Application of bone marrow stem cell engineered exosome containing BDNF gene in treatment of spinal cord injury

Abstract

The invention provides application of a bone marrow stem cell engineered exosome containing a BDNF gene in treatment of spinal cord injury, and belongs to the technical field of biology. Through an endogenous transfection technology, mRNA and protein of the BDNF are loaded into the exosome, and the bone marrow stem cell engineered exosome for endogenous carrying of the brain-derived neurotrophic factor BDNF is constructed for the first time. The bone marrow stem cell engineered exosome containing the BDNF gene has dual treatment effects of a natural exosome and BDNF, and can recover spinal cord tissue injury, and compared with the morphological structure of a normal spinal cord part, the recovery rate is close to 90%. Moreover, the side effects and limitations of the original drug treatment and surgical treatment are overcome, the recovery of the motion function of the spinal cord injury is remarkably promoted, a good treatment effect can still be kept on the 28th day of the spinal cord injury, and the problem that no drug or surgical method can be used for completely treating the spinal cord injury at present is solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of bone marrow stem cell engineered exosomes containing the BDNF gene in treating spinal cord injury. Background Art

[0002] SCI is defined as spinal cord injury that can cause temporary or permanent changes in spinal cord function, and SCI has a high incidence, high cost, high disability rate, and a low age of onset. SCI can be caused by high-intensity injuries such as traffic accidents, falls and violent injuries, or by infections, tumors, spinal degenerative diseases, ischemia-reperfusion injury and vascular causes. The annual incidence of SCI is approximately 17,000 people per year, and the first-year cost of a high-level quadriplegic patient exceeds US$1 million. There are approximately 930,000 new cases of spinal cord injury caused by accidents each year. Severe SCI is a huge physical, psychological and economic burden on patients and their families.

[0003] Although systemic methylprednisolone sodium succinate (MPSS) is the only approved treatment for acute SCI, its limited efficacy and severe side effects severely limit its clinical application. Surgery for spinal cord injury also serves only to decompress the injured area, with minimal efficacy.

[0004] Currently, spinal cord injuries can be treated with surgical procedures such as laminectomy and dural sac expansion and repair. Laminectomy primarily involves removing the diseased lamina, bone, and cartilage endplates to relieve spinal cord compression and restore spinal cord function. It is suitable for patients in the early stages of spinal cord injury, without significant spinal canal stenosis, and with mild spinal cord injury. Dural sac expansion and repair primarily involves surgically expanding the spinal cord and dural sac to achieve the desired effect. It is suitable for patients with dural sac compression but mild spinal cord impairment. However, surgical treatment of spinal cord injury disrupts the original physiological structure between the spine and spinal cord and carries significant risks, such as nerve damage. Improper surgical handling can damage the cervical nerves, leading to numbness, weakness, or even paralysis in the patient's limbs. Improper postoperative care can also allow pathogens to enter, causing wound infection and impaired wound healing.

[0005] Intravenous administration of mesenchymal stem cells (MSCs) has emerged as a novel approach to promote functional recovery after spinal cord injury. Stem cell therapy involves transplanting various stem cell types into the damaged area to address issues such as cell viability, cell retention at the lesion site, a supportive physical matrix, filling the lesion cavity, and mediating directional growth. Among various stem cell types, MSCs are widely used in cell therapy due to their ability to self-renew and differentiate into multiple lineages, which presents no ethical concerns. MSCs also offer the advantages of low immunogenicity and diverse sources. However, the lungs have the ability to clear over 60% of administered MSCs, resulting in a reduction in the number of MSCs available to host and exert therapeutic effects at the injury site. Although some cells in the lungs may survive for 2–3 days and release exosomes that can travel to the target injury site, patients may experience coughing as a side effect. However, the neuroinflammatory microenvironment of spinal cord injury lesions has been shown to be detrimental to the survival and function of MSCs, posing a significant obstacle to the clinical application of these cells.

[0006] Furthermore, based on stem cells, more and more studies have confirmed that mesenchymal stem cells mainly exert their therapeutic ability through their paracrine effects. Paracrine effects are also considered to be an important link in intercellular communication in maintaining the balance of the intracellular environment and the progression of disease.

[0007] Among these, exosomes play a significant role in the paracrine effects of cells. MSC-secreted exosomes (MSC-Exos), with diameters ranging from 30 to 100 nm, originate from the endosomal pathway. Inward budding of the endosomal membrane leads to the formation of intraluminal vesicles, which are then released as exosomes. Mesenchymal stem cell exosomes offer advantages such as ease of storage, stability, generally no immune rejection, and non-tumorigenicity. They serve as important mediators of intercellular communication, capable of targeted activation of downstream, long-range signaling molecules. They are released by cells through direct extrusion of the plasma membrane. EVs carry various RNA and protein molecules, such as mRNA and miRNA, as well as trophic factors. These molecules can be functionally transferred to recipient cells and are believed to play important roles in (patho)physiological processes. Exosomes can be used to promote functional recovery after spinal cord injury. However, while exosomes offer potential as therapeutic agents, their low loading capacity, difficulty in tracking in vivo, and inherent lack of functional specificity significantly limit their potential for clinical application.

