Application of neuropeptide S in promoting treatment of spinal cord injury by EMSCs

Abstract

The invention discloses application of neuropeptide S in promoting treatment of spinal cord injury by EMSCs, and belongs to the technical field of spinal cord injury drug repair. According to the invention, the EMSCs are used as a living cell carrier, so that NPS is overexpressed on the EMSCs for treating SCI; the result shows that the EMSCs with overexpressed NPS are migrated to the diseased region, and the highly expressed NPS is detected at the diseased region, so that the athletic ability of the SCI rat is remarkably improved, the axon regeneration potential, the myelin sheath regeneration potential, the endogenous neural stem cell differentiation capacity and the nerve regeneration capacity of the SCI rat are improved, and a better treatment effect is achieved.

Description

Technical Field

[0001] The invention relates to application of neuropeptide S in promoting EMSCs to treat spinal cord injury, belonging to the technical field of spinal cord injury drug repair. Background Art

[0002] Spinal cord injury (SCI) is a serious neurological disorder usually caused by trauma, resulting in complete or partial loss of neurological function. Due to the lack of effective treatment, SCI is a devastating disease and can even be fatal.

[0003] The fundamental reason for the limited therapeutic effect of spinal cord injury is due to a series of pathological events after spinal cord injury, including the death of a large number of neurons and glial cells, lack of neurotrophic factors, accumulation of inhibitory myelin fragments, infiltration of immune cells and glial scar formed by excessive proliferation of reactive astrocytes. The microenvironment of the SCI injury site is not suitable for nerve regeneration. Previous studies have shown that stem cell transplantation has considerable potential in promoting tissue repair and functional improvement after SCI.

[0004] Ectodermal mesenchymal stem cells (EMSCs) from the nasal mucosa are a special cell group that originates from the cranial neural crest. Compared with other mesenchymal stem cells, they have the significant advantage of being more convenient to obtain and are promising cells that can be used to repair nervous system injuries. However, due to the harsh microenvironment after spinal cord injury, transplanted cells are difficult to survive and grow in the injured area.

[0005] There are many kinds of neuropeptides, and their structures and sources are different. Neuropeptides are a class of small molecule proteins that play a role in the nervous system. They act as signal molecules to regulate a variety of physiological processes, including pain perception, appetite control, learning and memory, and mood regulation. There are many types of neuropeptides, including endogenous opioid peptides and neuropeptide Y family. These neuropeptides exert their effects by binding to specific receptors, which usually belong to the G protein-coupled receptor (GPCRs) family. Neuropeptides are widely distributed and play a role not only in the central nervous system, but also in the peripheral nervous system and endocrine system. Due to the complexity of the neuropeptide system, their distribution and function may vary in different species. For example, neuropeptide Y is composed of 36 amino acids and belongs to the glucagon family. Its tertiary structure is α-helical and can bind to Y-type receptors. It is widely distributed in the central nervous system and is mainly involved in appetite regulation, energy metabolism, stress response, and mood regulation. Neuropeptide P is a polypeptide composed of 11 amino acids and belongs to the tachykinin family. It is synthesized and released by neurons in the central and peripheral nervous systems and is involved in pain transmission, inflammatory response and mood regulation. Neuropeptide S is a peptide chain composed of 20 amino acids, synthesized in specific neurons in the brainstem and mainly concentrated in the central nervous system. It has wakefulness-promoting and anti-anxiety effects, and also regulates breathing and appetite and enhances memory function.

[0006] At present, the research on neuropeptide S is mostly focused on inhibiting oxidative stress, anxiety, food intake and pain. There are few studies related to the treatment of spinal cord injury. For example, the literature "Intra-amygdala microinfusion of neuropeptide S attenuates neuropathic pain and suppresses the response of spinal microglia and astrocytes after spinal nerve ligation in rats" discloses that neuropeptide S can produce anti-nociceptive effects at the level of the rat spinal cord, and NPS can produce anti-nociceptive perception through NPSR. There is no research on the use of neuropeptide S to treat SCI. Summary of the invention

[0007] To solve the above problems, the present invention uses mesenchymal stem cells to overexpress NPS and inoculate them into a hydrogel scaffold for the treatment of SCI.

