Neural stem cell exosome-loaded cerebrospinal fluid-rich fibroin hydrogel scaffold system, preparation method and application thereof

Abstract

The invention relates to a cerebrospinal fluid-rich fibroin hydrogel stent system loaded with neural stem cell exosomes as well as a preparation method and application of the cerebrospinal fluid-rich fibroin hydrogel stent system, in particular relates to cerebrospinal fluid-rich fibroin hydrogel loaded with neural stem cell-derived exosomes to promote repair of neurons after spinal cord injury, and belongs to the technical field of biological medicines. The engineered neural stem cell exosome of the targeted neuronal cells designed by the invention can play roles in improving the local ischemia environment after spinal cord injury, promoting vascular regeneration and inhibiting neuronal aging, and is more beneficial to repair and regeneration of local nerves. The exosome is combined with the fibroin hydrogel rich in cerebrospinal fluid to form an exosome sustained-release system to be applied to local spinal cord injury, so that the treatment efficiency of the exosome is improved, rapid release, large accumulative release amount and long action time are realized, the functional recovery after spinal cord injury is obviously improved, and the exosome has good practical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, a preparation method and an application thereof. Background Art

[0002] Spinal cord injury is a common disease in spinal surgery. It refers to a central nervous system disease characterized by abnormal physiological function or loss of sensory, motor and other functions due to complete or incomplete damage to the spinal cord tissue after direct or indirect violence. According to statistics, there are about 10.4-83 cases of spinal cord injury per million people in the world each year, and its incidence and mortality rate are rising year by year. Patients with mild spinal cord injury lose their labor force, while those with severe spinal cord injury suffer from limb paralysis, incontinence, and loss of self-care ability in daily life, which seriously affects the quality of life of patients. Unfortunately, due to the poor plasticity of the central nervous system and limited nerve regeneration ability, the regeneration ability of the spinal cord after injury is extremely weak. There is currently no effective treatment to completely restore neurological function after spinal cord injury.

[0003] Exosomes are a subcellular component secreted by cells. They are essentially nanoscale membrane vesicles with a diameter of about 30 to 100 nm and a double-layer membrane structure. The components of their contents are mainly proteins, lipids, and nucleic acids. Due to the advantages of high biocompatibility and low immunogenicity, exosomes often have better delivery capabilities than drug delivery systems based on artificial liposomes, so exosomes can be used as drug delivery carriers. However, natural exosomes are not targeted enough, so they are easily cleared quickly and taken up by non-target cells after entering the body, or interact with cell membranes, resulting in a short half-life of exosome drugs. In response to the above-mentioned shortcomings of natural exosomes, the academic community has developed a variety of engineered exosome modification strategies, such as exosome membrane surface engineering to improve the targeting and stability of exosomes, and the modification of the content load of exosomes loaded with drugs. Exosomes modified by these strategies are collectively referred to as engineered exosomes. Many studies have confirmed that engineered exosomes are expected to be used in the treatment of spinal cord injury.

[0004] Patent CN 113046316 B discloses the application of exosome-hydrogel to promote local angiogenesis after spinal cord injury. The invention establishes a mouse spinal cord injury model, observes the therapeutic effect of exosomes derived from M2 bone marrow macrophages on spinal cord injury, evaluates the therapeutic effect of exosomes combined with hydrogel to form a sustained-release system for spinal cord injury by observing various indicators after spinal cord injury, and observes the regeneration of blood vessels at the injured site of the mouse tail spinal cord by immunofluorescence staining.

[0005] The internal environment after spinal cord injury is mainly low in oxygen, low in nutrition and acidic. Oxygen clearance disorder will lead to abnormal nerve cell metabolism, greatly reducing the repair efficiency, and then evolving into aging nerve cells. Related studies have confirmed that injury can trigger the aging of nerve cells in the spinal cord tissue and the disorder of the extracellular matrix, which further confirms the importance of cell aging in the occurrence and development of spinal cord injury. At present, there are no exosome products that are mainly used to inhibit neuronal aging after spinal cord injury.

[0006] Cerebrospinal fluid (CSF) is a transparent fluid that surrounds the brain and provides mechanical support, transfers micronutrients and signaling molecules to neurons, and participates in the removal of unnecessary metabolites. CSF interacts directly with the extracellular space of the brain and can therefore reflect the occurrence of pathophysiological changes. Studies have found that the cerebrospinal fluid of young mice can reduce cell senescence in the cerebral cortex of Alzheimer's mice and improve memory impairment in Alzheimer's mice. The mechanism may be related to CSF ​​inhibiting the aging of nerve cells by regulating complement C3 / C3aR. At present, there are no reports on the use of CSF to prevent and treat neuronal aging after spinal cord injury and thus repair spinal cord injury.