[0008] Therefore, how to improve the therapeutic effect of exosomes by adjusting them is crucial for the treatment of spinal cord injury. Summary of the Invention

[0009] The purpose of the present invention is to provide an application of bone marrow stem cell engineered exosomes containing the BDNF gene in the treatment of spinal cord injury. The novel bone marrow stem cell engineered exosomes containing the BDNF gene prepared by the present invention can reduce the expression of inflammatory factors, promote the expression of anti-inflammatory factors, have excellent anti-inflammatory effects, and have good biocompatibility and low toxicity, and can repair spinal cord tissue and nerve function atrophied due to injury.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides an application of bone marrow stem cell engineered exosomes containing the BDNF gene as a new drug for treating spinal cord injury. The bone marrow stem cell engineered exosomes containing the BDNF gene can overexpress brain-derived neurotrophic factor (BDNF), thereby treating spinal cord injury.

[0012] The beneficial effects of the present invention compared with the prior art are:

[0013] (1) The present invention uses endogenous transfection technology to load BDNF mRNA and protein into exosomes, and for the first time constructs bone marrow stem cell engineered exosomes for endogenous delivery of brain-derived neurotrophic factor (BDNF).

[0014] (2) The engineered exosomes from bone marrow stem cells containing the BDNF gene of the present invention have the dual therapeutic effects of natural exosomes and BDNF, restoring spinal cord tissue damage, reducing vacuolation, and making the spinal cord tissue more intact. Compared with the morphological structure of the normal spinal cord, the recovery rate is close to 90%. In addition, the present invention overcomes the side effects and limitations of existing drug and surgical treatments, repairing spinal cord tissue and nerve function atrophied by injury, and significantly promoting the recovery of motor function in spinal cord injury to a level close to that before injury. The treatment can still maintain a good therapeutic effect on the 28th day after spinal cord injury, solving the problem that there are currently no drugs or surgical methods that can completely treat spinal cord injury.

[0015] (3) The bone marrow stem cell engineered exosomes containing the BDNF gene of the present invention can completely repair damaged neurons, solving the problem that the existing technology cannot completely repair damaged neurons, and has achieved extremely significant progress.

[0016] (4) The engineered exosomes of bone marrow stem cells containing the BDNF gene of the present invention can reduce the expression of inflammatory factors IL-6, IL-1β and TNF-α, promote the expression of anti-inflammatory factors, have a good anti-inflammatory effect, and have good biocompatibility and low toxicity, are non-toxic to cells, and can improve cell viability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a flow chart for the preparation of engineered exosomes of the present invention;

[0019] Figure 2 The transfection and identification results of engineered exosomes in Experimental Example 1, where A is the BCA assay for protein content in exosomes; B is the particle size assay for exosomes; C is the Western blot assay for exosome marker proteins; and D is the exosome marker.

[0020] Figure 3 The identification results of engineered exosomes in Experimental Example 1, where A is the immunofluorescence assay for bone marrow stem cell transfection efficiency; B is the Real-Time PCR assay for engineered exosome construction; C is the Western blot assay for engineered exosome construction; and D is a quantification graph of Western blot results.

[0021] Figure 4 This is the exosome absorption by BV2 microglia in Experimental Example 1;

[0022] Figure 5 The CCK8 method in Experimental Example 1 was used to detect the cytotoxicity of engineered exosomes overexpressing BDNF;

[0023] Figure 6 The expression changes of inflammatory factors and M1 / M2 polarization in BV2 microglia detected by Q-PCR in Experimental Example 1, where A is the expression level of ARG-1, B is the expression level of iNOS, C is the expression level of TNF-α; D is the expression level of IL-1β; E is the expression level of IL-6;

[0024] Figure 7 This is a diagram of the process of constructing the rat spinal cord injury model in Experimental Example 1;

[0025] Figure 8 This is the result of in situ hybridization of rat spinal cord injury tissue in Experimental Example 1;

[0026] Figure 9 This is the nuclear magnetic resonance examination of the rat spinal cord tissue in Experimental Example 1 (horizontal plane);

[0027] Figure 10 This is the MRI examination of the rat spinal cord tissue in Experimental Example 1 (sagittal plane);

[0028] Figure 11 This is the immunohistochemistry result of the rat spinal cord injury tissue in Experimental Example 1;

[0029] Figure 12 The ELISA test results of IL-6, IL-1β, and TNF-α expression in rat blood in Experimental Example 1;

[0030] Figure 13 This is the HE staining result of the injured spinal cord tissue of the rat in Experimental Example 1;

[0031] Figure 14 This is the electrophysiological result of MEPs in rats in Experiment 1;

[0032] Figure 15 HE staining of the bladder of rats in each group of Experimental Example 1;

[0033] Figure 16 The Nissl staining results of the spinal cord injury sites of rats in each group of Experimental Example 1;

[0034] Figure 17 This is the result of rat gait analysis in Experiment 2;

[0035] Figure 18 This is the result of the rat swimming experiment in Experimental Example 2;

[0036] Figure 19 This is Experimental Example 3: Effect of Exos-BDNF on the expression of BDNF, TrKB, p-PI3K, and p-Akt proteins in injured spinal cord tissue of SCI rats. DETAILED DESCRIPTION

[0037] 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.

[0038] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The present invention provides an engineered exosome, referring to Figure 1 The preparation process is as follows:

[0043] (1) Human bone marrow mesenchymal stem cell complete culture medium (Fuheng, PYH094) was used to seed bone marrow mesenchymal stem cells into six-well plates for culture.