[0008] The first object of the present invention is to provide an application of EMSCs cells overexpressing neuropeptide S or a cell preparation containing EMSCs cells overexpressing neuropeptide S in the preparation of a drug for treating and / or assisting in the treatment of spinal cord injury. In one embodiment, the amino acid sequence of neuropeptide S is: MIGSLKLNLILALSLSVVHVIWSYPVLSSKVPGKPDYFLILLSTCPARLEGSDGLAFLKPILEK TSMKRSFRNGVGSGVKKTSFRRAKQ (SEQ ID NO.1, amino acid sequence number is NP001258042.1; its corresponding mRNA number is NM_001271113.1).

[0009] In one embodiment, the number of viable cells of EMSCs cells overexpressing neuropeptide S in the drug is not less than 2×10 5 Pieces / mL.

[0010] In one embodiment, the spinal cord injury includes traumatic spinal cord injury and non-traumatic spinal cord injury.

[0011] In one embodiment, the drug is carried by a hydrogel scaffold.

[0012] In one embodiment, the method for preparing the hydrogel scaffold is: mixing a fibrinogen solution and thrombin to prepare a hydrogel.

[0013] In one embodiment, the average pore size of the porous three-dimensional structure of the hydrogel scaffold is 201.29±7.25 μm, which can serve not only as a carrier of cells but also as a conduit for nerve regeneration, promoting the connection of nerves after regeneration.

[0014] In one embodiment, the drug contains the EMSCs cells expressing transglutaminase TG2, a drug carrier and / or a pharmaceutical excipient.

[0015] In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes;

[0016] The pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants and / or flavoring agents;

[0017] The dosage form of the medicine is powder, granule, capsule, tablet, pill, injection or oral solution.

[0018] The second object of the present invention is to provide a drug for treating and / or assisting in the treatment of spinal cord injury, wherein the drug contains EMSCs cells overexpressing neuropeptide S as an effective ingredient.

[0019] In one embodiment, the amino acid sequence of neuropeptide S is shown as SEQ ID NO.1.

[0020] The number of viable cells of EMSCs overexpressing neuropeptide S in the drug was not less than 2×10 5 Pieces / mL.

[0021] In one embodiment, the spinal cord injury includes traumatic spinal cord injury and non-traumatic spinal cord injury;

[0022] Optionally, the drug is carried by a hydrogel scaffold;

[0023] Optionally, the drug contains the EMSCs cells expressing NPS, a drug carrier and / or a pharmaceutical excipient;

[0024] Optionally, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes;

[0025] Optionally, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants and / or flavoring agents;

[0026] Optionally, the drug is in the form of powder, granules, capsules, tablets, pills, injections or oral solutions.

[0027] Beneficial effects of the present invention:

[0028] The present invention uses EMSCs as living cell carriers to overexpress NPS on EMSCs for treating SCI; the results show that EMSCs overexpressing NPS migrate to the lesion site, and highly expressed NPS is detected at the lesion site, which significantly improves the motor ability of SCI rats, and improves the axon regeneration potential, myelin regeneration potential, endogenous neural stem cell differentiation ability, and nerve regeneration ability of SCI rats, and has a good therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of adenoviral vector.

[0030] Figure 2 The expression of NPS in different EMSCs groups; (A) Cell immunofluorescence staining was used to determine the expression levels of NPS protein (green) and m Scarlet (red) in NPS-EMSCs, ADV-EMSCs and normal EMSCs groups; immunofluorescence showed that about 95.57±2.32% of mesenchymal stem cells were m Scarlet positive); Scale bar = 100μm; (B) Western blot analysis of NPS expression in infected and uninfected nasal mucosal mesenchymal stem cells; (C) Fluorescence statistical graph; (D) Western blot result statistical graph.

[0031] Figure 3RNA sequencing revealed changes in the cellular gene expression profile of EMSCs overexpressing NPS; (A) RNA gene differential expression map; (B) differential gene volcano map; (C) clustering heat map of differentially expressed genes; (D) GO enrichment analysis; (E) KEGG enrichment analysis; (F) Wiki Pathways analysis.

