A biomarker for the diagnosis and prognosis of spinal cord injury

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to biomarkers for spinal cord injury prognosis and their applications. As key factors regulating IL-17+γδT cells, STAT3 and RORγt affect the secretion of IL-17. In mouse experiments, the mRNA expression levels of STAT3 and RORγt in spinal cord tissues were significantly increased compared with those in the SH group after SCI, and their expression levels were decreased compared with those in the SCI group after HBO treatment; in spinal cord injury subjects, the gene expression levels of STAT3 and RORγt in peripheral blood were significantly increased, and were significantly decreased after HBO treatment. The AUC of the STAT3 gene expression level as a biomarker for spinal cord injury diagnosis was 0.992; the AUC of the RORγt gene expression level as a biomarker for spinal cord injury diagnosis was 0.997, and the combined AUG of the two was 1.00. The AUC of the STAT3 gene expression level as a biomarker for predicting the efficacy of HBO treatment for SCI injury was 0.909; the AUC of the RORγt gene expression level as a biomarker for predicting the efficacy of HBO treatment for SCI injury was 0.933, and the combined AUG of the two was 0.993. Therefore, STAT3 and / or RORγt can be used as biomarkers for spinal cord injury diagnosis and predicting the efficacy of HBO treatment for SCI injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to biomarkers for spinal cord injury prognosis and their applications. Background Art

[0002] Spinal cord injury (SCI) can be divided into primary injury and secondary injury. The primary injury is the tissue damage, bleeding and necrosis caused by direct or indirect violence acting on the spinal cord tissue. The secondary injury is a series of "cascade" biochemical reactions such as local tissue edema, ischemia / hypoxia, inflammatory response, and apoptosis that occur several minutes after the primary injury, resulting in secondary cell injury and death lasting for several days to weeks. The degree of its injury is the main factor affecting the prognosis of spinal cord injury. Spinal cord injury can cause motor, sensory and autonomic nerve dysfunction. Inflammatory response plays an important role in the series of changes after SCI, and excessive inflammatory response may hinder the normal progress of nerve regeneration and repair (Stahel PF, Flierl MA. Targeted modulation of the neuroinflammatory response after spinal cord injury [J]. Am J Pathol, 2010, 177(6): 2685-2687.). Surgical treatment can relieve primary injuries such as spinal cord compression, but it cannot change the pathological changes such as ischemia / hypoxia and inflammation of the damaged spinal cord.

[0003] Hyperbaric oxygen (HBO) treatment refers to an intervention method in which 100% oxygen is intermittently breathed in a treatment chamber at a pressure higher than sea level pressure (1.0 atmospheric pressure ATA). As a safe and non-invasive physical treatment method, HBO uses multiple channels to reverse or prevent pathological changes after spinal cord injury, achieve neuron repair and regeneration, and plays an important role in the recovery of spinal cord function in subjects after spinal cord injury. Hyperbaric oxygen treatment can reduce secondary injury of SCI, maximize the retention of the remaining structure and function of the damaged spinal cord, effectively promote the recovery of spinal cord function, shorten the treatment time, reduce the disability rate, and significantly improve the quality of life of the subjects. It has become an important and indispensable method for the comprehensive treatment of SCI (Liu Ming, Sun Yongming, Su Peng, et al. Efficacy analysis of hyperbaric oxygen comprehensive treatment for spinal cord injury [J]. China Journal of Modern Medicine, 2011, 21(36): 4568-4570, 4574.).

[0004] The nuclear receptor (NR) superfamily is responsible for encoding a large number of transcription factors and plays a key regulatory role in important physiological processes such as metabolic inflammatory responses and circadian rhythms. The retinoic acid-related orphan receptor (ROR) belongs to the ligand-dependent transcription factor nuclear receptor superfamily and includes three subtypes: RORα, RORβ, and RORγ. Among them, RORα is expressed in various organs, with the highest expression level in the brain tissue, and is related to inflammatory responses and anti-tumor effects (Lee IK, Song H, Kim H, et al. RORα regulates cholesterol metabolism of CD8+ T cells for anticancer immunity [J]. Cancers, 2020, 12(7): 1733.); RORβ is expressed in certain regions of the brain and retina, and there is less research on the functions of this subtype receptor. Some reports show that it plays a key role in the connection and differentiation of mammalian long-distance neurons (Byun H, Lee HL, Liu H, et al. Rorβ regulates selective axon target innervation in the mammalian midbrain [J]. Development, 2019, 146(14): dev171926.); RORγ is mainly expressed in organs such as the thymus, pancreas, and liver. Among them, RORγ specifically expressed in the thymus is called RORγt and plays an important role in immune function (Zeng JP, Li MX, Zhao QY, et al. Small molecule inhibitors of RORγt for Th17 regulation in inflammatory and autoimmune diseases [J]. J Pharm Anal, 2023, 13(6): 545-562.).

[0005] If the RORγt protein is acetylated and modified by histone acetyltransferase p300, etc., it can stably bind to the Il17a locus and initiate the transcription process of IL-17 (Chen Y, Wang DX, Zhao Y, et al. p300 promotes differentiation of Th17 cells via positive regulation of the nuclear transcription factor RORγt in acute respiratory distress syndrome [J]. Immunol Lett, 2018, 202: 8-15.). During the transcription process of IL-17, first, the T cell receptor receives the presented antigen and activates T cells. Immediately afterwards, interferon regulatory factor 4 (IRF4) and basic leucine zipper transcription factor (BATF) open chromatin in the early stage of T cell activation. Hypoxia-inducible factor-1α (HIF-1α) directly binds to RORγt and recruits p300 to acetylate the RORγt protein. At the same time, with the assistance of transcription factors such as nuclear factor κb inhibitor ζ (Iκbζ) and runt-related transcription factor 1 (RUNX1), RORγt exerts its transcriptional activity and produces IL-17 (Ciofani M, Madar A, Galan C, et al. A validated regulatory network for Th17 cell specification [J]. Cell, 2012, 151(2): 289-303.). Due to the above regulatory process, RORγt is considered an important transcription factor in the differentiation of Th17 cells and the production of IL-17. Therefore, inhibiting RORγt can reduce the differentiation of Th17 cells and the production of IL-17, thereby achieving the purpose of treating autoimmune diseases, which is a hot area in the current research and development of drugs for autoimmune diseases.