[0044] (2) When the bone marrow mesenchymal stem cells were cultured to a cell density of 30%-50%, the recombinant lentivirus pLV[Exp]-EGFP / Puro-EF1A>hBDNF (Yunzhou Biological, NM_001143810.2) was inoculated into the culture medium. On the second day, the medium was changed, the culture medium containing the virus was aspirated, and fresh human bone marrow mesenchymal stem cell complete culture medium was added to change the medium.

[0045] (3) Continue culturing for more than 3 days until cell phenotype changes occur. During the culture process, 2 mL of human bone marrow mesenchymal stem cell complete medium supplemented with 2.5 μg / mL puromycin (Solarbio, P8230) is used to update the culture medium every other day. After screening bone marrow stem cells containing the target gene, the cells are expanded and cultured with human bone marrow mesenchymal stem cell complete medium without puromycin for subsequent extraction of endogenous engineered exosomes.

[0046] Example 1

[0047] Example 1 of the present invention provides a method for preparing engineered exosomes, which specifically includes the following steps:

[0048] (1) Human bone marrow mesenchymal stem cell complete culture medium (Fuheng, PYH094) was used to seed bone marrow mesenchymal stem cells into six-well plates for culture.

[0049] (2) When the bone marrow mesenchymal stem cells were cultured to a cell density of 30%-50%, the recombinant lentivirus pLV[Exp]-EGFP / Puro-EF1A>hBDNF (Yunzhou Biological, NM_001143810.2) was inoculated into the culture medium. On the second day, the medium was changed, the culture medium containing the virus was aspirated, and fresh human bone marrow mesenchymal stem cell complete culture medium was added to change the medium.

[0050] (3) Continue culturing for more than 3 days until cell phenotype changes occur. During the culture process, 2 mL of human bone marrow mesenchymal stem cell complete medium supplemented with 2.5 μg / mL puromycin (Solarbio, P8230) is used to update the culture medium every other day. After screening bone marrow stem cells containing the target gene, the cells are expanded and cultured with human bone marrow mesenchymal stem cell complete medium without puromycin for subsequent extraction of endogenous engineered exosomes.

[0051] (4) The supernatant of bone marrow stem cells containing the target gene was collected and centrifuged three times using a horizontal centrifuge (himac, CP100NX), first at 2000 rpm for 5 min and then at 5100 rpm for 20 min to remove cells and large cell debris, and the supernatant was collected.

[0052] (5) Using an ultrahigh-speed centrifuge, centrifuge at 11300 r / min for 90 min, remove the supernatant, and resuspend the precipitate with an appropriate amount of PBS. Centrifuge at 35700 r / min for 120 min, remove the supernatant, and resuspend the precipitate with a small amount of PBS to separate the exosomes, which are the engineered exosomes.

[0053] Test Example 1

[0054] Test Example 1 of the present invention identified the engineered exosomes prepared in Example 1, specifically including:

[0055] (1) Protein concentration was determined by BCA (Solarbio PC0020), particle size was determined by NTA (Brookhaven Instruments Omni), transmission electron microscopy (JEOL-2100Plus, Japan Electron), Western blot (Bio Red), and real-time fluorescence quantitative PCR (Real-Time PCR System (C1000 TM Thermal Cycler) experiment was used to identify marker proteins and BDNF to identify the engineered exosomes of Example 1. The results are as follows Figure 2 、 3 shown.

[0056] Figure 2 BCA assays showed that the protein content of the exosomes extracted in Example 1 was 1-2.5 mg / ml, and the average exosome particle size was approximately 90-100 nm. Western blot results showed that the exosome markers TSG101, Alix, and CD63 were all positive. These results indicate that the engineered exosomes isolated from bone marrow stem cells in this invention are highly pure and meet the characteristics of exosomes.

[0057] Figure 3 Immunofluorescence revealed that bone marrow stem cells successfully transfected with BDNF exhibited green fluorescence. Real-time PCR revealed significantly elevated BDNF expression in the engineered exosomes. Western blot analysis revealed significantly increased BDNF protein expression in the engineered exosomes, reaching five times the level of BDNF expressed in standard exosomes. These results demonstrate that both BDNF mRNA and protein were successfully transfected into bone marrow mesenchymal stem cells and packaged into exosomes.

[0058] (2) Verification of cytotoxicity and anti-inflammatory effects of engineered exosomes

[0059] A. BV2 microglia phagocytize exosomes

[0060] Staining reaction: Mix the cell culture supernatant with PKH26 staining solution, mix thoroughly and incubate for a certain period of time to allow the fluorescent dye to bind to the surface of the exosomes. PKH26 exosome staining is a commonly used technique for marking the membrane surface of exosomes. Exosomes are a type of small vesicles secreted by cells that contain a variety of biologically active substances, such as proteins, nucleic acids, and lipids, which can transmit signals in vivo and in vitro and participate in intercellular communication. Through PKH26 staining, the surface of exosomes can be marked to produce a red fluorescent signal, which facilitates the tracking and study of exosomes. The results are as follows Figure 4 The specific steps are as follows:

[0061] ① Cell culture: First, collect the cell culture supernatant to be studied, which contains exosomes.

[0062] ② Prepare PKH26 staining solution: Mix PKH26 dye with the corresponding labeling solution and adjust the dye concentration according to the number and concentration of exosomes.