[0032] Figure 4 Cell culture and identification of rat nasal mucosa-derived mesenchymal stem cells; (A) Immunofluorescence was used to identify the expression of positive markers (CD44, Vimentin, Sox2, Nestin), scale bar = 200 μm; (B) Phase contrast microscopy showed primary rat EMSCs cultured in vitro to the third generation and EMSCs overexpressing NPS, scale bar = 100 μm; (B) Alkaline phosphatase staining showed that nasal mucosa-derived mesenchymal stem cells had osteogenic differentiation ability; Oil red staining showed that nasal mucosa-derived mesenchymal stem cells had adipogenic differentiation ability, scale bar = 20 μm.

[0033] Figure 5 The following are the results of hind limb scoring and spinal cord staining; (A) is the BBB score of the hind limbs at 1, 2, 4, and 8 weeks after injury; (B) is the result of H&E staining of the spinal cord.

[0034] Figure 6 Representative immunofluorescence images of gross observation of injured spinal cord segments 4 weeks after surgery; (A) mScarlet is scattered throughout the spinal cord injury area, and NPS promotes the survival and migration of mesenchymal stem cell (EMSC)-derived cells in vivo. Red fluorescent protein-positive (m Scarlet) cells are derived from transplanted NPS-EMSCs cells; (B) Western blot analysis of NPS expression in the injury site; (C) Statistical graph of the results of Western blot analysis of NPS.

[0035] Figure 7 The cross-section overview of the spinal cord segments in each group is shown by NF200 immunofluorescence staining. The NF200-positive axons (green) strongly extend to the rostral (1), injury site (2), and caudal (3) regions near the injury site clearly show the distribution of NF200-positive axons (green), indicating that NPS-EMSCs can extend NF200-positive axons to the injury site.

[0036] Figure 8 NPS-EMSCs protect the myelin sheath of the injured spinal cord in rats after SCI and improve regeneration; (A) Horizontal sections of MBP (purple) and GFAP (green) immunofluorescence staining show the distribution of MBP; (B) Immunofluorescence staining results.

[0037] Fig. 9The following are the results of fluorescence staining of Nestin and Tuj-1; (A) Immunofluorescence staining of Nestin (green) and Tuj-1 (purple) in the beak near the injury; (B) Immunofluorescence staining of Nestin (green) and Tuj-1 (purple) in the cross section of the spinal cord in the SCI+NPS-EMSCs group.

[0038] Fig.10 Western blot analysis of PI3K, AKT, and GSK3β phosphorylation levels; (A) Western blot analysis of PI3K, AKT, and GSK3β phosphorylation levels in rats with spinal cord injury; (B), (C), and (D) Western blot results statistical graphs of each indicator.

[0039] Fig.11 Immunofluorescence staining results of MAP2 (green) and GFAP (red) in NSCs pretreated with MK2206 co-cultured with NPS-EMSCs. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0041] The reagents involved in the following examples are as follows:

[0042] Fetal bovine serum (Gibco), DMEM / F12 medium (Hyclone), nestin (Affinity, DF7754) and vimentin (Abclonal; A21648), CD44 (Abclonal; A24023), Sox2 (Proteintech, 11064-1-AP), GFAP (Proteintech, 60190-1-Ig), MBP (Proteintech, 10458-1-AP), NPS (Cloud-Clone, PAA796Ra01), BCIP / NBT (Source Leaf, R32154), AM / PI (APExBIO, K2247) staining kit.

[0043] The recombinant rat NPS adenovirus shuttle plasmid was constructed by Heyuan Biotechnology (Shanghai) Co., Ltd. and confirmed by sequencing.

[0044] The experimental methods involved in the following embodiments are as follows:

[0045] 1. Immunofluorescence (NPS-EMSCs immunofluorescence)

[0046] Cells cultured in vitro were fixed with 4% paraformaldehyde, and for immunofluorescence staining, samples were blocked in 5% BSA and 0.3% Triton X-100 for 30 minutes at room temperature, and then incubated with primary antibodies overnight at 4°C. NPS (Cloud-Clone, PAA796Ra01), nestin (Affinity, DF7754) and vimentin (Abclonal; A21648), CD44 (Abclonal; A24023), Sox2 (Proteintech; 11064-1-AP), DAPI (Invitrogen, YD3918381) were used for nuclear staining. Subsequently, samples were incubated with secondary antibodies for 1 hour at room temperature before nuclear staining.