[0006] IL-17+ γδ T cells are key cell types in the immune system, and they play important roles in defending against infections, regulating inflammatory responses, and participating in the development of autoimmune diseases and tumors. IL-17+ γδ T cells are an important source of IL-17 among various cell types, including CD3+ αβ T cells, CD4+ αβ T cells, NKT cells, and γδ T cells themselves. These cells participate in regulating inflammatory responses and immune responses by producing IL-17. Especially in certain disease models, γδ T cells are major producers of IL-17 (Rebecca L. O’Brien, Christina L. Roark et al. “IL-17-producing γδ T cells.” European Journal of Immunology (2009).). IL-17+ γδ T cells do not completely rely on the activation of the T cell receptor (TCR) to produce IL-17, but rather rely more on non-TCR signals, such as the stimulation of IL-1β and IL-23 (C. Sutton, L. Mielke et al. “IL-17-producing γδ T cells and innate lymphoid cells.” European Journal of Immunology (2012). Yu-ling Wei, A. Han et al. “A Highly Focused Antigen Receptor Repertoire Characterizes γδ T Cells That are Poised to Make IL-17 Rapidly in Naive Animals.” Frontiers in Immunology (2015).). This indicates that IL-17+ γδ T cells have unique immune characteristics that enable them to respond rapidly to inflammatory signals. The functions and development of these cells are regulated by complex molecular mechanisms, including cytokine signaling and intracellular metabolic pathways. Therefore, elucidating the response mechanisms of γδ T cells and their subtype IL-17+ γδ T cells under diseases helps develop targeted therapeutic strategies.

[0007] Signal transducer and activator of transcription 3 (STAT3) is a signal transcription protein. The abnormal activation of STAT3 is closely related to cell proliferation, differentiation, carcinogenesis, etc. It is abnormally expressed in cancer stem cells such as breast cancer, pancreatic cancer, lymphoma, and lung cancer (LIU Hang, ZHAO Qinjie, XU Wei. Research progress of STAT3 inhibitors [J]. Journal of Pharmaceutical Practice and Service, 2021, 39(1): 4-8.). RORγt is located in the classical JAK-STAT3 signaling pathway, and its transcriptional activity is affected by gene expression regulation and ubiquitination / acetylation modification. Therefore, both RORγt and STAT3 are involved in the regulation of IL-17. In recent years, more and more studies have shown that the excessive production of the inflammatory factor IL-17 promotes the occurrence of autoimmune diseases. γδT cells are one of the key factors for HBO to alleviate the inflammatory response of SCI; IL-17+γδT cells are involved in the inflammatory response after SCI; and both STAT3 and RORγt are key factors regulating IL-17+γδT cells and can affect the secretion of IL-17. Therefore, the treatment plan based on the mechanism of action of IL-17 has become a current research hotspot. Summary of the Invention

[0008] The present invention discovers that γδT cells and their subtype IL-17+γδT cells, as key immune regulatory factors, are involved in hyperbaric oxygen (HBO) treatment and can reduce the inflammatory response after spinal cord injury (SCI). STAT3 and / or RORγt can affect the secretion of IL-17 and are both key factors regulating IL-17+γδT cells. Based on this, the present invention is completed.

[0009] In a first aspect, the present invention provides a biomarker for diagnosing spinal cord injury, and the biomarker is STAT3 and / or RORγt. When the gene expression level of STAT3 and / or RORγt in the biological sample of the subject is significantly increased compared with that of a healthy person, the subject has spinal cord injury.

[0010] Furthermore, the biological sample of the subject is selected from one or more of blood, cerebrospinal fluid, and / or spinal cord tissue.

[0011] Even further, the blood of the subject is at least one of peripheral blood, plasma, and serum.

[0012] Second aspect, the present invention provides an application of the biomarker as described in the first aspect in the preparation of a reagent for diagnosing spinal cord injury, wherein the reagent is a reagent capable of detecting STAT3 and / or RORγt; when the gene expression levels of STAT3 and / or RORγt in the biological sample of the subject are significantly increased compared to those of healthy individuals, the subject has spinal cord injury.

[0013] Furthermore, the biological sample of the subject is selected from one or more of blood, cerebrospinal fluid, and spinal cord tissue.

[0014] Even further, the blood of the subject is at least one of peripheral blood, plasma, and serum.

[0015] Third aspect, the present invention provides a kit for diagnosing spinal cord injury, wherein the kit contains the reagent as described in the second aspect of the present invention; when the gene expression levels of STAT3 and / or RORγt in the biological sample of the subject are significantly increased compared to those of healthy individuals, the subject has spinal cord injury.

[0016] Furthermore, the kit can be an ELISA detection kit and / or a colloidal gold detection kit.

[0017] Even further, the diagnostic method of the kit includes a direct method, an indirect method, a sandwich ELISA method, and / or a competitive method.