[0063] ③Terminate staining: Add buffer to terminate the staining reaction to stop further binding of the fluorescent dye.

[0064] ④ Washing and precipitation: Wash the labeled exosomes with an appropriate buffer to remove unbound fluorescent dye.

[0065] PKH26 is a red fluorescent dye that can stably bind to the lipid region of the cell membrane and emit red fluorescence. The absorption of exosomes by BV2 cells was observed using a laser confocal microscope. Figure 4 It can be seen that BV2 microglia absorbed exosomes very well.

[0066] B. CCK8 assay to detect the toxicity of engineered exosomes on BV2 microglia

[0067] The cell experiments were divided into five groups: CON group (untreated BV2 cell control), LPS group (1 μg / ml LPS-treated BV2 cells), Exos group (common Exos-treated BV2 cells in the inflammatory model), Exos-NC group (no Exos-treated BV2 cells in the inflammatory model), and Exos-hBDNF group (engineered Exos-overexpressing BDNF-treated BV2 cells in the inflammatory model).

[0068] In order to detect the toxicity of engineered exosomes on BV2 microglia, the CCK8 method was used. Untreated normal BV2 microglia were used as the control group, and a BV2 microglia inflammation model was constructed with LPS (1 μg / ml) as the inflammation model group. BV2 microglia after inflammation were treated with ordinary exosomes, empty exosomes, and BDNF engineered exosomes as three treatment groups. Cell suspensions were prepared and inoculated into 96-well plates. CCK-8 reagent was added at 24 h, 48 h, and 72 h, respectively. The absorbance at 450 nm was measured after 1-4 h of culture to detect the cytotoxicity of the engineered exosomes. The results are shown in Figure 3. Figure 5 shown.

[0069] Figure 5 The results of CCK8 cytotoxicity analysis showed that neither engineered exosomes overexpressing the BDNF gene (Exo-hBDNF) nor empty exosomes (Exo-NC) had significant cytotoxicity, and after treatment with BDNF engineered exosomes, cell viability was significantly increased compared with that after treatment with ordinary exosomes and empty exosomes.

[0070] C. Real-time PCR detection of inflammatory factors and polarization of BV2 microglia

[0071] ① Using TRNzol Universal RNA Reagent (Tiangen Y2115), untreated normal BV2 microglia served as the control group. A BV2 microglial inflammation model was constructed using LPS (1 μg / mL) as the inflammation model group. Inflammatory BV2 microglia were treated with conventional exosomes, empty exosomes, and BDNF-engineered exosomes, and total RNA was extracted from the three treatment groups. For specific steps, please refer to the reagent instructions.

[0072] ② Use the miRNA cDNA reverse transcription kit (Tiangen 2052) to reverse transcribe the extracted RNA into cDNA. For specific steps, please refer to the reagent instructions.

[0073] ③ Using real-time fluorescence quantitative PCR (Thermal Cycler C1000 TM ) analyzed the expression changes of inflammatory factors in engineered exosomes and the polarization of BV2 microglia. Figure 6 shown.

[0074] result:

[0075] The cell experiments were divided into five groups: CON group (untreated BV2 cell control), LPS group (1 μg / ml LPS-treated BV2 cells), Exos group (common Exos-treated BV2 cells in the inflammatory model), Exos-NC group (no Exos-treated BV2 cells in the inflammatory model), and Exos-hBDNF group (engineered Exos-overexpressing BDNF-treated BV2 cells in the inflammatory model).

[0076] Figure 6 Figures A, B, and C respectively tested the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α. As can be seen from the figure, ordinary exosomes, empty exosomes, and engineered exosomes overexpressing BDNF can all reduce the expression levels of cellular proinflammatory factors and play an anti-inflammatory role. In addition, engineered exosomes overexpressing BDNF have a better anti-inflammatory effect than the other two exosomes. Figure 6 Figures D and E detected the expression levels of the M2 polarization marker Arg-1 and the M1 polarization marker iNOS, respectively. It can be seen that ordinary exosomes, empty exosomes and engineered exosomes overexpressing BDNF can all transform BV2 microglia from M1 (pro-inflammatory) phenotype to M2 (anti-inflammatory) phenotype, playing an anti-inflammatory role. In addition, engineered exosomes overexpressing BDNF have a better effect in promoting phenotypic transformation than the other two exosomes.

[0077] Test Example 2

[0078] Experimental Example 2 of the present invention tested the anti-inflammatory effect of the engineered exosomes prepared in Example 1 on the spinal cord injury site and the repair effect on the morphology of the injury site and neural function. The specific steps are as follows:

[0079] (1) Construction of spinal cord injury animal model and drug administration:

[0080] Six-week-old healthy female SPF rats were randomly divided into Sham group (sham operation group), SCI group (spinal cord injury model group), SCI+Exos group (normal exosome injection group), SCI+Exos-NC group (exosome empty load injection group) and SCI+Exos-hBDNF group (engineered exosome overexpressing BDNF injection group), a total of five groups, each with 6 rats. The rats in the model group were anesthetized with isoflurane, the back hair was trimmed and disinfected; an incision of about 2 cm was made at the 10th thoracic vertebra, the vertebral plate was cut and the spinal cord was exposed; a 10G solid ball was used to hit the rat spinal cord from a height of 10 cm, and the spinal cord was observed to be congested and edematous, and the rats had tail spasms and paralysis of the right lower limb, indicating that the modeling was successful. The modeling process is as follows Figure 7 The muscles and skin were sutured sequentially as shown, and penicillin was administered postoperatively to prevent infection. Rats in the sham group underwent laminectomy of the 10th thoracic vertebra only. Two hours after modeling, drugs were administered according to the method shown in Table 1.