[0047] 2. Alkaline phosphatase and Oil Red O staining

[0048] Alkaline phosphatase staining: add an appropriate amount of BCIP / NBT colorimetric solution to the cell slides that need to be stained, and incubate at room temperature in the dark for 10-20 minutes. Observe and control the color development time under a microscope. When the best color development effect is achieved, rinse with tap water to stop the color development. The sections after color development are counterstained, dehydrated and transparent, and can be stored for a long time after sealing.

[0049] Oil Red O staining: Mix Oil Red O staining solution with ddHO 2 O in a ratio of 3:2 and filter at 0.22um for later use. Incubate with 60% isopropanol for 2 minutes and discard. Add the stock solution prepared in the previous step and incubate for 10 minutes in a dark place. After incubation, discard the dye solution and add ddH 2 Wash until the water is transparent and take pictures using a microscope.

[0050] 3. Hydrogel characterization

[0051] 4. AM / PI staining (scaffold staining)

[0052] We used AM / PI (APExBIO, K2247) staining kit for live and dead double staining of cells.

[0053] 5. Basso, Beattie & Bresnahan locomotor rating scale (BBB scale)

[0054] Hindlimb motor function of rats was evaluated by BBB scoring. The evaluation index scores (0-21) including joint movement, stepping ability, coordination and trunk stability represent the sequential recovery stage, and the scores are positively correlated with the recovery stage. During the test, the rats were allowed to walk freely for 5 minutes under the close observation of two observers. The observers then evaluated according to the scoring system. The final score was the average of the scores calculated by the two observers. In this test, the observers were blinded to the group identity of the rats.

[0055] 6. GFAP and MBP

[0056] After sampling, frozen sections were prepared. For immunofluorescence staining, the samples were blocked in 5% BSA and 0.3% Triton X-100 at room temperature for 30 minutes, and then incubated with primary antibodies at 4°C overnight, GFAP (Proteintech, 60190-1-Ig), MBP (Proteintech, 10458-1-AP). DAPI (Invitrogen, YD3918381) was used for nuclear staining. Subsequently, the samples were incubated with secondary antibodies at room temperature for 1 hour before nuclear staining.

[0057] Example 1: Construction of EMSCs overexpressing NPS

[0058] 1. Construct EMSCs overexpressing NPS. The steps are as follows:

[0059] (1) Construction of adenoviral vector:

[0060] The following primers were designed for the synthesis of NPS gene (SEQ ID NO. 2), forward: CGCAAATGGGCGGTAGGCGTG (SEQ ID NO. 3), reverse: GAAATTTGTGATGCTATTGC (SEQ ID NO. 4);

[0061] The construction steps are as follows Figure 1 .

[0062] (2) Adenovirus transfection

[0063] The recombinant rat TG2 adenovirus (AdV NPS mScarlet) and the control EGFP adenovirus (AdV mScarlet) were constructed by Heyuan Biotechnology (Shanghai) Co., Ltd. and confirmed by sequencing. The specific construction was as follows: the NPS gene was inserted into the vector to prepare pcADV-EF1-mScarlet-CMV-Nps-3FLAG (e.g. Figure 1), the insertion site was between EcoRI and BamHI, and the GL2004 pcADV-EF1-mScarlet-CMV-MCS-3xFLAG vector was set as a parallel group. EMSCs cells (1×10 6 The cells / well (American Type Culture Collection) were plated in a 6-cm diameter culture dish supplemented with 10% FBS in DMEM / F12 and placed in a 5% CO 2 The cells were incubated in a humidified incubator for 12 h. The culture medium was replaced before transfection, and the cells were transfected at a confluence of 80-90% for 48 h (the amount of plasmid transfection was 40 ug), and EMSCs overexpressing the NPS (Adv-NPS-mScarlet) gene were harvested.

[0064] (3) Verification

[0065] NPS-overexpressed EMSCs (NPS-EMSCs), EMSCs transfected with empty plasmid (Adv-EMSCs) and untransfected EMSCs were taken for fluorescence detection.