[0018] Fourth aspect, the present invention provides a biomarker for predicting the efficacy of hyperbaric oxygen therapy for spinal cord injury, wherein the biomarker is STAT3 and / or RORγt. When the gene expression levels of STAT3 and / or RORγt in the biological sample of the subject are detected after hyperbaric oxygen therapy, if the gene expression levels of STAT3 and / or RORγt are significantly higher than or equal to those before treatment, the efficacy of hyperbaric oxygen therapy for the spinal cord injury subject is poor; if the gene expression levels of STAT3 and / or RORγt are significantly lower than those before treatment, the hyperbaric oxygen therapy for the spinal cord injury subject is effective.

[0019] Fifth aspect, the present invention provides an application of the biomarker as described in the fourth aspect in the preparation of a reagent for predicting the efficacy of hyperbaric oxygen therapy for spinal cord injury, wherein the reagent is a reagent capable of detecting STAT3 and / or RORγt; if the gene expression levels of STAT3 and / or RORγt in the biological sample of the subject are significantly higher than or equal to those before treatment, the efficacy of hyperbaric oxygen therapy for the spinal cord injury subject is poor; if the gene expression levels of STAT3 and / or RORγt are significantly lower than those before treatment, the hyperbaric oxygen therapy for the spinal cord injury subject is effective.

[0020] Furthermore, the biological sample of the subject is selected from one or more of blood, cerebrospinal fluid, and spinal cord tissue.

[0021] Furthermore, the blood of the subject is at least one of peripheral blood, plasma and serum.

[0022] In a sixth aspect, the present invention provides a kit for predicting the efficacy of hyperbaric oxygen therapy for spinal cord injury, the kit containing the reagent as described in the fifth aspect of the present invention; if the gene expression levels of STAT3 and / or RORγt in the biological sample of the subject are significantly higher than or equal to those before treatment, the efficacy of hyperbaric oxygen therapy for the spinal cord injury subject is poor, and if the gene expression levels of STAT3 and / or RORγt are significantly lower than those before treatment, the hyperbaric oxygen therapy for the spinal cord injury subject is effective.

[0023] Further, the kit can be an ELISA detection kit and / or a colloidal gold detection kit.

[0024] Furthermore, the diagnostic methods of the kit include direct method, indirect method, sandwich ELISA and / or competitive method.

[0025] Beneficial effects

[0026] The present invention discovers that STAT3 and RORγt, as key factors regulating IL-17+γδT cells, affect the secretion of IL-17. After spinal cord injury, the contents of inflammation-related factors increase and decrease after hyperbaric oxygen therapy.

[0027] In the mouse experiment, the mRNA expression levels of STAT3 and RORγt in the spinal cord tissue after SCI were significantly higher than those in the SH group, and their expression levels decreased after HBO treatment compared with the SCI group; in the spinal cord injury subjects, the gene expression levels of STAT3 and RORγt in peripheral blood both increased significantly and decreased significantly after HBO treatment. The AUC of the STAT3 gene expression level as a biomarker for spinal cord injury diagnosis is 0.992; the AUC of the RORγt gene expression level as a biomarker for spinal cord injury diagnosis is 0.997, and the combined AUG of the two is 1.00. The AUC of the STAT3 gene expression level as a biomarker for predicting the efficacy of HBO treatment for SCI injury is 0.909; the AUC of the RORγt gene expression level as a biomarker for predicting the efficacy of HBO treatment for SCI injury is 0.933, and the combined AUG of the two is 0.993. Therefore, STAT3 and / or RORγt can be used as biomarkers for spinal cord injury diagnosis and predicting the efficacy of HBO treatment for SCI injury. Description of the drawings

[0028] Figure 1 HBO treatment significantly promotes the recovery of the hind limb motor function of SCI mice.

[0029] Figure 2 HBO treatment significantly reverses the injury of motor neurons in SCI mice.

[0030] Figure 3 HBO treatment significantly reduced the proportion of γδT cells in the injured spinal cord tissue of SCI mice.

[0031] Figure 4 HBO treatment significantly inhibited the highly expressed inflammatory factors in the injured spinal cord tissue after SCI.

[0032] Figure 5 Protein expressions of STAT3, P-STAT3 and RORγt and gene expressions of STAT3 and RORγt in the injured spinal cord tissue of SCI mice before and after HBO treatment.

[0033] Figure 6 TCRδ - / - Gel electrophoresis results of mouse gene typing.

[0034] Figure 7 Comparison of motor function, ChAT-positive motor neurons and inflammatory factors between WT and TCRδ- / - mice.

[0035] Figure 8 IL-17 - / - Gel electrophoresis results of mouse gene typing.

[0036] Figure 9 Comparison of motor function, ChAT-positive motor neurons and inflammatory factors between WT and IL-17- / - mice.

[0037] Figure 10 mRNA expression levels of each gene in normal controls and spinal cord injury subjects (Control: normal control, SCI: spinal cord injury, ****P<0.0001).

[0038] Figure 11 ROC curve of STAT3 and / or RORγt as a diagnosis for spinal cord injury.

[0039] Figure 12 mRNA expression levels of each gene in spinal cord injury subjects before and after hyperbaric oxygen treatment (SCI: spinal cord injury, HBO: hyperbaric oxygen, ****P<0.0001).

[0040] Figure 13 ROC curve of STAT3 and / or RORγt as a prediction for the efficacy of HBO treatment of SCI injury. Detailed implementation manners

[0041] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is for helping to understand the present invention, but does not limit the present invention. In addition, the technical features involved in the following described implementation manners can be combined with each other as long as they do not conflict with each other.

[0042] In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples are commercially available through conventional channels unless otherwise specified.

[0043] Term

[0044] PVDF (polyvinylidene fluoride) membrane: Most commonly used for protein transfer from gel to membrane in immunoblot (i.e., Western blot), and can also be used for adsorption analysis, amino acid analysis, N-terminal protein sequencing, dot and slot blot detection, glycoprotein chromogenic reaction, and lipopolysaccharide analysis, etc.