[0081] Table 1 Dosage

[0082]

[0083]

[0084] (2) In situ hybridization assay to detect the therapeutic effect of engineered exosomes:

[0085] 1) Prepare the probe (brain-derived neurotrophic factor (Bdnf), transcript variant 1, mRNA), Probe sequence (SEQ ID NO. 1):

[0086] 5′-GGTTGACAGTATTAGCGAGTGGGTC-3′;

[0087] 2) Take a larger humidified box and place the slices crosswise;

[0088] 3) Drop 10 μL of probe on the sliced ​​tissue and cover with a glass slide;

[0089] 4) Cover the humidified chamber and incubate at 37°C for 12 to 16 hours.

[0090] 5) Post-hybridization wash: Carefully remove the coverslip with tweezers; wash the sections with 40 ml of preheated post-hybridization wash solution at 43°C for 15 minutes.

[0091] 6) Wash twice in 2×SSC (37°C), 10 min each time;

[0092] 7) Place the slices in 1×PBS in a staining jar for testing. Do not allow the slices to dry out.

[0093] 8) Detection: Remove the sections from the 1× PBS and remove excess water to prevent drying. Place the sections in a humidified chamber and process four sections simultaneously.

[0094] 9) Incubate each section with 30 μL to 60 μL of rhodamine anti-digoxigenin antibody at room temperature for 20 min.

[0095] 10) Remove the plastic cover and place the sections in a staining jar containing 1× PBS. Wash three times with 1× PBS at room temperature, 2 minutes each time.

[0096] 11) Remove the slices from the 1× PBS and tilt them to allow the liquid to drain.

[0097] 12) Add DAB color development solution / DAPI staining solution to each section, cover with a coverslip and incubate at room temperature for 2-5 minutes;

[0098] 13) Use hematoxylin to stain, alcohol to dehydrate, xylene to make it transparent, and then seal the slides with resin and take photos under a microscope. Or seal the slides with anti-fading mounting medium and take photos under a fluorescence microscope. The results are as follows: Figure 8 shown.

[0099] Depend on Figure 8 It can be seen that after spinal cord injury in animals, the SCI group experienced a significant loss of trophic factors at the injury site. After exosome treatment, trophic factor expression increased, but after engineered exosome treatment, BDNF accumulated significantly at the injury site. This suggests that the therapeutic effect in injured rats is due to the injection of engineered exosomes.

[0100] (3) Anti-inflammatory effect of engineered exosomes and their effect on the morphological repair of spinal cord injury sites:

[0101] A. Nuclear magnetic resonance imaging

[0102] After spinal cord injury in rats, severe local inflammation and spinal cord atrophy occurred. Two hours after modeling, the rats were treated with engineered exosomes and subjected to nuclear magnetic resonance imaging (Aspect Imaging LTD, Israel) to detect the recovery of spinal cord tissue injury and inflammation. HE staining was also performed on the spinal cord tissue injury site to observe the integrity of the spinal cord structure. Figure 9 、 10 shown.

[0103] Magnetic resonance imaging (MRI) was performed on the rats in each group to detect the level of inflammation and tissue recovery at the injury site. Figure 9 、 10The results showed that there was no obvious luminescence in the spinal cord of rats in the Sham group (luminescence indicates inflammation), and the spinal cord structure was intact. The spinal cord tissue of rats in the SCI group was significantly atrophied, the diameter was reduced, and the luminescence was obvious, indicating that they had severe inflammation. The rats treated with ordinary exosomes and empty exosomes had their spinal cord atrophy restored, and the inflammation was alleviated compared to the SCI group. Exos-hBDNF is a rat treated with engineered exosomes. It can be clearly seen from the figure that after treatment with engineered exosomes, the spinal cord tissue injury of the rats was restored to its original state, with a smooth morphological structure. Compared with the morphological structure of the normal spinal cord, the recovery was close to 90%, close to the Sham group, and there was no obvious inflammatory luminescence. This shows that engineered exosomes have excellent anti-inflammatory effects and repair functions on the morphology of the spinal cord injury site.

[0104] B. Immunohistochemistry

[0105] Immunohistochemistry (IHC) is a method that uses the principle of antigen-antibody reaction, that is, the specific binding of antigen and antibody, to identify antigens (peptides and proteins) in tissue cells through chemical reactions to make the color developing agent (fluorescein, enzyme, metal ion, isotope) labeled with antibody develop color, and to conduct localization, qualitative and quantitative research on them. Figure 11 The method is as follows:

[0106] 1) Dewaxing: 60°C, 30 min; xylene I 10 min, xylene II 10 min;

[0107] 2) Hydration: Dewaxed sections were hydrated with 100% alcohol, 95% alcohol, 85% alcohol, 75% alcohol, and double-distilled water for 3 min each.

[0108] 3) Antigen retrieval: Retrieve in 0.01M sodium citrate buffer at high pressure for 20-30 minutes. Avoid boiling the solution during microwave heating and allow it to cool naturally. Wash with 0.02M PBS for 3 minutes three times.