[0066] Immunofluorescence test results Figure 2 As shown in A, the results showed that 48 hours after transfection with NPS recombinant adenovirus, 90% of the cells were positive and emitted red fluorescence (m Scarlet), indicating that the adenovirus transfection was successful. At the same time, green fluorescence (NPS) was emitted, indicating that EMSCs overexpressed NPS.

[0067] WB verification results are as follows Figure 2 B and Figure 2 As shown in C, the results showed that the NPS expression level in the NPS-EMSCs group was increased and significantly higher than that in the other groups. That is, EMSCs overexpressing NPS (NPS-EMSCs) were successfully constructed.

[0068] 2. Detection of gene expression profile of NPS-EMSCs cells

[0069] To further identify the gene expression changes of NPS-EMSCs, total cellular RNA was extracted using TRIzol and subjected to RNA sequencing.

[0070] The gene expression results are as follows Figure 3 As shown in A and 3B, the results showed that 1341 genes were down-regulated and 1120 genes were up-regulated, and the volcano plot results also showed that NPS had the largest difference;

[0071] Functional pathway enrichment analysis was performed using the Gene Ontology (GO) database, and the results are shown in Figure 3As shown in C and 3D, the results showed that among the biological functions, the cell adhesion, axon guidance and other related pathways were enriched in the main enriched gene expression; among the molecular functions, the ion channel activity, extracellular matrix structural components, growth factor activity, fibronectin binding, collagen binding and other related pathways were enriched; among the cellular components, the neuronal cell body, axon, cell surface, extracellular matrix, basement membrane and other related pathways were enriched.

[0072] After obtaining the differentially expressed genes, KEGG enrichment analysis was performed on the differentially expressed genes. The results are as follows Figure 3 As shown in E and 3F, the results showed that compared with NPS-EMSCs and EMSCs, the 20 pathways with the most significant changes are shown in the figure. Wiki PathwaysEnrichment Top20 found that EMSCs overexpressing NPS can be used for the treatment of spinal cord injury.

[0073] 3. Characterization of NPS-EMSCs

[0074] The morphology, surface marker expression, and differentiation potential of NPS-EMSCs were characterized. Figure 4 shown.

[0075] Cell marker results Figure 4 As shown in A, the results showed that the expression of markers CD44, Vimentin, Sox2, and Nestin in NPS-EMSCs was also similar to that in EMSCs;

[0076] The osteogenic and adipogenic differentiation of EMSCs and NPS-EMSCs were induced by their respective induction medium. The results of cell morphology, alkaline phosphatase, and Oil Red O staining were shown in Figure 4 As shown in B, the results showed that the morphology of NPS-overexpressing EMSCs did not change compared with that of EMSCs, indicating that EMSCs and NPS-EMSCs had similar differentiation potential.

[0077] Example 2: Construction of scaffolds seeded with NPS-EMSCs

[0078] 1. Characterization of hydrogel scaffolds

[0079] Preparation of hydrogel scaffold: Fibrinogen solution (concentration of 100 mg / mL, solvent is PBS solution) was mixed with thrombin (enzyme dosage of 20 U / mL, solvent is PBS solution) in a volume ratio of 1:1 to obtain a hydrogel (wherein the fibrinogen concentration is 50 mg / mL). After the obtained hydrogel scaffold was vacuum freeze-dried, the sample was attached to a metal short column using a carbon sticker and sputtered with gold for 30 seconds to obtain a scanning electron microscope image. The results are shown in Figure 2. Figure 4As shown in C, the results show that the average pore size of the porous three-dimensional structure of the scaffold is 201.29±7.25μm, which can not only serve as a carrier of cells, but also as a conduit for nerve regeneration to promote the connection of nerves after regeneration.

[0080] 2. Take the NPS-EMSCs cells constructed in Example 1 and inoculate them into the hydrogel scaffold, and the steps are as follows:

[0081] The successfully transfected NPS-EMSCs cells and hydrogel scaffolds were plated in a 24-well culture plate and sterilized by ultraviolet light. 5 The cells were inoculated on the hydrogel and cultured with culture medium for 48 h for subsequent experiments.