[0045] Spinal cord injury (SCI): It is caused by the displacement of vertebral bodies or the protrusion of bone fragments into the spinal canal, resulting in varying degrees of damage to the spinal cord or cauda equina nerve. It is a central nervous system injury with a relatively high disability and fatality rate, and can lead to a series of complications such as varying degrees of limb paralysis, sensory or autonomic function loss, etc.

[0046] Choline acetyltransferase (ChAT): An enzyme that synthesizes the neurotransmitter acetylcholine and is widely used as a marker for cholinergic neurons. In the human basal forebrain tissue, cholinergic neurons also express the low-affinity nerve growth factor receptor p75 and calbindin D28K (D28K).

[0047] Example

[0048] Example 1 Mouse Test Samples

[0049] 1.1 Experimental Mice

[0050] Specific pathogen-free (SPF) grade female C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0051] TCRδ - / - The mice were provided by Shanghai Model Organisms Center, Inc.

[0052] IL-17 - / - The mice were provided by Cyagen Biosciences (Suzhou) Inc.

[0053] The mice were housed in a temperature-controlled negative-pressure animal room where they had easy access to food and water. The mouse experiments were carried out after being approved by the Animal Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University.

[0054] 1.2 Establishment of Mouse SCI Model

[0055] Using the MASCIS III spinal cord injury impactor, a spinal cord impact injury model was made using the modified Allen's method: Experimental mice were anesthetized with chloral hydrate. With the spinous process of T10 as the center of the surgical area, the spinous process of T10 and the lamina were resected to expose the dura mater, and SCI was caused using the MASCIS spinal cord injury impactor. The signs of successful impact were: spinal cord tissue edema, bleeding, and the intact dura mater bulging and showing a purplish-red color; the mice showed a tail-flick reflex, retraction and flapping of both lower limbs, and flaccid paralysis. The mice in the SH group only had their spinal cords exposed and then the wounds were sutured layer by layer without receiving spinal cord impact (Table 1).

[0056] Table 1. Moderate contusion SCI model at the T10 level in WT mice

[0057] Before injury After injury SH group Normal activity Normal activity SCI group Normal activity Complete paralysis of both hindlimbs HBO group Normal activity Complete paralysis of both hindlimbs

[0058] Example 2 Mouse test method

[0059] 2.1 HBO treatment for mice

[0060] The experimental mice in the above HBO group were placed in an animal hyperbaric oxygen chamber for HBO treatment 6 h after modeling. The chamber was washed with pure oxygen, the pressure was increased and maintained for a period of time, the pressure was stabilized at 2 ATA, oxygen was inhaled continuously for several minutes, the pressure was decreased and maintained for a period of time. During HBO treatment, continuous ventilation was maintained, the oxygen flow rate was maintained at 8 - 10 L / min, the oxygen concentration in the chamber was above 95%, once a day.

[0061] 2.2 Hindlimb motor function scoring of mice

[0062] At the corresponding time points after injury, the mice were scored using the Basso Mouse Scale (BMS) and the BMS sub-score to evaluate the recovery of hindlimb motor function in the mice. The BMS score aims to quantitatively evaluate the severity of motor dysfunction, with 0 points representing complete paralysis and 9 points representing normal activity. The BMS sub-score can better distinguish the subtle differences in hindlimb motor function in mice, and the cumulative score ranges from 0 to 11 points. The higher the cumulative score, the more stable the hindlimb trunk of the mouse. The scoring was performed by two researchers who were unaware of the experiment. The mice were observed walking freely in an open area and the behavioral scores were evaluated. The average score of the two researchers was the final score.

[0063] 2.3 Immunofluorescence detection

[0064] Seven days after surgery, the mice were anesthetized by intraperitoneal injection, the heart was exposed, paraformaldehyde was perfused through the left ventricle, the skin on the back of the mice was cut open, other tissues were separated, the spinal cord at the T10 segment was exposed, and the spinal cord tissue containing the injury site was collected.

[0065] Prepare paraffin sections, dewax to water, and perform antigen repair on tissue sections. After serum blocking, add ChAT fluorescent antibody and TCR fluorescent antibody for incubation, wash, and then add goat anti-rabbit IgG H&L fluorescent antibody for incubation. Drop DAPI staining solution, incubate for nuclear counterstaining, mount the slides, take pictures, and calculate the average optical density.

[0066] 2.4 Flow cytometry detection

[0067] Seven days after surgery, anesthetize the mice by intraperitoneal injection. Take spinal cord tissue, prepare single-cell suspension, after cell stimulation, resuspend in staining buffer, and stain with CD45, CD3, gdTCR, and CD4 antibodies (1:100). After cell fixation, perform intracellular staining with fluorescently labeled IFN-γ and IL-17A antibodies (1:100), resuspend the cells, wash, and then perform flow cytometry analysis of the cells on the machine.

[0068] 2.5 ELISA detection

[0069] Seven days after surgery, anesthetize the mice by intraperitoneal injection. Perfuse the left ventricle with normal saline and then take the damaged spinal cord tissue. Use ELISA kits for inflammatory factors IL-17, IFN-γ, IL-1β, IL-6, and TNF-α to measure. Calculate the expression of the above-mentioned inflammatory factors in each sample according to the standard curve.

[0070] 2.6 Gel test

[0071] Used to verify whether the knockout of related genes in mice is successful.

[0072] a. Prepare the gel: Prepare the buffer. Prepare the agarose gel according to the target band, dissolve the agarose in the buffer, heat in a microwave oven until the agarose melts and becomes transparent, add nucleic acid dye, shake well, pour into the electrophoresis template, and insert the comb. Use after condensation.

