Engineering scaffold for spinal cord injury repair and construction method thereof

Abstract

The invention provides an engineering scaffold for spinal cord injury repair and a construction method thereof.The engineering scaffold comprises a columnar scaffold body and a tubular mantle wrapping the outer side wall of the columnar scaffold body, and the columnar scaffold body is of a porous structure made of porous gelatin sponge; neural stem cells transfected by AdV-TrkC and Schwann cells transfected by AdV-NT-3 are distributed in the porous structure, and the neural stem cells and the Schwann cells are distributed in the porous structure. AAV-CMV-MuSK-GFP (DJ type) adeno-associated vector viruses and rAAV-hSyn-agrin-mCherry adeno-associated vector viruses are further distributed in the porous structure in a dispersed mode. The engineering scaffold can activate a target neuronal mitogen activated protein kinase adenosine cyclophosphate effect element binding protein signal channel and promote transmission of synaptic signals between neurons, so that repair of spinal cord injury is promoted, connection recovery of neurons and an injury part is effectively promoted, and the treatment effect of the spinal cord injury is further enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of nerve repair materials, and specifically relates to an engineering scaffold for repairing spinal cord injury and a construction method thereof. Background Art

[0002] Spinal cord injury (SCI) is a serious central nervous system injury disease. Neuronal death and axonal rupture can lead to motor, sensory and autonomic motor dysfunction. In the past 30 years, its global prevalence has increased from 236 cases per million to 1298 cases. According to data from The Lancet, the two main causes of spinal cord injury are falls and traffic accidents. The incidence increases with age and remains above 50,000 cases between about 20 and about 84 years old. Cervical spinal cord injury accounts for 54.2% of the total incidence, while injuries below the cervical spinal cord account for 45.8% of the total incidence. According to statistics from relevant Canadian institutions, the total cost of each SCI patient in his lifetime exceeds 3 million US dollars, and the total economic burden caused by this cause is as high as nearly 2.67 billion US dollars. At present, when encountering SCI patients in clinical practice, it is limited to borrowing some drugs and combining a small amount of physical therapy to slow down the degree of paralysis. Therefore, the treatment of SCI has always been one of the world's problems that plague the medical community.

[0003] At the same time, the current research in the field of spinal cord injury repair is more focused on the repair scheme of constructing artificial scaffold materials and implanting them into the spinal cord injury area. For example, the Chinese invention patent with the authorization announcement number CN101278865B discloses a partitioned tissue engineering spinal cord, which includes an extraspinal catheter and a central catheter. A gray matter catheter with the same shape as the spinal cord gray matter is set in the extraspinal catheter, and corresponding catheters are set in the white matter area according to the geometric forms of the ascending fiber bundle and the descending fiber bundle. The seed cells constituting the partitioned tissue engineering spinal cord are one or more of bone marrow stromal stem cells, neural stem cells, Schwann cells, olfactory ensheathing cells, and embryonic stem cells. Although this partitioned tissue engineering spinal cord can guide the migration of neurons and glial cells through the fiber components in the partitioned area, it plays a role in facilitating the extension of nerve fibers and realizes the repair of spinal cord injury to a certain extent. However, since SCI can cause the death of spinal cord injury and its surrounding neurons and glial cells, and cause the interruption of nearby axons, it triggers changes in the entire nervous system, including changes in the principle injury circuits above and below the spinal cord injury. Although SCI disrupts the communication flow between the circuits that regulate movement and autonomic function and forms an inflammatory microenvironment, there is still local regeneration of residual descending fibers in the uninjured area. In the case of incomplete injury, a stimulus source can be applied to the brain or the injured area to reactivate the circuit in the spinal cord or strengthen the connection strength between the brain and the spinal cord. When SCI is severe, the reconstruction of the circuit requires the regrowth of severed axons at the injured site to restore the connection. CST is composed of descending fibers emitted by the fifth layer of large pyramidal neurons in the cerebral cortex, which directly or indirectly terminate in the anterior horn motor neurons of the spinal cord and control the autonomous movement of the limbs and trunk. CST is considered to be a key element in restoring autonomous movement function. However, the aforementioned engineered spinal cord cannot effectively promote the restoration of connection between neurons and the injured site, thus greatly affecting the treatment effect of spinal cord injury. Summary of the invention

[0004] In order to solve the above technical problems, the present invention first provides an engineering scaffold for repairing spinal cord injury, which can effectively promote the restoration of connection between neurons and injured parts, and further enhance the treatment effect of spinal cord injury.