[0109] 4) Blocking: Add 3% H2O2 to block endogenous peroxidase and incubate in a wet chamber for 10 minutes. Wash with 0.02M PBS for 3 minutes x 3 times;

[0110] 5) Blocking: Remove PBS, add non-immune, normal goat serum to block nonspecific antigens, and incubate at 37°C for 30 minutes;

[0111] 6) Wash with PBS three times, 3 minutes each time, add primary antibody (select the optimal dilution ratio), and incubate at 4°C overnight. Remove and place at room temperature for 40 minutes, then wash with PBS three times, 3 minutes each time. Add HRP-conjugated secondary antibody (rabbit anti-antibody) and incubate at 37°C for 60 minutes.

[0112] 7) Wash with PBS three times, 3 min each time; stain with DAB, rinse with tap water, counterstain with hematoxylin, differentiate with 0.1% hydrochloric acid alcohol, and observe under a microscope to control the degree of staining.

[0113] 8) Dehydration: 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol. Gradient dehydration, 3 min each.

[0114] 9) Transparency: Use xylene to make it transparent for 3 minutes x 2 times.

[0115] 10) Seal the slides with neutral gum.

[0116] The immunohistochemical experiment of the present invention detected three indicators, namely IL-6, TNF-α, and IL-10. Figure 11 It can be seen that IL-6, TNF-α, and IL-10 were diffusely distributed in the surrounding tissues of the injury under the microscope, and the positive expression was brown. IL-6 and TNT-α are pro-inflammatory factors. The IL-6 and TNF-α values ​​of the SCI group were significantly higher than those of the other groups. After treatment with Exos and Exos-NC, the values ​​of IL-6 and TNF-α decreased. After treatment with Exos-hBDNF, the values ​​of IL-6 and TNF-α decreased significantly. IL-10 is an anti-inflammatory factor. The IL-10 expression level in the SCI group was significantly lower than that of the other groups. After treatment with Exos and Exos-NC, the value of IL-10 increased. After treatment with Exos-hBDNF, the value of IL-10 increased significantly. This shows that engineered exosomes have more orderly anti-inflammatory ability than ordinary exosomes.

[0117] C. Elisa test

[0118] Blood samples from rats in each group were taken for Elisa test to detect three pro-inflammatory factors, namely IL-6, IL-1β, and TNF-α. The results are as follows Figure 12 The steps of Elisa detection are as follows:

[0119] 1) Sample Addition: Set 10 wells at 5 concentrations of the standard, with replicate wells for each concentration. Add 50 μl of each standard at different concentrations. Set one blank well to add 50 μl of distilled water (blank control wells do not add sample or enzyme-labeled reagent; all other steps remain the same). For the test sample wells, first add 40 μl of sample diluent to the test sample wells on the enzyme-labeled plate, then add 10 μl of the test sample (final sample dilution is 5x). Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix.

[0120] 2) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0121] 3) Liquid preparation: dilute 30 (20 times of 48T) times of concentrated washing solution with 30 (20 times of 48T) times of distilled water and set aside.

[0122] 4) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds, then discard, repeat this 5 times, and pat dry.

[0123] 5) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0124] 6) Incubation: Same as step 2).

[0125] 7) Washing: The operation is the same as step 4).

[0126] 8) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0127] 9) Termination: Add 5 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0128] 10) Measurement: Use the blank well as the zero setting and measure the absorbance (OD) of each well at 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0129] Depend on Figure 12 It can be seen that the levels of three proinflammatory factors IL-6, IL-1β, and TNF-α in the blood of rats in the Sham group were the lowest. After spinal cord injury modeling, the expression levels of the three inflammatory factors in the blood of rats in the model group were significantly increased. After Exos and Exos-NC treatment, the three proinflammatory factors in the blood of rats were significantly reduced, but after engineered exosome treatment, the three proinflammatory factors had a more significant reduction effect than ordinary exosome treatment.

[0130] D. HE staining

[0131] The spinal cord tissue injury sites of rats in each group were taken for HE staining. Figure 13 As shown, the injured area of ​​rat spinal cord tissue was observed under 4x, 20x, and 40x microscopes, respectively.

[0132] Depend on Figure 13 It can be seen that the spinal cord tissue structure of rats in the Sham group was intact without obvious vacuoles. A large number of vacuoles appeared in the tissues of rats in the SCI group, the spinal cord tissue was severely damaged, and the structure was destroyed. After Exos and Exos-NC treatment, the vacuoles in the spinal cord tissue were reduced. After treatment with engineered exosomes Exos-hBDNF, the spinal cord tissue recovered to a level similar to that before injury, with fewer vacuoles than those treated with ordinary exosomes, and the spinal cord tissue was more intact.

[0133] (4) Repair of neural function at the site of spinal cord injury by engineered exosomes

[0134] A. Electrophysiological testing

[0135] Electrophysiological testing was performed on rats after spinal cord injury. Under general anesthesia, the rats underwent craniotomy and then implanted stimulating electrodes at AP-1mm, ML±2 mm and AP+4 mm, ML±2 mm. At the same time, two hook electrodes were inserted into the exposed sciatic nerve, and a ground electrode was connected to the foot pad. Electrical stimulation was performed based on the determination of compound action potentials and hindlimb muscle contraction, and the intensity of electrical stimulation was maintained in all rats. Under stimulation with an SD9 stimulator (Grass Technologies), the signal was amplified, bandpass filtered (0.1-3kHz), digitized, and acquired at 10kHz. The results are shown in Figure 14 .