[0073] b. Loading: Add an appropriate amount of DNA to the agarose gel, and select a suitable DNA marker according to the size of the target band and add it to the gel.

[0074] c. Electrophoresis: Set the voltage to 100V and perform electrophoresis.

[0075] d. Imaging: Observe and take pictures using a gel imaging system.

[0076] 2.7 Determination of protein expression level

[0077] 2.7.1 Protein extraction from spinal cord tissue

[0078] Cut the spinal cord tissue into pieces, homogenize, ultrasonically lyse, centrifuge, and aspirate the supernatant for standby.

[0079] 2.7.2 Determination of protein concentration by BCA method

[0080] Prepare a protein standard solution at a concentration of 25 mg / ml using a protein standard preparation solution. Dilute the protein standard solution to a final concentration of 0.5 mg / ml using lysis buffer and dispense into aliquots.

[0081] Prepare BCA working solution for later use.

[0082] Add the standard to the standard well and the sample to the sample well. At the same time, add BCA working solution and react. Measure the absorbance using an ELISA reader and calculate the protein concentration of the sample based on the standard curve and the volume of the sample used. After the protein concentration measurement is completed, add RIPA and 6×loading buffer, and use a metal bath to denature the protein to prevent its degradation.

[0083] 2.7.3 SDS-PAGE Polyacrylamide Gel Electrophoresis

[0084] Prepare the separating gel: Place the glass plates on the fixing rack, inject the separating gel between the glass plates, add anhydrous ethanol to form a layer, and let it stand. After the separating gel solidifies, pour out the anhydrous ethanol.

[0085] Prepare the stacking gel: Fill the space between the glass plates with stacking gel and insert the corresponding comb, then let it stand.

[0086] After the gel solidifies, place the glass plates in the electrophoresis tank, fill it with electrophoresis buffer, rinse the sample wells with electrophoresis buffer, and keep 1 / 2 electrophoresis buffer outside. Load the protein sample and the marker. Run the upper gel at 80 V, and when the sample migrates into the separating gel, adjust the voltage to 120 V and continue electrophoresis until the sample migrates to the bottom of the separating gel.

[0087] Transfer the membrane using a semi-dry transfer system. After electrophoresis, peel off the gel. Activate a nitrocellulose membrane similar in size to the gel in methanol and place it on a soaked filter paper. Then place the gel on the PVDF membrane, cover it with filter paper, and put it into the transfer tank. Transfer the membrane using a constant current. After the transfer is completed, take out the PVDF membrane and cut the PVDF according to the target protein. Place the PVDF membrane in the blocking solution and block it at room temperature. After blocking, pour out the blocking solution, add the primary antibody diluted with the blocking solution, and incubate. After the incubation of the primary antibody, recover the antibody, add PBST for washing. Add the secondary antibody labeled with HRP diluted with the blocking solution and incubate. After the incubation of the secondary antibody, pour out the antibody, add PBST for washing. After washing, use an ECL detection reagent and add the A and B developers. Cover the developing solution on the PVDF membrane and expose the protein bands using a chemiluminescence imager. Use Image J to measure the protein gray value to calculate the relative protein expression level.

[0088] 2.8 Tissue RNA Extraction

[0089] Cut the spinal cord tissue into tissue fragments. After homogenization, add RNAiso Plus, add chloroform and mix well until it becomes milky, then centrifuge. Aspirate the supernatant, add isopropanol and let it stand, discard the supernatant after centrifugation, and the precipitate is RNA. Centrifuge to wash the precipitate. After drying the precipitate, add an appropriate amount of RNase-free water to dissolve the precipitate, and measure the concentration and purity of RNA.

[0090] 2.9 mRNA Reverse Transcription and qPCR Detection

[0091] 2.9.1 mRNA Reverse Transcription

[0092] The kit used for mRNA reverse transcription is PrimeScriptTM reagent Kit.

[0093] Genomic DNA removal reaction, the reaction system is shown in Table 2, react at 42 °C for 2 min and then place at 4 °C.

[0094] Table 2. Genomic DNA Removal Reaction System

[0095]

[0096] The mRNA reverse transcription system is shown in Table 3.

[0097] Table 3. mRNA Reverse Transcription System (TB Green qPCR Method)

[0098]

[0099] The reverse transcription reaction conditions are: 37 °C for 15 min; 85 °C for 5 sec; 4 °C ∞.

[0100] 2.9.2 PCR Reaction

[0101] GAPDH is the internal reference gene in mRNA detection.

[0102] The kit used for qPCR detection is TB Green Prim mix Ex Taq TM II; The PCR reaction system is shown in Table 4.

[0103] Table 4. qPCR Reaction System (TB Green qPCR Method)

[0104]

[0105] Use the Applied Biosystems 7500Fast Real-Time PCR System to perform the PCR reaction. The PCR reaction program is set as follows in Table 5:

[0106] Table 5. qPCR Reaction Program (Two-step PCR Reaction)

[0107]

[0108] The primer sequences used in the PCR reaction are shown in Table 6 below:

[0109] Table 6. Primers required for qPCR detection

[0110]

[0111]

[0112] Example 3 HBO treatment reduces spinal cord tissue injury

[0113] 3.1 Relative content of γδT cells in spinal cord tissue before and after HBO treatment

[0114] It was confirmed by immunofluorescence detection that γδT cells were present in the injured spinal cord tissue (as shown in Figure 3 A). Flow cytometry analysis showed that after spinal cord injury, compared with 1.17±0.28% in the SH group, the proportion of γδT cells in the injured spinal cord tissue in the SCI group was significantly increased to 6.29±1.2% of CD45+ cells (vs SH, P<0.001). HBO treatment significantly reduced the proportion of γδT cells to 2.45±0.45% (vs. SCI, P<0.001).