[0007] The regenerative capacity of the central nervous system is limited, so effective treatments are urgently needed to promote the repair of damaged nerves. Summary of the invention

[0008] The purpose of the present invention is to provide a cerebrospinal fluid-rich silk protein hydrogel scaffold system, preparation method and application thereof loaded with neural stem cell exosomes in order to solve the above problems. The silk protein hydrogel rich in cerebrospinal fluid and loaded with neural stem cell-derived exosomes are used for the repair of neurons after spinal cord injury. The present invention designs engineered neural stem cell exosomes targeting neuronal cells, which can improve the local ischemic environment after spinal cord injury, promote angiogenesis and inhibit neuronal aging, and are more conducive to the repair and regeneration of local nerves. By combining with a silk protein hydrogel rich in mouse cerebrospinal fluid to form an exosome sustained-release system for application to local spinal cord injury, the exosome treatment efficiency is improved, sustainable sustained release is achieved, and long-term action is achieved, which significantly improves the functional recovery of mice after spinal cord injury, and has good practical application value.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] In a first aspect, the present invention provides an engineered exosome, which is a neural stem cell exosome transfected with RVG-LAMP2B and is secreted by a neural stem cell.

[0011] Furthermore, the engineered exosomes contain specific markers, including CD63, CD9 and TSG101.

[0012] Furthermore, the engineered exosomes, namely RVG-LAMP2B-Exos, are obtained by isolating and culturing primary neural stem cells, then incubating them with complete culture medium (DMEM complete culture medium) containing exosome-free serum, and collecting them by differential centrifugation.

[0013] Furthermore, the engineered exosomes are prepared into a local administration (injection) preparation or a local external preparation.

[0014] The second aspect of the present invention provides a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, the hydrogel scaffold system comprising: a composite hydrogel scaffold, and engineered exosomes loaded on the composite hydrogel scaffold; the preparation method of the composite hydrogel scaffold comprises the following steps: after the silk protein is modified by methacrylation, cerebrospinal fluid is added to form a composite hydrogel scaffold under light conditions.

[0015] Furthermore, the engineered exosomes are extracted from neural stem cells after transfection of RVG-LAMP2B plasmid, and are used to increase the aging phenotype of neuronal cells that occur after spinal cord injury, and to target and inhibit the aging of neuronal cells.

[0016] Furthermore, the composite hydrogel scaffold is prepared by the following method: after the silk protein is modified by methacrylation, cerebrospinal fluid is added, and a composite hydrogel scaffold is formed under 405 nm light conditions.

[0017] Furthermore, the cerebrospinal fluid is the cerebrospinal fluid of young mice. The cerebrospinal fluid can also be commercially available artificial cerebrospinal fluid.

[0018] The third aspect of the present invention provides a method for preparing a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, the method for preparing the hydrogel scaffold system comprising:

[0019] Preparation of composite hydrogel scaffolds and engineered exosomes;

[0020] The engineered exosomes are added into a composite hydrogel scaffold to prepare a hydrogel scaffold system.

[0021] Furthermore, the method for constructing a hydrogel scaffold system after adding the engineered exosomes to the composite hydrogel scaffold comprises the following steps: mixing the engineered exosomes with the composite hydrogel scaffold, wherein the concentration of the engineered exosomes in the composite hydrogel scaffold is 100-200 ug / mL, co-culturing for 1-2 hours under light-proof conditions to form an aqueous exosome-hydrogel preparation, namely the hydrogel scaffold system, and storing it at 4°C in the dark.

[0022] Furthermore, the composite hydrogel scaffold is an aqueous photo-crosslinked hydrogel.

[0023] Furthermore, the hydrogel scaffold system is a topical preparation hydrogel scaffold system, and the preparation method of the topical preparation hydrogel scaffold system includes an exosome-hydrogel preparation with a photo-crosslinked cerebrospinal fluid-rich silk protein hydrogel as a carrier. The specific method of preparing the hydrogel scaffold includes: the silk protein is modified by methacrylation and then added to the cerebrospinal fluid of young mice to form a composite hydrogel scaffold under 405nm light conditions.