[0082] 3. Detection after scaffold inoculation with NPS-EMSCs

[0083] After the cells were seeded on the scaffolds and cultured in vitro, AM / PI staining was used to determine the survival status of each group of cells on the scaffolds on the 1st, 3rd, and 5th days. Figure 4 As shown in D, the results show that EMSCs can adhere to the scaffold and survive; when cultured in vitro, the cells are evenly distributed and grow along the scaffold. It can be seen that the hydrogel scaffold has very good biocompatibility and can ensure the proliferation and survival of cells growing on the hydrogel scaffold.

[0084] Example 3: Application of NPS-EMSCs in the treatment of SCI

[0085] 1. Construction of SCI rat model

[0086] A complete transection model of spinal cord injury was made using SD rats. Anesthesia was maintained by inhalation of isoflurane through a mask. The rats were prone on the operating table, their limbs were fixed, their backs were shaved and disinfected with iodine. A longitudinal incision was made centered on the spinous process of T8, the soft tissue was separated, the corresponding lamina was removed, and the spinal cord was exposed. A 2 mm long T8-T9 spinal cord slice was removed under a microscope. After the injury, an appropriate amount of hemostatic sponge was given to stop bleeding, the hydrogel scaffold was transplanted to the SCI transection area, the wound was sutured, the spinal cord meninges were dissected, the muscle and skin layers were carefully sutured, and the rats were transferred to a 37 ° C thermostat after disinfection, and the rats were observed until anesthesia recovered. In the sham operation group, only the chest plate was removed and then closed. To avoid infection, antibiotics were injected daily for 1 week after surgery, and the rats were manually urinated twice a day until spontaneous urination was restored. The specific groups are as follows:

[0087] Sham operation group: only the corresponding lamina was removed.

[0088] Model group (SCI group): 2 mm long T8-T9 spinal cord was removed, and the blood was stopped and then disinfected and sutured.

[0089] Treatment group (SCI+NPS-EMSCs): After resection of 2 mm long T8-T9 spinal cord, hydrogel loaded with NPS-EMSCs was transplanted (the number of transplanted cells was 2×10 5 ) At the injured site, stop bleeding, disinfect and suture.

[0090] Negative control group (SCI+EMSCs group): After resection of 2 mm long T8-T9 spinal cord, the hydrogel loaded with EMSCs was transplanted (the number of transplanted cells was 2×10 5 ) At the injured site, stop bleeding, disinfect and suture.

[0091] Positive control group (SCI+Fibrin group): After resection of 2 mm long T8-T9 spinal cord, the hydrogel was transplanted into the injured area, and the injured area was disinfected and sutured after hemostasis.

[0092] 2. Assessment of rat motor ability

[0093] The Basso, Beattie & Bresnahan locomotor rating scale (BBB scale) was used to evaluate the motor function recovery of the hind limb behavior of SCI rats after treatment, and the BBB locomotor rating scale was used for behavioral analysis every week.

[0094] The results of BBB exercise analysis are as follows Figure 5 As shown in A, the results showed that after spinal injury, the BBB scores of the four groups dropped to 0 on the first day of surgery, indicating that the SCI model was successfully established. Compared with the SCI group, the BBB score of the SCI+NPS-EMSCs treatment group was significantly increased 8 weeks after transplantation (P<0.05).

[0095] The results of H&E staining of the spinal cord (12 weeks) are as follows Figure 5 As shown in B, the results showed that the tissue structure of the spinal cord injury site in the control group was loose and there was an obvious cavity; compared with the SCI+NPS-EMSCs group, the amount of free tissue at the injury site in the SCI+EMSCs group was greater; the cavity area in the SCI+NPS-EMSCs group was the smallest, indicating that the NPS-EMSCs group had the best integration effect in the transected spinal cord.

[0096] 3. Detection of NPS-EMSCs transplantation effect

[0097] At 4 weeks after injury, the m Scarlet-positive cell area in the damaged area was detected by immunofluorescence.

[0098] Immunofluorescence results Figure 6As shown in A, the results showed that in the SCI+NPS-EMSCs group, m Scarlet-positive cells (i.e., NPS-EMSCs) were observed throughout the injury site as well as in the rostral and caudal regions of the injury site;

[0099] The results of WB verification of tissue proteins in the spinal cord injury area are as follows: Figure 6 As shown in B, the results showed that the expression of NPS in the SCI+NPS-EMSCs group was significantly higher than that in the other groups, which was consistent with the results of immunofluorescence, indicating that the intervention of NPS-EMSCs did increase the expression of NPS in the spinal cord injury site.