[0136] Figure 14 High-amplitude motor evoked potentials (MEPs) propagated from the motor cortex to the sciatic nerve, with signals in the right hindlimb of mice in the spinal cord injury group reduced nearly threefold compared to the sham-operated group. The signal amplitudes in the Exos and Exos-NC groups were significantly higher than those in the SCI group, while the signal amplitudes in the Exos-BDNF group were significantly higher than those in the Exos and Exos-NC groups. These data suggest that Exos-BDNF is superior to Exos and Exos-NC in improving motor function after spinal cord injury.

[0137] B. HE staining of bladder tissue

[0138] After spinal cord injury, the rats lose their ability to urinate autonomously due to damaged spinal cord nerve function. Their bladders will proliferate and thicken. The bladder tissues of the rats in each group were taken for HE staining. The results are as follows: Figure 15 shown.

[0139] Figure 15 Bladder H&E staining showed that the bladder thickness of rats treated with Exos and Exos-NC was smaller, indicating that the bladder function of the Exos and Exos-NC treatment groups recovered faster, indicating that their neural function had recovered. However, the bladder thickness after Exos-hBDNF treatment was significantly smaller and similar to that of the Sham group, indicating that the neural function of the bladder was better restored.

[0140] C. Nissl staining

[0141] The spinal cord tissue of each group of rats was taken for Nissl staining. The samples after Nissl staining turned dark blue, which is often used to show the basic neural structure of the spinal cord. The large and numerous Nissl bodies indicate that the nerve cells have a strong ability to synthesize proteins; on the contrary, when the nerve cells are damaged, the number of Nissl bodies will decrease or even disappear. Figure 16The Nissl staining steps are as follows:

[0142] 1) Sample processing

[0143] For paraffin sections: Dewax the sections in xylene for 5-10 minutes, dewax again in fresh xylene for 5-10 minutes, and then dewax the sections in anhydrous ethanol for 5 minutes.

[0144] 2) Nissl staining: Stain the treated samples with Nissl stain for 2-5 minutes (the time can be adjusted according to your staining requirements). Rinse with water until the fragments are colorless.

[0145] 3) Shake off excess water from the slices and bake them in a 65°C oven to dry.

[0146] 4) After fully drying, seal the slides with neutral gum.

[0147] Figure 16 Nissl staining results showed that the number of neurons in the anterior horn of the spinal cord of rats in the SCI+Exos and SCI+Exos-NC groups was greater than that in the SCI group, and the number of neurons in the anterior horn of the spinal cord of rats in the SCI+Exos-hBDNF group was greater than that in the SCI+Exos and SCI+Exos-NC groups. This proves that although ordinary exosomes can play a role in restoring neurological function, the effect of engineered exosomes is better than that of ordinary exosomes. After injection of engineered exosomes, the number of neurons in the anterior horn of the spinal cord of rats recovered to a level similar to that before injury.

[0148] Test Example 3

[0149] Experimental Example 3 of the present invention tested the effect of the engineered exosomes of Example 1 on the recovery of motor function in rats, specifically including:

[0150] (1) Gait analysis experiment

[0151] Within 28 days after the SCI animal model surgery, the present invention conducted a gait analysis experiment on each group of rats using an animal gait analysis system (Noldus Information Technology CatWalk XT). The experimental steps are as follows:

[0152] Preliminary preparation: A few days before the experiment, the animals need to undergo necessary training to acclimate them to the experimental environment. The goal of the training is to enable the animals to walk from one side of the passage to the other at a relatively constant speed without any noticeable pauses or stops in between.

[0153] Experimental setup: Adjust the width of the runway to suit the experimental animals. Also, adjust the intensity of the green and red lights to achieve the best recognition effect.

[0154] Data Acquisition: Place the animal into the experimental channel from the left side and start the software to begin data acquisition. Data acquisition stops when the animal walks normally to the other side. Typically, three consecutive trails are acquired for each animal.

[0155] Subsequent processing: After the experiment, gently flip up the lid to remove the current animal, and wipe the glass surface with 1% alcohol to ensure that the glass surface is smooth.

[0156] The results are as follows Figure 17 As shown, the gait of rats in the Sham group was normal, with clear footprints and no errors in step sequence. The rats in the SCI group showed obvious confusion in step sequence and slight tail dragging. After treatment with Exos and Exos-NC, the step sequence errors of the rats were significantly improved, and correct steps occasionally appeared. Compared with all other treatment groups, the step sequence of the rats was significantly improved and the footprints were clear after Exos-hBDNF treatment. Ordinary exosomes and empty exosomes can achieve slight but not significant motor improvement. However, engineered exosomes can significantly promote the recovery of motor function in rats, and their gait, step sequence, and footprint clarity are restored to a level similar to that before injury.

[0157] (2) Swimming experiment

[0158] The rats in each group underwent a swimming test to assess their motor recovery. The rats were taught to swim from one end of a glass water tank to the other, and their swimming performance was observed during the process.