[0024] The fourth aspect of the present invention also provides the use of the hydrogel scaffold system in the preparation of spinal cord injury repair drugs (spinal cord injury treatment preparations).

[0025] Furthermore, the engineered exosomes were mixed with the composite hydrogel scaffold, the concentration of the engineered exosomes in the composite hydrogel scaffold was 100-200 ug / mL, and co-cultured for 1-2 hours under light-proof conditions to form an exosome-hydrogel sustained-release preparation, i.e., the spinal cord injury repair drug, which was stored at 4°C in the dark.

[0026] Furthermore, the hydrogel scaffold system was implanted in situ into the spinal cord injury defect.

[0027] Furthermore, when the exosome-gel sustained-release preparation is used, it is applied locally to the injured spinal cord in the form of a dressing at a rate of 80-120ul / cm 2 Covered exosome-gel sustained-release preparation, at 405nm 60mW / cm 2 Under the right conditions, the hydrogel is exposed to light for 30s-1min to transform from a liquid phase to a solid phase, and the wound is sutured layer by layer.

[0028] Furthermore, the spinal cord injury repair drug includes one or more of the following uses:

[0029] a) Enhance neuronal angiogenesis after spinal cord injury;

[0030] b) inhibiting neuronal senescence after spinal cord injury;

[0031] c) Improve motor function recovery after spinal cord injury.

[0032] The technical concept of this application is as follows:

[0033] Exosomes derived from neural stem cells (NSC) have been shown to have specific neurorepair effects. A large number of studies have shown that engineered exosomes can promote the treatment of spinal cord injury. The engineered modification of exosomes can be given powerful therapeutic capabilities by targeted delivery of transfected plasmid fragments. Based on this, RVG-LAMP2B is transfected into exosomes to achieve targeted neurons and repair damage. The present invention overcomes the existing technical defects and prepares a neuron-targeted neural stem cell exosome, namely RVG-LAMP2B-Exos, which can promote angiogenesis after spinal cord injury and inhibit neuronal aging, which is more conducive to neural repair and regeneration. By combining with a photo-crosslinked cerebrospinal fluid-rich silk protein hydrogel to form an exosome scaffold system for application to local spinal cord injury, the exosome treatment efficiency is improved, and sustained release can be achieved to achieve long-term action, significantly improving the functional recovery of mice after spinal cord injury.

[0034] The present application has found that the cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes can alleviate spinal cord injury and play a repairing role. In cell experiments, the material has the effect of reducing lipopolysaccharide (LPS)-induced neuronal cell inflammatory response and inhibiting neuronal cell aging. The specific mechanism of action is: engineered exosomes inhibit the decomposition of glutamine and reduce the aging level of neuronal cells, thereby reducing spinal cord injury. At the same time, the present invention also found through animal experiments that injecting the material into the damaged spinal cord of mice can effectively repair the injury.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] 1) The present invention provides a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes for in situ treatment of spinal cord injury, which can inhibit the decomposition of glutamine and reduce the aging level of neuronal cells, thereby alleviating spinal cord injury.

[0037] 2) The present invention uses cerebrospinal fluid-rich silk protein hydrogel as an exosome carrier. Generally, the hydrogel is easily affected by the environment, which can easily change the gel state, thereby affecting the culture effect of the exosomes and making it difficult for the exosomes to be evenly distributed in the gel. The silk protein hydrogel will only become a solid phase under irradiation within a specific wavelength range, and has good biocompatibility, allowing the exosomes to better combine with the hydrogel in the aqueous phase, greatly increasing its effect. The addition of cerebrospinal fluid to the hydrogel can inhibit the aging of neurons to a greater extent.

[0038] 3) The present invention uses a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes to repair spinal cord injury, which provides a new use for the material, greatly improves its application value, expands the application scope of the exosome-hydrogel, and opens up new areas of application, which has broad application prospects and commercial value.

[0039] 4) The present invention utilizes a hydrogel rich in cerebrospinal fluid of young mice to provide a sustained-release environment for exosomes. The cerebrospinal fluid in the cerebrospinal fluid-rich silk protein hydrogel serves as a carrier to delay the degradation of exosomes, thereby relying on exosomes to exert a therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results of flow cytometry for the identification of neural stem cells include (a) the expression of Nestin, and (b) the expression of SOX2.

