An exosome miR-105-5p and its application in the preparation of drugs for treating spinal cord injury.

Abstract

This invention discloses an exosome miR-105-5p and its application in the preparation of drugs for treating spinal cord injury, belonging to the field of molecular biology. The nucleotide sequence of the exosome miR-105-5p is shown in SEQ ID NO: 1. Through the construction of a rat model of spinal cord injury, it was found that the levels of inflammatory factors such as IL-6 in the spinal cord tissue of the model group were significantly increased. Bioinformatics results showed that miR-105-5p can regulate IL-6, and dual-luciferase reporter gene assays confirmed a direct regulatory relationship between the two. In vitro experiments on cultured nerve cells and animal experiments showed that miR-105-5p in exosomes downregulated IL-6 expression. This invention discovers a novel exosome-mediated non-coding RNA-related regulatory pathway in rats with spinal cord injury, providing an experimental basis and research ideas for future drug development, disease diagnosis, and prognosis prediction.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to an exosome miR-105-5p and its use in the preparation of drugs for treating spinal cord injury. Background Technology

[0002] Spinal cord injury (SCI) is a serious central nervous system disorder caused by various factors. It can cause severe sensory, motor, and autonomic dysfunction, characterized by high disability, high mortality, low cure rate, and a high incidence of serious complications. SCI is a complex pathophysiological process involving multiple pathways and factors. It involves not only the primary injury, penetration, or compression that damages fragile nerve tissue, but also subsequent edema, lipid oxidation, inflammation, cytotoxicity, and microglial activation, which can induce secondary injuries. As a result, millions of patients worldwide suffer from limb dysfunction, which not only greatly impacts the quality of life of the patients themselves, but also places a heavy burden on society and families.

[0003] The pathophysiological process of spontaneous intraepithelial neoplasia (SCI) is regulated by multiple pathways and factors, involving various biological factors such as inflammatory factors (e.g., TNF-α, IL, CAM, NF-κB), apoptosis factors (e.g., Bax, Bcl-2, Caspase), and neurotransmitters (e.g., 5-HT, Glu, NA). In recent years, scholars have explored the role of exosomes and their contained miRNAs in SCI, reporting that exosomes derived from mesenchymal stem cells can repair traumatic SCI. Ding et al. found that 78 miRNAs were upregulated and 54 were downregulated in exosomes from a rat SCI model. After screening, the authors considered miR-30b-5p, miR-152-3p, and miR-200c-3p to be specific markers of acute SCI in serum exosomes. Currently, scholars both domestically and internationally generally recognize the important role of inhibiting inflammatory responses in controlling the occurrence and development of SCI, but there are currently no reports of a correlation between miR-105-5p and the treatment of SCI. Summary of the Invention

[0004] The purpose of this invention is to provide an exosome miR-105-5p and its application in the preparation of drugs for treating spinal cord injury, so as to solve the problems existing in the prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an exosome miR-105-5p associated with the treatment of spinal cord injury, the nucleotide sequence of which is shown in SEQ ID NO: 1: 5'-ucaaaugcucagacuccuguggu-3'.

[0007] Preferably, the exosome miR-105-5p achieves the purpose of treating spinal cord injury by downregulating the expression level of IL-6 in the spinal cord tissue.

[0008] The present invention also provides the use of exosome miR-105-5p in the preparation of a drug for treating spinal cord injury, wherein the nucleotide sequence of the exosome miR-105-5p is shown in SEQ ID NO: 1.

[0009] Preferably, the exosome miR-105-5p achieves the purpose of treating spinal cord injury by downregulating the expression level of IL-6 in the spinal cord tissue.

[0010] The present invention also provides the application of exosome miR-105-5p in the preparation of an inhibitor of inflammatory factors in spinal cord injury tissue, wherein the nucleotide sequence of exosome miR-105-5p is shown in SEQ ID NO: 1.

[0011] Preferably, the exosome miR-105-5p downregulates inflammatory factors in the spinal cord injury tissue.

[0012] Preferably, the inflammatory factor is IL-6.