[0115] 3.2 Relative content of IL-17+γδT cells in spinal cord tissue before and after HBO treatment

[0116] Analysis of the γδT cell subtypes in the injured spinal cord tissue found that the γδT cells in the injured spinal cord tissue were mainly IL-17+γδT cells.

[0117] In the SCI group, the proportion of IL-17+γδT cells in γδT cells was 9.51±1.28%, which was significantly higher than 4.12±0.68% in the SH group; the proportion of IFN-γ+γδT cells in γδT cells was 3.26±0.53%, which was significantly higher than 1.85±0.38% in the SH group (vs.SH, P<0.001). HBO treatment significantly reduced the proportion of IL-17+γδT cells in γδT cells and the proportion of IFN-γ+γδT cells in γδT cells, which decreased to 5.61±0.98% and 2.17±0.38% respectively (vs.SCI, P<0.001, P<0.01). However, compared with the SH group, the proportion of IL-17+γδT cells in γδT cells in the HBO group was still slightly higher than that in the SH group (vs.SH, P<0.05), while there was no significant difference in the proportion of IFN-γ+γδT cells in γδT cells between the SH group and the HBO group (vs SH, p>0.05) (as shown in Figure 3 C).

[0118] 3.3 Levels of inflammatory factors in spinal cord tissue before and after HBO treatment

[0119] ELISA detection of inflammatory factors was performed using homogenates of injured spinal cord tissue. As Figure 4 shown, the expression levels of IL-17, IFN-γ, IL-1β, IL-6, and TNF-α in the injured spinal cord tissue were significantly upregulated after SCI (vs. SH, P<0.0001). HBO treatment significantly downregulated the expression levels of IL-17, IFN-γ, IL-1β, IL-6, and TNF-α (vs. SCI, P<0.01, P<0.0001), but the expression levels of these 5 cytokines were still higher than those in the SH group (vs. SH, P<0.05, P<0.001, P<0.0001). These results showed that the elevated levels of γδT cells and inflammatory cytokines during the inflammatory response after SCI could be significantly inhibited by HBO treatment (as Figure 4 shown).

[0120] 3.4 Relative protein expression levels of STAT3, P-STAT3, and RORγt before and after HBO treatment

[0121] Protein expression of STAT3, P-STAT3, and RORγt in each group was analyzed by western blotting.

[0122] The relative protein expression levels of STAT3, P-STAT3, and RORγt in the spinal cord tissue after SCI were 1.610±0.064, 1.694±0.033, and 1.398±0.036, respectively, which were significantly higher than those in the SH group (0.929±0.048, 0.635±0.053, 0.973±0.078) (vs. SH, P<0.01, P<0.001, P<0.0001). Compared with the SCI group, HBO treatment significantly reduced the expression of these proteins. The relative protein expression levels of STAT3, P-STAT3, and RORγt in the HBO treatment group were 1.297±0.097, 1.326±0.056, and 0.790±0.036, respectively (vs. SCI, P<0.01, P<0.001) (as Figure 5 shown).

[0123] 3.5 Gene expression levels of STAT3 and RORγt before and after HBO treatment

[0124] The expression level of STAT3 mRNA in the spinal cord tissue after SCI was 3.828 ± 0.662, which was significantly higher than that in the SH group (1.001 ± 0.038) (vs. SH, P < 0.0001). After HBO treatment, its expression level was 2.172 ± 0.635, which was lower than that in the SCI group (vs. SCI, P < 0.001), and still slightly higher than that in the SH group (vs SH, P < 0.01). At the same time, the expression level of RORγt mRNA after SCI was 1.864 ± 0.531, which was also significantly higher than that in the SH group (1.015 ± 0.064) (vs. SH, P < 0.01). And after HBO treatment, the expression level was 0.877 ± 0.286, which was significantly decreased compared with the SCI group (vs. SCI, P < 0.001) (as Figure 5 shown).

[0125] Example 4 Proportion and related traits of TCRδ - / - mouse γδT cells after HBO treatment

[0126] 4.1 Establishment of mouse model

[0127] Table 7. WT mice and mouse models with TCRδ gene knockout

[0128] Before surgery After surgery WT group Normal Complete paralysis <![CDATA[TCRδ - / - group]]> Normal Complete paralysis

[0129] TCRδ - / - mice, that is, TCRδ gene knockout, led to the depletion of γδT cells, Figure 6 for TCRδ - / - mouse gene typing gel electrophoresis results, a single band represents TCRδ - / - .

[0130] 4.2 BMS score and BMS sub-score

[0131] TCRδ - / - and WT mice were completely paralyzed after surgery, and the BMS score and BMS sub-score were both 0. After HBO treatment, the hind limb motor function of the two groups of mice gradually improved. The BMS score and BMS sub-score of TCRδ - / - mice were significantly higher than those of WT mice, and the difference was statistically significant (vs. WT SCI, P < 0.001). And the recovery speed of the injured hind limb motor function of TCRδ - / - mice was also faster. However, HBO treatment had no significant promoting effect on the recovery of the injured motor function of TCRδ - / - mice. There was no statistical difference in the BMS score and BMS sub-score between the TCRδ - / - SCI group and the TCRδ - / - HBO group (as Figure 7 a shown).

[0132] 4.3 Number of ChAT-positive motor neurons in spinal cord tissue

[0133] As Figure 7 shown in b, c, in TCRδ - / - SH mice, the motor neurons in the anterior horn of the spinal cord were arranged orderly. In contrast, in the TCRδ - / - SCI group, the arrangement of ChAT-positive motor neurons was disordered, and the ChAT expression was significantly decreased (vs. TCRδ - / - SH, P < 0.0001), but was significantly higher than that in the WT SCI group (vs. WT SCI, P < 0.0001). However, HBO treatment could not significantly increase the expression of ChAT in the spinal cord tissue of TCRδ - / - mice (vs. TCRδ - / - SCI, P > 0.05).