[0041] Figure 2 These are test images of engineered exosomes, including (a) transmission electron microscopy image, which shows that the exosomes have a spherical morphology; (b) particle size-concentration distribution diagram of nanoparticle tracking analysis (NTA); (c) volume particle size distribution diagram of nanoparticle tracking analysis. NTA measurement shows that the average diameter of exosomes is about 100 nm.

[0042] Figure 3 These are the Western blot images of the NSCs group, NSCs-Exos group, Exos-NC group, and RVG-LAMP2B-Exos group. Western blot verified the expression of engineered exosome markers including CD63, CD9, TSG101, and LAMP2B.

[0043] Figure 4 The staining results of RVG-LAMP2B-Exos binding to mouse spinal cord neurons, where (a) the phalloidin staining results show that RVG-LAMP2B-Exos bind most tightly to neurons, and (b) the quantitative expression of immunofluorescence staining.

[0044] Figure 5 Comparison of the exosome release rate of silk hydrogel before and after the addition of cerebrospinal fluid. (a) The exosome release rate of the composite hydrogel after the addition of cerebrospinal fluid is higher, and the exosome release time of the cerebrospinal fluid-exosome group is longer than that of the simple silk hydrogel-exosome group. (b) The cumulative release efficiency of the exosomes of the composite hydrogel after the addition of cerebrospinal fluid is higher.

[0045] Figure 6 The results of verifying the effect of RVG-LAMP2B-Exos on endothelial cell and neuronal aging include: (a) angiogenesis diagram of Control group, LPS, and LPS+RVG-LAMP2B-Exos group; (b) Western blot band diagram of Control group, LPS, and LPS+RVG-LAMP2B-Exos group.

[0046] Figure 7 These are the staining results of cerebrospinal fluid-rich silk protein hydrogel-exosomes in alleviating spinal cord injury in vivo, including: (a) HE staining results, (b) quantitative expression of HE staining, (c) NF, GFAP, DAPI, Merge staining results, and (d) quantitative expression of NF, GFAP, DAPI, Merge staining.

[0047] Figure 8 Diagram of the mechanism of action of cerebrospinal fluid-enriched silk protein hydrogel loaded with engineered exosomes derived from neural stem cells in repairing spinal cord injury. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific drawings and embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection of the present invention.

[0049] The present invention relates to a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, a preparation method and its application, specifically a cerebrospinal fluid-rich silk protein hydrogel loaded with neural stem cell-derived exosomes to promote the repair of neurons after spinal cord injury, belonging to the field of biomedical technology. The present invention designs engineered neural stem cell exosomes targeting neuronal cells, which can improve the local ischemic environment after spinal cord injury, promote angiogenesis and inhibit neuronal aging, and are more conducive to the repair and regeneration of local nerves. By combining with cerebrospinal fluid-rich silk protein hydrogel to form an exosome sustained-release system for application to the local spinal cord injury, the exosome treatment efficiency is improved, rapid release is achieved, the cumulative release amount is large, the action time is long, and the functional recovery after spinal cord injury is significantly improved, which has good practical application value.

[0050] Any features such as preparation methods, materials, structures or composition ratios that are not clearly described in this technical solution shall be deemed as common technical features disclosed in the prior art.

[0051] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] Example 1

[0053] Neural stem cell isolation and culture:

[0054] Primary neural stem cells were extracted using 6-week-old C57BL / 6 mice. The specific steps are as follows: First, 6-week-old pregnant female SPF-grade C57BL / 6 mice purchased from the Experimental Animal Department of Fudan University were placed in an animal incubator, and the items related to cell extraction were placed in a clean bench and irradiated with ultraviolet light for half an hour. All instruments were sterilized by high-pressure steam. The mice were first anesthetized by inhalation of 3% isoflurane (RWD, Shenzhen, Guangdong), and then maintained in anesthesia with 1.8% isoflurane. Subsequently, a cesarean section was performed, and the uterus and placenta were carefully dissected to extract the pups. Both pregnant and pups were killed by overdose of anesthesia. Both pregnant and pups were killed by overdose of anesthesia. The pups were immersed in 75% ethanol, a dorsal incision was made on the skin and soft tissue, and the spinal cord was obtained. Next, the excised spinal cord was rinsed in pre-cooled culture medium (Gibco, Gardner, CA), cut into small pieces, and transferred to a 100 μm pore size mesh (Falcon, Corning, NY, USA), and the resulting cell suspension was centrifuged at 300 rpm for 5 minutes at 4°C. The precipitated cells were resuspended in culture medium (Gibco, Gardner, CA) supplemented with 2% B27 (Gibco), glutamine (0.5 mM, Gibco), penicillin (100 IU / mL), streptomycin (100 mg / mL, Gibco), epidermal growth factor (20 ng / mL, Peprotech) and basic fibroblast growth factor (20 ng / mL, Peprotech) and cultured for 48 hours at 37°C and 5% CO2 to generate neurospheres. The culture medium was collected. Flow cytometry experiments verified the surface markers of neural stem cells. The results showed that Nestin and SOX2 were expressed at high levels in cells ( Figure 1 ).