[0013] The present invention discloses the following technical effects:

[0014] This invention explores the role of exosome-mediated miR-105-5p in spinal cord injury (SCI) and its interaction with inflammatory factors. Experiments revealed a significant increase in the levels of inflammatory factors IL-6, IL-16, TNF-α, and NF-κB in spinal cord tissue after SCI, indicating an accompanying inflammatory response. Bioinformatics queries and dual-luciferase reporter gene assays further validated the regulatory relationship between the inflammatory factor IL-6 and miR-105-5p. In vitro experiments with cultured neural cells and animal studies further confirmed that exosomal miR-105-5p can downregulate IL-6 expression, thus having a positive intervention effect on the inflammatory stress following spinal cord injury. This invention discovers a novel exosome-mediated non-coding RNA-related regulatory pathway in SCI rats, providing an experimental basis and research ideas for future drug development, disease diagnosis, and prognosis prediction. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 A diagram showing the structure of exosomes observed under an electron microscope;

[0017] Figure 2 Results of Western blot analysis of exosome marker proteins;

[0018] Figure 3 The results show the determination of the binding site of miR-105-5p to IL-6;

[0019] Figure 4 The effect of miR-105-5p on IL-6 expression;

[0020] Figure 5 Shapes of spinal cord neurons cultured for 48 hours (×200);

[0021] Figure 6 Immunofluorescence staining results for MAP2, a hallmark protein of neuronal dendrites; A: DAPI nuclear staining (×200); B: Immunofluorescence MAP2 antibody staining (×200); C: Composite image (×200);

[0022] Figure 7 The results of Western blot analysis of IL-6 in the spinal cord tissue of rats in each group;

[0023] Figure 8 The expression of IL-6 in the spinal cord tissue of rats in each group was shown; compared with the normal control group, **P<0.01; compared with the model group, ▲▲ P<0.01;

[0024] Figure 9 The apoptosis of nerve cells in each group of rats was observed; compared with the normal control group, ** P<0.01; compared with the model group, ▲▲ P<0.01. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms used in this invention are only for describing specific embodiments and are not intended to limit the invention. Additionally, for the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Experimental materials

[0032] (1) Animals: 40 adult male SPF-grade SD rats, weighing 200 - 240 g, provided by the Henan Provincial Experimental Animal Center, animal license number: SCXK(Yu)2019 - 0002. The animals were housed in the SPF-grade animal breeding room of Henan Orthopaedic Hospital (Luoyang Orthopaedic Hospital of Henan Province). The temperature in the breeding room was 20 - 25 °C, the relative humidity was 50% - 70%, the light and dark periods were each half, the light was from 7:00 to 19:00, and the environmental noise was <50 dB. 4 1-month-old SPF-grade SD rats, half male and half female, provided by the Henan Provincial Experimental Animal Center, animal license number SCXK(Yu)2019 - 0002.

[0033] (2) Drugs and reagents: RNA extraction buffer (Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G3013); RIPA lysis buffer (Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G2002); BCA protein quantitative detection kit (Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G2026); anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10009218); chloroform (Sinopharm Chemical Reagent Co., Ltd., catalog number: 10006818); isopropanol (Sinopharm Chemical Reagent Co., Ltd., catalog number: 80109218); 2×SYBR Green qPCR Master Mix (None ROX) (Wuhan Saiweier Biotechnology Co., Ltd., catalog number: G3320); RT First Strand cDNA Synthesis Kit (Wuhan Saiwell Biotechnology Co., Ltd., Catalog No.: G3330); Phosphorylated Protease Inhibitor (Wuhan Saiwell Biotechnology Co., Ltd., Catalog No.: G2007); SDS-PAGE Gel Preparation Kit (Wuhan Saiwell Biotechnology Co., Ltd., Catalog No.: G2003); Exo-Fect™ Exosome Transfection Kit (System Biosciences, USA, Catalog No.: EXFT10A-1).