[0005] In order to achieve the above-mentioned purpose, the present invention provides an engineering scaffold for repairing spinal cord injury, the engineering scaffold comprises a columnar scaffold body and a tubular mantle wrapped around the outer wall of the columnar scaffold body, the columnar scaffold body is a porous structure made of porous gelatin sponge, and the material of the tubular mantle is a biodegradable material; neural stem cells and Schwann cells are distributed in the porous structure of the columnar scaffold body, the neural stem cells are transfected with AdV-TrkC, and the Schwann cells are transfected with AdV-NT-3; AAV-CMV-MuSK-GFP (DJ type) adeno-associated vector virus and rAAV-hSyn-agrin-mCherry adeno-associated vector virus are also diffusely distributed in the porous structure of the columnar scaffold body.

[0006] The inventors have found through a large number of experimental studies that the adhesion properties between agrin and tyrosine receptor kinase (MuSK) can connect the synapses between the unreconstructed regenerated CST and the transplanted target neurons through agrin and tyrosine receptor kinase. Agrin on the presynaptic membrane is prone to specific adhesion to the tyrosine receptor kinase on the postsynaptic membrane, and activates the formation of the acetylcholine receptor (Acetylcholine Receptor, AChR) / MuSK / low-density lipoprotein receptor-related protein 4 (low-density lipoprotein receptor-related protein 4, LRP4) complex on the postsynaptic membrane, causing tyrosine phosphorylation, activating the target neuron mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) / cyclic adenosine monophosphate response element binding protein (cAMP-response element binding protein, CREB) signaling pathway, and promoting the transmission of synaptic signals between neurons. The engineering scaffold of the present invention uses AAV-CMV-MuSK-GFP (DJ type) adeno-associated virus to make neural stem cells overexpress tyrosine receptor kinase (MuSK), and transfects neuronal cells in the injured area with rAAV-hSyn-agrin-mCherry adeno-associated virus with a sustained release effect in the inner columnar porous structure, thereby promoting the overexpression of agrin. The agrin of the presynaptic membrane activates the formation of acetylcholine receptor low-density lipoprotein receptor-associated protein complex on the postsynaptic membrane, causing tyrosine phosphorylation, activating the target neuron mitogen-activated protein kinase cyclic adenosine monophosphate effector element binding protein signaling pathway, promoting the transmission of synaptic signals between neurons, thereby promoting the repair of spinal cord injury, and providing a new strategy for the clinical treatment of spinal cord injury.

[0007] In addition, since the tubular mantle covers the porous gelatin sponge, it can promote the porous gelatin sponge to absorb moisture between tissues and expand, thereby reducing the gap between the porous gelatin sponges and reducing outward expansion. That is, the tubular mantle can improve the swelling effect of the columnar stent body, so that the engineering stent will not cause compression to the surrounding tissues after transplantation, thereby avoiding secondary damage to the spinal cord.

[0008] Specifically, the vector of the AAV-CMV-MuSK-GFP (DJ type) adeno-associated vector virus can also be adenovirus and lentivirus. Adenovirus and lentivirus, as vector viruses for overexpressing tyrosine receptor kinase (MuSK), have strong advantages in vector construction and expression.

[0009] Specifically, the rAAV-hSyn-agrin-mCherry adeno-associated vector virus carries the DNA sequence of the agrin gene as shown in SEQ ID NO: 1. After the vector virus is transfected into neurons, it causes the neurons to overexpress agrin.

[0010] Preferably, the vector of the rAAV-hSyn-agrin-mCherry adeno-associated vector virus may also be adenovirus and lentivirus.

[0011] Preferably, the inner columnar porous structure is loaded with NT-3. NT-3 can bind to receptor tyrosine kinase (RTK), thereby supporting the survival and development of neurons.

[0012] Preferably, the material of the tubular mantle is methacrylated gelatin, and preferably, the tubular mantle is made of acellular optic nerve epimembrane. Acellular optic nerve epimembrane has no immunogenicity and has similar physical and chemical properties to the recipient epimembranous tissue, which is more conducive to the degradation and absorption of the tubular mantle in the recipient.