[0055] RVG-LAMP2B-Exos acquisition:

[0056] RVG-LAMP2B plasmid was constructed by GeneChem (Shanghai). To obtain engineered exosomes, neural stem cells were further transfected with RVG-LAMP2B plasmid using Lipofectamine 2000 (Invitrogen, USA). Centrifugation was performed in sequence: 37°C, 300 rpm for 10 min, 4°C, 2000 rpm for 10 min, 4°C, 10,000 rpm for 60 min. After centrifugation, the supernatant was passed through a 0.22 μm sterile filter (Falcon) to remove cell debris. The filtrate was ultracentrifuged at 120,000 rpm for 70 min, the supernatant was discarded, and the precipitate was resuspended in 200 μL phosphate buffer (PBS, Gibco) to isolate RVG-LAMP2B-Exos (engineered exosomes). Characterization was performed by electron microscopy, flow cytometry, and Western blot. Transmission electron microscopy and NTA measurements showed that the isolated exosomes exhibited a double-membrane spherical morphology with a diameter in the range of about 100 nm (see Figure 2 ). Western blot confirmed the expression of exosome-specific markers CD63, CD9, and TSG101 as well as transfected LAMP2B (see Figure 3 ), DAPI, PKH26 and Phalloidine staining, verifying that RVG-LAMP2B-Exos bind most significantly to mouse spinal cord neurons compared with NSCs-Exos (exosomes obtained by the same method as RVG-LAMP2B-Exos without transfection of neural stem cells with RVG-LAMP2B plasmid) and Exos-NC (natural exosomes collected from neural stem cells without transfection of any plasmid) ( Figure 4 ).

[0057] Preparation of cerebrospinal fluid-rich silk protein hydrogel-exosomes (cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes):

[0058] A certain amount of silk cocoons were weighed using an electronic balance, and silk protein fibers were extracted by degumming. The silk was dissolved by lithium bromide and then methacrylated (40 g of silkworm cocoons were placed in 8 L of 0.05 M Na2CO3 solution and boiled at 100 °C for 45 min to remove serine. The silk was washed with warm water, cold water, and deionized water in turn, and dried in an oven at 40 °C for 24 h to obtain degummed silk fibroin. Subsequently, 20 g of degummed silk fibroin was dissolved in 9.3 M LiBr solution, dissolved at 60°C for 1h, stirred at 300rpm. Then, 6mL of glycidyl methacrylate (GMA) was added to the solution and stirred at 60°C and 300rpm for 4h. The obtained solution was dialyzed in deionized water with a dialysis membrane (14000Da cutoff) for 5 days to remove salt and unreacted GMA. For further measurement, part of the SilMA solution was frozen in liquid nitrogen for 30min, freeze-dried for 3d, and stored at -20°C. ), concentrated to obtain a silk protein hydrogel precursor solution with a mass fraction of 5% to 30% (30% in this embodiment). The cerebrospinal fluid of mice was obtained after the mice were anesthetized and their heads were fixed on the orientation instrument. The hair of the head and neck was trimmed and disinfected, and a longitudinal incision (about 1cm) was made along the longitudinal axis with a scalpel. The dorsal muscles of the neck were bluntly separated with scissors to expose the foramen magnum. The cerebrospinal fluid was directly extracted through the foramen magnum. After the extraction, the outer muscles and skin were sutured. Then, cerebrospinal fluid was added to the precursor solution at a mass ratio of 100:1 to 1:100 (100:1 in this embodiment), 405 nm, 60 mW / cm 2 The mixture was illuminated for 30s-1min (1min in this embodiment) under the conditions of light, and then RVG-LAMP2B-Exos was mixed into the liquid hydrogel at a ratio of 500μg:50μL to form a suspension to obtain CSF-SilkMA-RVG-LAMP2B-Exos. In addition, RVG-LAMP2B-Exos was added to the simple silk protein hydrogel (without adding cerebrospinal fluid to the precursor solution) at the same ratio to obtain SilkMA-RVG-LAMP2B-Exos as a comparative example, which was compared with the cerebrospinal fluid-rich hydrogel used in this embodiment. Through this example, it was found that the composite hydrogel after adding cerebrospinal fluid in the present invention released more (engineered) exosomes per day and had a higher cumulative release efficiency. At the same time, compared with the simple silk protein hydrogel-exosome group, the exosome release time of the cerebrospinal fluid-exosome group was longer ( Figure 5 ), it can be seen that the addition of cerebrospinal fluid can release more and faster exosomes while achieving a longer release time, extending the release time of exosomes and increasing the amount of exosomes released.