[0034] (3) Instruments: Real-time PCR instrument, ABI (Stepone Plus); NanoDrop 2000 spectrophotometer, Thermo Fisher Scientific (NanoDrop 2000); Tabletop high-speed refrigerated microcentrifuge, DragonLab (D3024R); Digital pendulum shaker, Servicebio (DS-S100); Vertical electrophoresis apparatus, Servicebio (SVE-2); Ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd. (JY92-11N); Nanodropone nucleic acid and protein concentration analyzer, Thermo Fisher Scientific (nanodropone); Grayscale analysis software, Alpha Innotech (alphaEaseFC); Image analysis software, Adobe Systems Incorporated (Adobe...). PhotoShop); Chemiluminescence analyzer, Shanghai Qinxiang Scientific Instruments Co., Ltd. (model: 6100); Flow cytometer, Miltenyi GmbH, Germany (model: 130-092-197).

[0035] 2. Experimental methods

[0036] 2.1 Construction of the SCI rat model

[0037] Twenty SD rats were randomly divided into two groups: a normal control group and a model group, with ten rats in each group. The model group animals were intraperitoneally anesthetized with 3% sodium pentobarbital (40 mg / kg), and a spinal cord hemisection method was used to establish the SCI rat model. The wound was sutured, and penicillin sodium (12 U units) was administered intramuscularly for anti-infection treatment. Immediately after the animals regained consciousness from anesthesia, they exhibited tail wagging, bilateral hind limb twitching, or urinary incontinence. Three days after surgery, seven rats in the model group and all rats in the normal control group survived. The degree of muscle spasm was assessed and recorded using the BBB scoring method from J Neurotrauma "BASSO DM, BEATTIEeattie MS, BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats," confirming successful model establishment.

[0038] 2.2 Determination of the content of inflammatory factors in spinal cord tissue

[0039] After anesthetizing rats in both the normal control group and the model group, all rats were sacrificed with cervical dislocation. A portion of the spinal cord tissue from the surgical site was taken, weighed, and homogenized with 9 times the amount of PBS. The tissue was then transferred to a centrifuge at a centrifuge radius of 3000 r / min for 20 min. The supernatant was collected, and the levels of inflammatory factors IL-6, IL-16, TNF-α, and NF-κB were measured according to the instructions of the ELISA kit.

[0040] 2.3 Extraction and identification of plasma exosomes

[0041] Exosomes from rat plasma were obtained by high-speed centrifugation and identified by the following methods: ① measuring exosome particle size; ② observing exosome morphology using transmission electron microscopy; ③ detecting exosome-specific proteins CD9 and TSG101 using Western blot.

[0042] 2.4 Literature search and bioinformatics query

[0043] Existing literature was searched to summarize miRNAs that can regulate the expression of the inflammatory factor IL-6. At the same time, miRDB (http: / / www.mirdb.org / ), TargetScan (https: / / www.targetscan.org / vert-80 / ), and miRWalk (http: / / 129.206.7.150 / ) were searched to collect miRNAs that can regulate the expression of the inflammatory factor IL-6. The intersection of the target miRNAs obtained by the two methods was calculated.

[0044] 2.5 Dual-luciferase reporter gene assay to verify the regulatory relationship between miR-105-5p and IL-6

[0045] Based on the miR-105-5p binding site to IL-6 predicted by a miRNA target gene prediction website, mutant and target sequences were designed. After adding restriction enzyme sites, the target fragment IL-6-3'UTR wild-type (WT, sequence see below) was synthesized by PCR. Figure 3 ) and mutant (MUT, sequence see Figure 3 The miR-105-5pscramble recombinant plasmid was then cloned into the pmirGLO vector. 293T cells were co-transfected with the miR-105-5pscramble negative control and the pmirGLO-IL-6-WT / MUT recombinant plasmid. The control group was co-transfected with either the wild-type or mutant IL-6 recombinant plasmid. After transfection, cells were cultured at 37°C and 5% CO2 for 48 hours, then the medium was replaced with 1 mL of standard DMEM containing 10% fetal bovine serum and no antibiotics, and cultured for another 48 hours to induce cell lysis. After these steps, 5 μL of cell lysate was collected from each group, and fluorescence intensity was measured according to the instructions of the luciferase activity assay kit. Using the Renal globulin fluorescence value as an internal control, the fluorescence intensity of each group was measured using a fluorometer, and the relative fluorescence intensity of each group was calculated (the relative fluorescence intensity of each group is the ratio of its fluorescence intensity value to the Renal globulin fluorescence intensity value). By analyzing and comparing the fluorescence intensity differences between the mutant group and the wild group, the regulatory relationship of miR-105-5p on IL-6 was confirmed.