[0100] 4. Axon regeneration potential detection

[0101] In SCI, axon regeneration is considered an important step in tissue repair after injury. At 4 weeks after injury, immunofluorescence was used to evaluate the expression of a key marker (neurofilament 200, an organizational protein present only in neurons) in the rostral, injured, and caudal regions.

[0102] The results are as follows Figure 7 As shown, the results showed that in the SCI group, axons were completely lost in the center of the injury; the number of NF200-positive axons passing through the injury area increased slightly in the SCI+EMSCs group and the SCI+Fibrin group; in contrast, the SCI+NPS-EMSCs-treated group presented a large number of NF200-positive axons in the middle area of ​​the injury. At the same time, the nerve continuity treated with SCI+NPS-EMSCs was also more obvious than that of the other groups, indicating that axons grew inside the spinal cord injury area. The above results indicate that SCI+NPS-EMSCs can promote axonal growth in the injury area, thereby contributing to the recovery of hindlimb function in rats after SCI and increasing the possibility of axon regeneration after SCI.

[0103] 5. Myelin regeneration potential detection

[0104] In SCI, demyelination is a common pathology leading to functional impairment. Myelin basic protein (MBP) is a protein on the myelin sheath that is essential for the conduction of nerve impulses along the axon and can maintain the stability of the myelin sheath in the central nervous system.

[0105] At 8 weeks after injury, the effect of NPS-EMSCs on myelin regeneration was detected by double immunofluorescence staining of GFAP and MBP in the injured spinal cord area. Figure 8 shown.

[0106] The results showed that in the SCI+NPS-EMSCs group, MBP-positive cells were clearly visible at the injury site, but not obvious in the other groups, indicating that NPS-EMSCs treatment can promote myelin regeneration and have a significant inhibitory effect on glial scars. The results of co-staining GFAP were also consistent with previous results, and NPS-EMSCs intervention significantly inhibited the expression of GFAP.

[0107] 6. Detection of differentiation ability of endogenous neural stem cells

[0108] After SCI, a large number of spinal cord neurons undergo apoptosis, axonal rupture, glial scars and cavities, which seriously hinder neuronal regeneration and repair. Endogenous neural stem cells (NSCs) have the potential to differentiate into neurons, astrocytes and oligodendrocytes. Compared with the other two differentiation types, neurons play a vital role in the body's normal electrophysiological activities and have sufficient self-renewal potential to provide cells.

[0109] Nestin is a marker of NSC and can be used to mark neural stem cells, while Tuj-1 is a marker of early neurons. At 8 weeks after injury, immunofluorescence was performed on Nestin and Tuj-1. The results are as follows Fig. 9 , indicating that after spinal cord injury, regardless of whether there is treatment or not, neural stem cells and neurons will appear in the rostral and caudal parts near the spinal cord injury, indicating that after spinal cord injury, neural stem cells will recruit and differentiate neurons to the injured site; and the Tuj-1 positive cells in the injured spinal cord of the SCI+NPS-EMSCs group were significantly more than those near the injury and caudal parts, indicating that there are more newborn neurons in the injured site; Nestin immunofluorescence results showed that NPS-EMSCs can recruit more neural stem cells to the vicinity of the injury site, and more Tuj-1 positive cells were also found near the injured spinal cord in the SCI+NPS-EMSCs group and the SCI+EMSCs group, and the Tuj-1 positive cells in the SCI+NPS-EMSCs group were more than those in the SCI+EMSCs group, indicating that the recruited NSCs differentiated into neurons rather than astrocytes.

[0110] 7. Nerve regeneration ability detection

[0111] At the second week after injury, spinal cord samples were collected from the injury site of each group to study the mechanism of promoting neuronal differentiation.