[0059] Effects of RVG-LAMP2B-Exos on endothelial cell and neuronal aging:

[0060] This example provides an experimental process of RVG-LAMP2B-Exos reducing the aging level of in vitro neural cells and promoting angiogenesis. In this example, three experimental groups were set up, namely: a control group (Control, spinal cord neuronal cells without any treatment), an LPS group (LPS, mouse spinal cord neuronal cells were adjusted to a concentration of 2×10 7 / L of high glucose Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (10%, FBS) and penicillin-streptomycin solution (100U / ml and 100mg / ml, respectively), 100μL of the prepared cell suspension was inoculated into a 96-well plate per well for 24h, and then intervened with 5μg / ml LPS for 12h), LPS+RVG-LAMP2B-Exos group (LPS+RVG-LAMP2B-Exos, mouse spinal cord neuron cells were adjusted to a concentration of 2×10 7 / L of cell suspension, the prepared cell suspension was inoculated into a 96-well plate at 100 μL per well, placed in a cell culture incubator for 24 hours, replaced with a culture medium containing RVG-LAMP2B-Exos at a concentration of 10 μmol / L and incubated for 48 hours, and then intervened with LPS at a concentration of 5 μg / ml for 12 hours). Through this embodiment, it was found that RVG-LAMP2B-Exos compared with the control group and the LPS group, and the angiogenesis experiment showed that RVG-LAMP2B-Exos has the effect of promoting angiogenesis ( Figure 6 a), WB results showed that LPS-induced neuronal cell senescence was alleviated ( Figure 6 b).

[0061] Detection of cerebrospinal fluid-enriched silk protein hydrogel-exosomes in alleviating spinal cord injury in mice:

[0062] The present embodiment provides an experimental process of reducing the level of neuroinflammation and inhibiting neurofibrosis in vivo by using silk protein hydrogel-exosomes rich in cerebrospinal fluid. In order to study the in vivo therapeutic effect, 40 6-week-old SD rats were taken. In the present embodiment, 5 experimental groups (embodiments or comparative examples) were set, namely: Sham group (sham operation group), SCI group (simple spinal cord injury group), SCI+cerebrospinal fluid-rich silk protein hydrogel group (SCI+CSF-SilkMA, injected with 1 μL of 200 μg / mL CSF-SilkMA), SCI+silk protein hydrogel-exosome group (SCI+SilkMA-RVG-LAMP2B-Exos, injected with 1 μL of 200 μg / mL SilkMA and 1 μL of 20 μM / LRVG-LAMP2B-Exos mixture), SCI+cerebrospinal fluid-rich silk protein hydrogel-exosome group (SCI+CSF-SilkMA-RVG-LAMP2B-Exos, injected with 1 μL of 200 μg / mL CSF-SilkMA and 1 μL of 20 μM / LRVG-LAMP2B-Exos mixture) (Example). After 4 weeks, the long-term recovery effect of mice was evaluated. HE and NF / GFAP staining were performed. HE was used to observe neuroinflammation in spinal cord neurons. Our results showed that the SCI group had the strongest neuroinflammation reaction, while the simple silk hydrogel-exosome group (SCI+silk hydrogel-exosome group) and the hydrogel-exosome group rich in cerebrospinal fluid (SCI+silk hydrogel-exosome group rich in cerebrospinal fluid) had significantly reduced neuroinflammation (P<0.001). Among them, the hydrogel-exosome group rich in cerebrospinal fluid had the mildest inflammatory reaction, which was almost indistinguishable from the control group (Sham group). NF fluorescence staining was used to observe the axonal expression and growth of neurons, reflecting the repair and regeneration ability of neurons. Similarly, the results showed that the cerebrospinal fluid-rich hydrogel-exosome group had the highest NF expression, indicating that the axonal repair effect of this group was the strongest (P<0.001). GFAP staining is a marker that mainly marks astrocytes and is used to evaluate the degree of fibrosis after nerve injury. The higher the degree of fibrosis, the worse the prognosis of the nerve will be. Scar repair. The experimental results showed that the SCI group had the most obvious neurofibrosis, while compared with other groups, the cerebrospinal fluid-rich silk protein hydrogel-exosomes could significantly reverse this phenomenon, especially compared with the simple silk protein hydrogel-exosomes group, the addition of cerebrospinal fluid further promoted the recovery of neurons (P<0.001), while the simple cerebrospinal fluid-rich silk protein hydrogel group (SCI+cerebrospinal fluid-rich silk protein hydrogel group) was weaker than the cerebrospinal fluid-rich hydrogel-exosomes group (SCI+cerebrospinal fluid-rich silk protein hydrogel-exosomes group) in inhibiting neuroinflammatory response, neurofibrosis and promoting axonal growth. Figure 7 ).