[0046] 2.6 Culture and Identification of Spinal Cord Neurons

[0047] One-month-old SPF-grade SD rats were sterilized by immersion in 75% ethanol and then placed in PBS in an ice bath to obtain spinal cord tissue for cell culture. After cell adhesion, the culture medium was aspirated, coverslips were washed with PBS, and cells were fixed with 4% PFA (paraformaldehyde solution) at 4°C for 30 min. The cells were then washed three times with PBS, 5 min each time. The slides were removed of water and placed on a support. 50 μL of blocking buffer was added and the cells were blocked for 2 h. Then, 50 μL of primary antibody (diluted 1:100 in PBS) was added, and the cells were incubated overnight at 4°C. The next day, the cells were washed three times with PBS, 5 min each time, followed by the addition of secondary antibody (diluted 1:500 in PBS). The cells were incubated at room temperature in the dark for 2 h, and then washed three times with PBS, 5 min each time, in the dark. DAPI (diluted 1:1000 in PBS) staining was performed for 5 min, followed by three washes with PBS, 5 min each time. Finally, the cells were embedded in Fluoromount-G, and specific proteins were identified by immunofluorescence.

[0048] 2.7 Construction of SCI cell model

[0049] Referring to Huang Yu et al.'s "An Improved Method for Constructing an In Vitro Cell Model of Rat Spinal Cord Injury", rat spinal cord neurons were used at a rate of 1×10⁻⁶. 4 The cells were seeded at a density of / wells in 96-well plates pretreated with lysine, and treated with serum-free cell culture medium containing 700 μM freshly prepared H2O2 for 3 h to construct an in vitro rat SCI cell model.

[0050] 2.8 Exosome Transfection

[0051] According to the instructions of the Exo-Fect™ Exosome transfection kit, miR-105-5p mimics (used to mimic the expression of miR-105-5p in exosomes; this sequence is the synthetic miR-105-5p sequence, synthesized by Hanheng Biotechnology Co., Ltd.) and miR-105-5p inhibitor (used to inhibit the expression of miR-105-5p in exosomes; this sequence is the synthetic miR-105-5p antisense sequence, synthesized by Hanheng Biotechnology Co., Ltd.) were transfected into exosomes, and subsequent experiments were carried out after 48 hours.

[0052] 2.9 Determination of miR-105-5p expression in spinal cord tissue

[0053] Rat spinal cord tissue was collected and placed in a mortar. Trizol reagent was added to extract total RNA from each group of spinal cord tissue. cDNA was prepared by reverse transcription using the Taqman microRNART Kit according to the manufacturer's instructions. The housekeeping gene β-actin was used as an internal control for quantitative real-time PCR amplification to detect the expression level of miR-105-5p. Reaction conditions: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 10 s, 60℃ annealing for 60 s, 40 cycles. Primer sequences are shown in Table 1. (The last sentence appears to be incomplete and possibly refers to a different topic.) －△△ The Ct method represents the relative expression of miR-105-5p.

[0054] Table 1. PCR Primer Sequence List

[0055]

[0056] 2.10 Effect of miR-105-5p in exosomes on IL-6 expression in SCI neurons

[0057] Normal neural cells and SCI neural cells cultured in vitro were added to different types of culture media for further culture, divided into a normal control group (normal neural cells cultured in DMEM medium), a model group (SCI neural cells cultured in ordinary medium), an exo group (SCI neural cells co-cultured with exosomes transfected with miR-105-5p mimics), and an exo inhibitor group (SCI neural cells co-cultured with exosomes transfected with miR-105-5p inhibitor). IL-6 expression in each group was detected using ELISA: the cell suspension was diluted with PBS (pH 7.2-7.4) to a concentration of approximately 1 million / mL. Cells were disrupted and intracellular components released by repeated freeze-thaw cycles or sonication. The cells were then centrifuged at 3000 rpm for 20 minutes at 2-8°C, and the supernatant was carefully collected. IL-6 expression was detected according to the ELISA instructions.