[0159] The results are as follows Figure 17 As shown, the sham group rats had a normal swimming posture, with their bodies level with the water, their tails tilted upward, and their lower limbs strong. The SCI group rats had a large angle between their bodies and the water surface, their tails drooped, their lower limbs weak when swimming, no obvious swimming movements, poor balance, and a noticeable body tilt. After treatment with conventional exosomes and exosome-free, the angle between the rats' bodies and the water surface decreased, their tails tilted slightly upward, and their lower limbs showed a visible power-up movement during swimming, with reduced body tilt. Compared with the other treatment groups, rats treated with engineered exosomes had a smaller angle between their bodies and the water surface, a more tilted tail, a coordinated swimming posture, no noticeable body tilt, and stronger lower limbs. This indicates that engineered exosome treatment has a superior recovery effect on rat motor ability compared to conventional exosome treatment, and the swimming posture of rats injected with engineered exosomes is closer to that of the sham group.

[0160] Test Example 4

[0161] Experimental Example 4 of the present invention detected the relevant pathways of BDNF's protection against spinal cord injury in rats, specifically comprising the following steps:

[0162] BDNF is often used as an important protein to assess motor function recovery after nervous system injury and plays a key role in neuronal survival, differentiation, and regeneration. TrkB is a high-affinity receptor that binds to BDNF and plays a positive role in maintaining the physiological function of neurons and promoting motor function recovery after injury. BDNF is an important neurotrophic factor. Its concentration decreases in adulthood but is upregulated through autocrine and paracrine pathways after injury. After binding to its specific receptor, TrkB, BDNF forms a homodimeric complex structure, promoting autophosphorylation of tyrosine receptor residues.

[0163] Subsequently, phosphorylated TrkB and BDNF activate downstream signaling pathways, protecting damaged neurons. BDNF release is crucial for mitigating tissue damage and promoting motor recovery. Its expression increases the excitability of spinal cord neurons. Exogenous BDNF promotes axonal regeneration in the distal injured spinal cord and influences spinal circuit function. Furthermore, BDNF can be delivered to the lesion site, reorganizing motor circuits and enhancing walking ability.

[0164] TrkB can reduce neuronal degeneration and is an effective target for spinal cord injury. When BDNF binds to TrkB, it initiates intracellular signaling pathways and increases the adaptability of neural circuits. PI3K is an intracellular phosphatidylinositol kinase. Triggering the PI3K pathway can alter actin and microtubule dynamics. Following PI3K activation, Akt initiates signal transduction by binding to specific domains at anchor sites. The PI3K / Akt pathway plays a crucial role in regulating the growth, development, and differentiation of neurons in the central and peripheral nervous systems. BDNF expression can be actively mediated by the PI3K / Akt pathway.

[0165] Western blot was used to detect the expression of TrkB, PI3K, p-PI3K, Akt, p-Akt, Bcl-2, and Bax in brain tissue:

[0166] (1) Place the spinal cord tissue in a cryopreservation tube, quickly freeze it in liquid nitrogen, and then store it in a -80°C refrigerator.

[0167] (2) Remove the tissue from the refrigerator and add 0.3 mL of RIPA lysis buffer containing 1% PMSF and 1% phosphatase inhibitors. After homogenization, place on ice for 30 minutes and centrifuge at 12,000 g for 15 minutes at 4°C. The supernatant is the total protein.

[0168] (3) Determine the protein concentration according to the BCA kit instructions. Balance the sample concentration according to the concentration, then add the loading buffer and denature the protein at 95°C. Select an appropriate separation gel for electrophoresis based on the protein molecular weight. After the gel is prepared, add the sample and marker to the corresponding lane for electrophoresis.

[0169] (4) Next, transfer the target to a nitrocellulose membrane (PVDF). The length of the membrane can be adjusted according to the molecular weight of the selected protein. Block the membrane at room temperature for 2 hours. Then incubate the membrane with the primary antibody at 4°C overnight.

[0170] (5) Subsequently, the membrane was incubated with IRDye 800CW goat anti-rabbit secondary antibody (1:1,5000, USA, C80925-05) were incubated at room temperature for 30 minutes and washed with TBST for 10 minutes three times.

[0171] (6) Using dual-color infrared laser imaging system ( USA) were used for Western blot detection.

[0172] TrKB is a highly specific endogenous receptor for BDNF. Upon binding to TrKB, BDNF autophosphorylates its intracellular tyrosine residues, leading to ligand-induced dimerization of each receptor and subsequent activation of multiple intracellular signaling pathways with diverse functions. Figure 19 The results showed that compared with the sham operation group, the expression of TrkB protein in the SCI group was significantly downregulated. Compared with the SCI group, Exos-BDNF could significantly upregulate the expression of TrkB protein in the spinal cord tissue. As downstream proteins of TrKB, p-PI3K and pAkt were downregulated in the SCI model.

[0173] Compared with the sham operation group, the levels of p-PI3K / PI3K and p-Akt / Akt in the SCI group were significantly lower than those in the sham operation group. Compared with the SCI group, Exos-BDNF could significantly increase the levels of P-PI3K / PI3K and P-Akt / Akt.

[0174] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. The use of bone marrow stem cell engineered exosomes containing BDNF gene as a new type of drug for treating spinal cord injury, characterized in that: The bone marrow stem cell engineered exosomes containing the BDNF gene can overexpress brain-derived neurotrophic factor BDNF, thereby treating spinal cord injury.

Images