[0134] 4.4 Expression levels of inflammatory factors in spinal cord tissue

[0135] The expression levels of inflammatory factors in the spinal cord tissue of mice in different groups were detected by ELISA. The results showed that the expression levels of IL-17, IFN-γ, IL-1β, IL-6, and TNF-α in TCRδ - / - SCI mice were significantly lower than those in WT SCI mice (vs. WT SCI, P < 0.0001). Compared with the TCRδ - / - SH group of mice, the expression levels of inflammatory factors in the TCRδ - / - SCI group of mice were significantly increased (vs. TCRδ - / - SH, P < 0.01, P < 0.0001), but there was no significant difference in the levels of inflammatory factors between the TCRδ - / - SCI group and the TCRδ - / - HBO group (vs. TCRδ - / - SCI, P > 0.05) (as Figure 7 shown in d).

[0136] 4.5 Experimental results

[0137] When the γδT cells of mice were depleted, HBO treatment could not significantly improve their motor function after SCI, nor could it significantly reduce the highly expressed inflammatory cytokines induced by SCI, suggesting that γδT cells are one of the key factors in HBO inhibiting the inflammatory response after SCI.

[0138] Example 5 Proportion of IL-17 - / - +γδT cells in mice and related traits after HBO treatment

[0139] 5.1 Establishment of mouse model

[0140] Table 8. WT mice and mice models with IL-17A gene knockout

[0141] Before surgery After surgery WT group Normal Complete paralysis <![CDATA[IL-17 - / - group]]> Normal Complete paralysis

[0142] IL-17 - / - The mice were knocked out of the IL-17A gene, resulting in the absence of IL-17+γδT cells and a decrease in IL-17 secretion during the immune process. Figure 8 For IL-17 - / - Gel electrophoresis results of mouse gene genotyping, a single band represents IL-17 - / - , a double band represents IL-17 - / + . 5.2 BMS score and BMS sub-score

[0143] After spinal cord injury, both IL-17 - / - and WT mice showed complete hindlimb paralysis, and both the BMS score and BMS sub-score were 0. As the motor function gradually improved, the functional recovery of IL-17 - / - mice was significantly faster than that of WT mice (vs. WT SCI, P<0.001, P<0.0001). However, HBO treatment did not significantly accelerate the motor function recovery of IL-17 - / - mice after injury. There was no significant difference in the BMS score and BMS sub-score between the IL-17 - / - SCI group and the IL-17 - / - HBO group (vs. IL-17 - / - SCI, P>0.05) (as shown in Figure 9 a).

[0144] 5.3 Number of ChAT-positive motor neurons in spinal cord tissue

[0145] Evaluate the number of ChAT-positive motor neurons in the spinal cord tissue of mice in different groups. In the IL-17 - / - SH group, the motor neurons in the anterior horn of the spinal cord were arranged neatly. In contrast, the ChAT-positive motor neurons in the IL-17 - / - SCI group were arranged disorderly, and the ChAT expression was significantly decreased (vs. IL-17 - / - SH, P<0.0001). However, the ChAT expression level was still higher than that in WT SCI (vs. WT SCI, P<0.0001). HBO treatment did not significantly increase the ChAT expression in the spinal cord tissue of IL-17 - / - mice (vs. IL-17 - / - SCI, P>0.05) (as shown in Figure 9 b, c).

[0146] 5.4 Expression levels of inflammatory factors in spinal cord tissue

[0147] The expression levels of inflammatory factors in the spinal cord tissues of mice in different groups were detected by ELISA. Through ELISA detection, it was found that IL-17 - / - The expression levels of IFN-γ, IL-1β, IL-6 and TNF-α in mice after SCI were significantly lower than those in the WT SCI group (vs. WT SCI, P<0.0001). Compared with the IL-17 - / - SH group of mice, the expression levels of these 4 inflammatory factors in the spinal cord tissues of mice in the IL-17 - / - SCI group were significantly increased (vs. IL-17 - / - SH, P<0.0001, P<0.001). Similarly, for the IL-17 - / - mice treated with HBO, the levels of inflammatory factors were detected again, and it was found that compared with the IL-17 - / - SCI group, the expression of IL-6 in the IL-17 - / - HBO group was slightly decreased (vs. IL-17 - / - SCI, P<0.05), but there were no significant differences in the expression levels of IFN-γ, IL-1β, TNF-α (vs. IL-17 - / - SCI, P>0.05) (as shown in Figure 9 Figure d).

[0148] 5.5 Results

[0149] After the deletion of IL-17+γδT cells in mice, the levels of highly expressed inflammatory factors induced by SCI were significantly lower than those in WT mice, and the motor function recovered faster. However, HBO treatment could not significantly inhibit the expression of inflammatory factors in these mice after SCI, nor could it significantly promote the recovery of their motor function. These research results indicate that IL-17+γδT cells are involved in the inflammatory response after SCI and are the key target for HBO to inhibit the inflammatory response after SCI.

[0150] Example 8 Detection of the expression genes of STAT3 and RORγt in the blood of spinal cord injury subjects

[0151] 8.1 Sample inclusion

[0152] The research subjects were 18 normal healthy controls and 21 spinal cord injury subjects. The normal healthy controls were employees from Beijing Chaoyang Hospital, Capital Medical University (aged 18 - 75 years old). The subjects were spinal cord injury patients admitted to Beijing Chaoyang Hospital, Capital Medical University from July 2021 to July 2024, aged 18 - 75 years old, with clear history of trauma and surgery, and were diagnosed as spinal cord injury subjects (the diagnosis met the diagnostic criteria formulated by the American Spinal Injury Association), and were confirmed by CT or MRI examinations. Excluded were those with unstable vital signs due to craniocerebral injury or combined thoracoabdominal organ injury, those with contraindications to hyperbaric oxygen therapy, and those with severe heart, liver, and kidney insufficiency.