[0058] 2.11 Effects of exosome-mediated miR-105-5p on IL-6 expression in spinal cord tissue of SCI rats and its role in apoptosis

[0059] Twenty SD rats were randomly divided into four groups: a normal control group, a model group, an exo group, and an exo inhibitor group, with five rats in each group. Except for the normal control group, SCI rat models were established in all other groups according to the method described in "Construction of SCI Rat Model". The normal control group and the model group were administered physiological water by gavage. The exo group received a tail vein injection of exosomes transfected with miR-105-5p mimics, and the exo inhibitor group received a tail vein injection of exosomes transfected with miR-105-5p inhibitor. The expression of miR-105-5p in the spinal cord tissue of each group was detected according to the method described in section 2.9 above. IL-6 expression was detected by Western blot, and cell apoptosis was detected by flow cytometry to verify the effect of exosome-derived miR-105-5p on IL-6 expression in the spinal cord tissue of SCI rats and its role in cell apoptosis.

[0060] 2.12 Effects of miR-105-5p in exosomes on spinal cord repair in SCI rats

[0061] Rats were randomly divided into a normal control group, a model group, an exo group, and an exo inhibitor group, with 5 rats in each group. The normal control group was fed normally. Fifteen rats that successfully developed the model were randomly divided into the model group, the exo group, and the exo inhibitor group. The model group received no treatment. The exo group received tail vein injections of plasma exosomes rich in miR-105-5p, while the exo inhibitor group received tail vein injections of plasma exosomes without miR-105-5p. Seven days after injection, the rats were assessed using the BBB score to observe the effect of miR-105-5p on spinal cord repair in SCI rats.

[0062] 2.13 Statistical Methods

[0063] Data was entered using Excel spreadsheets, and statistical analysis was performed using SPSS 22.0. Quantitative data were presented as follows: The comparison between two groups was performed using a t-test, and the comparison between multiple groups was performed using analysis of variance. P < 0.05 indicates that the difference is statistically significant.

[0064] 3. Results and Analysis

[0065] 3.1 Confirmation of successful model establishment and comparison of the content of inflammatory factors in rat spinal cord tissue

[0066] In the normal control group, all rats exhibited normal activity and a BBB score of 20. In the model group, hind limb movement was weak, with scores ranging from 2 to 4 points, a statistically significant difference compared to the normal control group (P<0.01), demonstrating successful model establishment. The levels of inflammatory factors IL-6, IL-16, TNF-α, and NF-κB in the spinal cord tissue of the model group were significantly higher than those in the normal control group (P<0.05, P<0.01, P<0.01, P<0.01), as shown in Table 2.

[0067] Table 2 Comparison of BBB scores and inflammatory factor content in spinal cord tissue among different groups of rats

[0068]

[0069] 3.2 Extraction and identification of plasma exosomes

[0070] The particle size of exosomes in the normal control group and the model group was measured, and the particle size of exosomes in both groups was within the range of 70-120 nm. Electron microscopy revealed that the exosomes exhibited a typical lipid bilayer structure with similarly sized bilayer vesicles, consistent with the typical morphological characteristics of exosomes. Furthermore, Western blot experiments confirmed the positive expression of their marker proteins CD9 and TSG101. Figure 1 and Figure 2 .

[0071] 3.3 Bioinformatics query results

[0072] This invention uses the Rnahybrid website (https: / / bibiserv.cebitec.uni-bielefeld.de / rnahybrid / submission.html / ) to predict the binding sites of the above miRNAs with IL-6. It was found that miR-105-5p binds well with IL-6. Therefore, this invention selects miR-105-5p for further research.

[0073] 3.4 Results of Dual-Luciferase Reporter Gene Experiment

[0074] The prediction results suggest that miR-105-5p and IL-6 have complementary binding sites, see [link to relevant documentation]. Figure 3 The results of dual-luciferase reporter gene assays showed that the relative fluorescence intensity of wild-type IL-6 binding to miR-105-5p was significantly lower than that of the IL-6 mutant group (P<0.01). Figure 4 This indicates that after the mutation of IL-6, fluorescence is released and detected because it no longer binds to miR-105-5p. Before the mutation, wild-type IL-6 binds tightly to miR-105-5p, so fluorescence is not released, resulting in a lower fluorescence value. This suggests that there is a targeted regulatory relationship between IL-6 and miR-105-5p.