[0112] (1) Detection of AKT signaling pathway proteins

[0113] In recent years, the AKT signaling pathway, as a signaling pathway related to various cell functions such as cell growth, reproduction and differentiation, has been used in spinal cord injury diseases (see references such as "Enhanced differentiation of human neural stem cells into neurons on graphene", "Neural Stem Cell Grafts Form Extensive Synaptic Networks that Integrate with Host Circuits after Spinal Cord Injury"). The results of KEGG enrichment analysis are as follows Figure 3 As shown, the results indicated that the differentially altered genes between NPS-EMSCs and EMSCs were involved in the PI3K / AKT signaling pathway;

[0114] WB test results Fig.10 As shown, the results showed that the phosphorylation levels of PI3K, AKT and GSK3β in the SCI+NPS-EMSCs group were significantly increased, indicating that NPS-EMSCs promoted the proliferation and neuronal differentiation of NSCs by activating the PI3K / AKT / GSK3β phosphorylation pathway.

[0115] (2) AKT signaling pathway verification

[0116] MK2206 is an AKT pathway inhibitor, and MK2206 treatment blocks the conditioned medium-induced phosphorylation of AKT and GSK3β. In order to verify the mechanism of NPS-EMSCs promoting neuronal differentiation in vitro, the phosphorylation levels of AKT and GSK3β in NSCs were detected to determine whether NPS-EMSCs treatment promoted the proliferation and neuronal differentiation of NSCs by activating the AKT-GSK3β signaling pathway.

[0117] The proliferation and neuronal differentiation of NSCs after pretreatment were examined by immunofluorescence staining. Fig.11 As shown in the results, the neuronal differentiation of NSCs was reduced after pretreatment with MK2206. It can be seen that NPS-EMSCs promote the proliferation and neuronal differentiation of NSCs by activating the PI3K / AKT / GSK3β signaling pathway.

[0118] NPS nucleotide sequence (SEQ ID NO.2):

[0119] ATGATTGGCTCATTAAAACTCAACCTCATCTTAGCTCTGTCGCTGTCCGTGGTACACGTGATTTGGAGTTATCCGGTCCTCTCTTCCAAGGTGCCTGGGAAGCCTGATTACTTTCTCATTTTGCTGAGTACCTGC CCAGCCAGGCTGGAGGGGAGCGACGGGCTAGCTTTTCTAAAGCCAATTTTGGAGAAGACGTCGATGAAAAGGTCCTTTCGCAACGGAGTCGGCTCAGGGGTGAAAAAAACTTCATTTCGAAGAGCAAAGCAATGA

[0120] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of EMSCs cells overexpressing neuropeptide S or cell preparations containing EMSCs cells overexpressing neuropeptide S in the preparation of drugs for treating and / or assisting in the treatment of spinal cord injury.

2. The use according to claim 1, characterized in that: The amino acid sequence of neuropeptide S is shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that: The number of viable cells of EMSCs overexpressing neuropeptide S in the drug was not less than 2×10 5 Pieces / mL.

4. The use according to any one of claims 1 to 3, characterized in that: Spinal cord injury includes traumatic spinal cord injury and non-traumatic spinal cord injury.

5. The use according to any one of claims 1 to 4, characterized in that: The drug is carried by a hydrogel scaffold.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine contains the EMSCs cells expressing transglutaminase TG2, a medicine carrier and / or a pharmaceutical excipient.

7. The use according to claim 6, characterized in that: The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; The pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants and / or flavoring agents; The dosage form of the medicine is powder, granule, capsule, tablet, pill, injection or oral solution.

8. A drug for treating and / or assisting in the treatment of spinal cord injury, characterized in that: The medicine takes EMSCs cells over-expressing neuropeptide S as an effective ingredient.

9. The drug according to claim 8, characterized in that The amino acid sequence of neuropeptide S is shown in SEQ ID NO.1; The number of viable cells of EMSCs overexpressing neuropeptide S in the drug was not less than 2×10 5 Pieces / mL.

10. The drug according to claim 8, characterized in that Spinal cord injury includes traumatic spinal cord injury and non-traumatic spinal cord injury; Optionally, the drug is carried by a hydrogel scaffold; Optionally, the drug contains the EMSCs cells expressing neuropeptide S, a drug carrier and / or a pharmaceutical excipient; Optionally, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; Optionally, the pharmaceutical excipients include fillers, binders, wetting agents, disintegrants, lubricants and / or flavoring agents; Optionally, the drug is in the form of powder, granules, capsules, tablets, pills, injections or oral solutions.

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