[0153] 8.2 Hyperbaric oxygen therapy

[0154] All subjects received hyperbaric oxygen therapy after trauma or surgery. It was a multi - person air - pressurized oxygen chamber. After pressurization, the pressure was stabilized at 2 ATA, continuous oxygen inhalation was carried out for a period of time, and decompression was carried out for several minutes. Continuous ventilation was maintained during hyperbaric oxygen therapy, once a day, for 10 consecutive days.

[0155] 8.3 Blood specimen detection

[0156] 8.3.1 Blood specimen collection

[0157] Peripheral venous blood was collected from all subjects before hyperbaric oxygen therapy and after 10 times of hyperbaric oxygen therapy. Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation and lysed.

[0158] 8.3.2 qPCR detection of the expression of genes STAT3 and RORγt in the blood of spinal cord injury subjects

[0159] RNA was extracted and reverse - transcribed into cDNA according to the instructions. The reaction system was prepared to contain TB Premix ExTaq TM II, cDNA, and a pair of primers (as shown in Table 9), and qPCR analysis was performed. The 2 -ΔCT method was used to calculate the relative expression levels of the above - mentioned genes in the blood of spinal cord injury subjects.

[0160] Table 9. Primer sequences required for qPCR detection

[0161]

[0162] 8.4 Comparison of the expression of STAT3 and RORγt genes in peripheral blood between spinal cord injury subjects and normal healthy people

[0163] The qPCR results of peripheral blood showed that the expression levels of STAT3 (4.590 ± 0.770) and RORγt (2.621 ± 0.436) in the blood of spinal cord injury subjects were significantly higher than those of STAT3 (2.793 ± 0.431) and RORγt (1.497 ± 0.215) in the normal population, with statistically significant differences (P<0.0001) (as Figure 10 shown).

[0164] The ROC curve was used to analyze the gene expression levels of STAT3 and / or RORγt as markers for the diagnosis of spinal cord injury (as Figure 11 and Table 10 shown).

[0165] Table 10. Performance of the above genes as diagnostic markers for spinal cord injury analyzed by ROC curve

[0166] Gene AUC(95%CI) Sensitivity(%,95%CI) Specificity(%,95%CI) STAT3 0.992(0.973–1.000) 100.0(83.9–100.0) 94.4(72.9–99.9) RORγt 0.997(0.989–1.000) 95.2(76.2–99.9) 100.0(81.5–100.0) Combined 1.000(1.000–1.000) 100.0(83.9–100.0) 100.0(81.5–100.0)

[0167] 8.5 Gene expression in spinal cord injury subjects before and after hyperbaric oxygen therapy

[0168] The qPCR results showed that after hyperbaric oxygen therapy in spinal cord injury subjects, the gene expression levels of STAT3 and RORγt in their blood were 3.168 ± 0.637 and 1.690 ± 0.384 respectively, which were significantly lower than those before hyperbaric oxygen therapy (4.590 ± 0.770 and 2.621 ± 0.436), with statistically significant differences (P<0.0001) (as Figure 12 shown).

[0169] The ROC curve was used to analyze the gene expression levels of STAT3 and RORγt as markers for predicting the efficacy of HBO in treating SCI injury (as Figure 13 and Table 11 shown).

[0170] Table 11. Performance of the above genes as markers for treatment monitoring analyzed by ROC curve

[0171] Gene AUC(95%CI) Sensitivity(%,95%CI) Specificity(%,95%CI) STAT3 0.909(0.811–1.000) 76.2(52.8–91.8) 100.0(83.9–100.0) RORγt 0.933(0.848–1.000) 81.0(58.1–94.6) 90.5(69.6-98.8) Combined 0.993(0.978–1.000) 100.0(83.9–100.0) 90.5(69.6-98.8)

Claims

1. A biomarker composition for diagnosing spinal cord injury, the biomarker composition consisting of two effective detection components, STAT3 and RORγt. When the gene expression levels of STAT3 and RORγt in the biological sample of a subject are significantly increased compared with those of healthy individuals, the subject is diagnosed with spinal cord injury.

2. The biomarker composition according to claim 1, wherein The biological sample of the subject is selected from one or more of blood, cerebrospinal fluid, and / or spinal cord tissue.

3. The biomarker composition as described in claim 2, wherein The blood is at least one of peripheral blood, plasma, and serum.

4. Use of a detection reagent for the biomarker composition according to claim 1 in the preparation of a reagent for diagnosing spinal cord injury, the detection reagent being a reagent for detecting STAT3 and RORγt; when the gene expression levels of STAT3 and RORγt in the biological sample of a subject are significantly increased compared with those of healthy individuals, the subject is diagnosed with spinal cord injury.

5. The application according to claim 4, wherein The biological sample of the subject is selected from one or more of blood, cerebrospinal fluid, and / or spinal cord tissue.

6. The application according to claim 5, characterized in that, The blood is at least one of peripheral blood, plasma, and serum.

7. A kit for diagnosing spinal cord injury, the kit containing the detection reagent described in claim 4; when the gene expression levels of STAT3 and RORγt in the biological sample of a subject are significantly increased compared with those of healthy individuals, the subject is diagnosed with spinal cord injury.

8. The kit according to claim 7, the kit being an ELISA detection kit and / or a colloidal gold detection kit.

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