[0075] 3.5 Results of culture and identification of spinal cord neurons

[0076] Freshly inoculated cells are dispersed in round shapes, with uniform cytoplasm, translucent cell bodies, and a halo around the periphery; after 2 days, the protrusions elongate. They exhibit distinct nerve cell morphology, as shown in [the image / description]. Figure 5 Immunofluorescence staining showed that both the neuronal dendritic marker MAP2 and the neuronal dendritic protein MAP2 were positive. Figure 6 This indicates that the cultured cells are nerve cells.

[0077] 3.6 Effects of miR-105-5p in exosomes on IL-6 expression in SCI neurons

[0078] The results of the ELISA experiment are shown in Table 3. Compared with the normal control group, the expression of IL-6 in the model group and the exo inhibitor group was significantly increased, and the difference was statistically significant (P<0.01); compared with the model group, the expression of IL-6 in the normal control group and the exo group was significantly decreased, and the difference was statistically significant (P<0.01).

[0079] Table 3. Effects of miR-105-5p in exosomes on IL-6 expression in SCI neurons.

[0080]

[0081] Note: Compared with the normal control group, ★★ P<0.01; compared with the model group, ▲▲ P<0.01.

[0082] 3.7 Effects of exosome-mediated miR-105-5p on IL-6 expression in spinal cord tissue of SCI rats and its role in apoptosis

[0083] The results of the qRT-PCR experiment are shown in Table 4. The expression of IL-6 in the spinal cord tissue of rats in the model group and the exo inhibitor group was significantly higher than that in the normal control group (P<0.01); while the expression of IL-6 in the normal control group and the exo group was significantly lower than that in the model group (P<0.01). Western blot results showed that, compared with the normal control group, both the model group and the exo inhibitor group induced high expression of IL-6, with statistically significant differences (P<0.01); while the expression of IL-6 in the normal control group and the exo group was significantly lower than that in the model group (P<0.01). Figure 7 , Figure 8 Meanwhile, compared with the normal control group, the apoptosis phenomenon was significantly more severe in the model group and the exo inhibitor group (P<0.01), while the apoptosis phenomenon in the exo group was significantly alleviated compared with the model group (P<0.01). Figure 9 .

[0084] Table 4. Effects of miR-105-5p in exosomes on IL-6 expression in spinal cord tissue of SCI rats.

[0085]

[0086] Note: Compared with the normal control group, ★★ P<0.01; compared with the model group, ▲▲ P<0.01.

[0087] 3.8 Effects of miR-105-5p in exosomes on spinal cord repair in SCI rats

[0088] Rats were randomly divided into a normal control group, a model group, an EXO group, and an EXO inhibitor group. Seven days after treatment, the BBB scores of the rats were assessed. The results showed that the EXO group recovered the BBB score for muscle spasticity after spinal cord injury, with a statistically significant difference compared to the model control group (P<0.01). However, the BBB score of the EXO inhibitor group showed no statistically significant difference compared to the model group.

[0089] Table 5. Effects of miR-105-5p-rich plasma exosomes on functional recovery in rats with spinal cord injury.

[0090]

[0091] Note: Compared with the normal control group, ★★ P<0.01; compared with the model group, ▲▲ P<0.01; compared with the exo group, ■■ P<0.01.

[0092] The above results indicate that miR-105-5p can be used to treat spinal cord injury, which is of great significance for the clinical treatment or drug development of spinal cord injury.

[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of exosomes transfected with miR-105-5p in the preparation of drugs for treating spinal cord injury, characterized in that, The nucleotide sequence of miR-105-5p is shown in SEQ ID NO: 1; The exosomes transfected with miR-105-5p achieve the purpose of treating spinal cord injury by downregulating the expression level of IL-6 in the spinal cord tissue.

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