeks later, and DRGs were collected and CD22 mRNA expression was detected by real-time fluorescence quantitative PCR. Figure 5 As shown in Figure A, compared with mice injected with the control virus (AAV-PHP.S-Negative control), the expression of CD22 mRNA in DRG of mice injected with AAV-PHP.S-CD22 virus was significantly increased, and the difference was statistically significant ( *** P<0.001).

[0084] like Figure 5As shown in Figure B, compared with SNL mice injected with the control virus (AAV-PHP.S-Negative control), the mechanical hyperalgesia threshold of SNL mice injected with AAV-PHP.S-CD22 virus to von Frey filament stimulation was significantly increased, and the difference was statistically significant ( ** P<0.01).

[0085] The above experimental results indicate that overexpression of CD22 inhibits SNL-induced mechanical hyperalgesia.

[0086] Studies have reported that CD22 is also an important target for tumor treatment. Both the tumor itself and chemotherapy drugs used to treat the tumor can cause hyperalgesia and produce neuropathic pain. The experimental results of the present invention show that in the treatment of neuropathic pain caused by cancer, using CD22 as a drug target may have the effect of killing two birds with one stone.

[0087] The above embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the protection scope of the present invention.

Claims

1. Application of cell surface adhesion molecule CD22 as a target in screening non-addictive drugs for analgesia, characterized in that: The NCBI number of the full-length sequence of CD22 is NM_001043317.2; the type of pain for analgesia is neuropathic pain induced by spinal nerve ligation.

2. The use according to claim 1, characterized in that: The drug is an agent that promotes the expression or function of CD22.

3. The use according to claim 2, characterized in that: The reagent is a CD22 overexpression vector or a CD22 protein.

4. The use according to claim 3, characterized in that: The CD22 overexpression vector is a lentivirus or adeno-associated virus containing the full-length sequence of CD22.

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