[0063] Construct a mouse spinal cord injury model. Implant the silk protein hydrogel-exosomes rich in cerebrospinal fluid (or not rich in cerebrospinal fluid) into the injured part of the spinal cord. Apply a dressing at 80-120ul / cm 2 Covering exosome-gel preparation, 60mW / cm at 405nm 2 Under the condition of light for 30s-1min, the hydrogel is transformed from liquid phase to solid phase, and the wound is sutured layer by layer. Figure 8 shown.

[0064] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes, characterized in that: The hydrogel scaffold system comprises: a composite hydrogel scaffold, and engineered exosomes loaded on the composite hydrogel scaffold; The preparation method of the composite hydrogel scaffold comprises the following steps: after the silk protein is modified by methacrylation, cerebrospinal fluid is added to form the composite hydrogel scaffold under light conditions.

2. The cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes according to claim 1, characterized in that: The engineered exosomes are extracted after RVG-LAMP2B plasmid is transfected into neural stem cells.

3. The cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes according to claim 1, characterized in that: The composite hydrogel scaffold is prepared by the following method: after the silk protein is modified by methacrylation, cerebrospinal fluid is added, and the composite hydrogel scaffold is formed under 405nm light conditions.

4. A method for preparing a cerebrospinal fluid-rich silk protein hydrogel scaffold system loaded with neural stem cell exosomes as described in claims 1-3, characterized in that: The preparation method of the hydrogel scaffold system comprises: Preparation of composite hydrogel scaffolds and engineered exosomes; The engineered exosomes are added into a composite hydrogel scaffold to prepare a hydrogel scaffold system.

5. The preparation method according to claim 4, characterized in that: The method for constructing a hydrogel scaffold system by adding the engineered exosomes to a composite hydrogel scaffold comprises the following steps: mixing the engineered exosomes with the composite hydrogel scaffold, wherein the concentration of the engineered exosomes in the composite hydrogel scaffold is 100-200 ug / mL, and co-culturing for 1-2 hours under light-proof conditions to form an aqueous exosome-hydrogel preparation, which is the hydrogel scaffold system.

6. Use of the hydrogel scaffold system according to any one of claims 1 to 3 in preparing a drug for repairing spinal cord injury.

7. Use of the hydrogel scaffold system according to claim 6 in preparing a spinal cord injury repair drug, characterized in that: The hydrogel scaffold system was implanted in situ into the spinal cord injury defect.

8. The use according to claim 7, characterized in that: When the hydrogel scaffold system is used, it is applied as a dressing on the surface of the injured spinal cord at a rate of 80-120ul / cm 2 Covered hydrogel scaffold system, 60mW / cm at 405nm 2 Under the right conditions, the hydrogel is exposed to light for 30s-1min to transform from the aqueous phase to the solid phase, and the wound is sutured layer by layer.

9. The use according to claim 7, characterized in that: The hydrogel scaffold system is formulated as a local injection.

10. The use according to claim 6, characterized in that: The spinal cord injury repair drug includes one or more of the following uses: a) Enhance neuronal angiogenesis after spinal cord injury; b) inhibiting neuronal senescence after spinal cord injury; c) Improve motor function recovery after spinal cord injury.

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