[0013] The present invention also provides a method for constructing an engineering scaffold for repairing spinal cord injury, and the construction method comprises the following steps:

[0014] (1) Construction of the main columnar support

[0015] A porous gelatin sponge is used to prepare a columnar stent body with a porous structure, and a biodegradable material is used to prepare a tubular mantle membrane. The tubular mantle membrane and the columnar stent body are sterilized and then used for standby;

[0016] (2) Seed cell culture

[0017] A recombinant adenovirus carrying the tyrosine kinase C gene, AdV-TrkC, was constructed, neural stem cells were cultured in vitro, and the neural stem cells were transfected with AdV-TrkC to prepare a TrkC gene-modified neural stem cell suspension; a recombinant adenovirus carrying the neurotrophin-3 gene, AdV-NT-3, was constructed, Schwann cells were cultured in vitro, and Schwann cells were transfected with AdV-NT-3 to prepare a Schwann cell suspension overexpressing NT-3;

[0018] (3) Scaffold implantation and culture of seed cells

[0019] Injecting a suspension of TrkC gene-modified neural stem cells into the scaffold; injecting a suspension of NT-3-overexpressing Schwann cells around the TrkC gene-modified neural stem cells, culturing the scaffolds on which the seed cells have been planted, allowing the TrkC gene-modified neural stem cells to differentiate into functional neurons, allowing the NT-3-overexpressing Schwann cells to differentiate into oligodendrocytes, and further culturing the scaffolds in vitro, allowing the oligodendrocytes surrounding the neurons to continue to differentiate into spinal cord nerve fiber structures;

[0020] (4) Overexpression of MuSK in the scaffold

[0021] A recombinant adenovirus AAV-CMV-MuSK-GFP (DJ type) expressing a tyrosine receptor kinase gene was constructed, and AAV-CMV-MuSK-GFP (DJ type) was added to the culture medium of the scaffold that differentiated into the spinal nerve fiber structure. After culturing for 24 hours to form the spinal nerve scaffold overexpressing MuSK, the culture medium was replaced with serum-free medium.

[0022] (5) Constructing a recombinant adenovirus rAAV-hSyn-agrin-mCherry carrying the aggregation protein gene, adding the rAAV-hSyn-agrin-mCherry adeno-associated virus culture medium to the culture medium of the spinal cord nerve scaffold overexpressing MuSK, and then wrapping the tubular mantle around the columnar scaffold body to form a scaffold for culture and standby use.

[0023] Specifically, the volume ratio of the TrkC gene modified neural stem cell suspension to the NT-3 overexpressing Schwann cell suspension is 3:1.

[0024] Specifically, the concentration of the AAV-CMV-MuSK-GFP (DJ type) adeno-associated virus is MOI=100.

[0025] Specifically, the virus concentration of the rAAV-hSyn-agrin-mCherry adeno-associated virus culture solution is MOI=100.

[0026] The beneficial effects of the present invention are:

[0027] The engineering scaffold of the present invention enables neural stem cells to overexpress tyrosine receptor kinase (MuSK) through AAV-CMV-MuSK-GFP (DJ type) adeno-associated virus, and transfects neuronal cells in the injured site through rAAV-hSyn-agrin-mCherry adeno-associated virus with a sustained release effect in the inner columnar porous structure, thereby promoting the overexpression of agrin, and then the agrin of the presynaptic membrane activates the formation of acetylcholine receptor low-density lipoprotein receptor-related protein complex on the postsynaptic membrane, causing tyrosine phosphorylation, activating the target neuron mitogen-activated protein kinase cyclic adenosine monophosphate effector element binding protein signaling pathway, promoting the transmission of synaptic signals between neurons, thereby promoting the repair of spinal cord injury, and providing a new strategy for the clinical treatment of spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a physical picture of an engineering scaffold for repairing spinal cord injury according to an embodiment of the present invention;

[0029] Figure 2 This is a scanning electron microscope image of an engineering scaffold for repairing spinal cord injury according to an embodiment of the present invention;

[0030] Figure 3 This is a schematic diagram of cell distribution of an engineering scaffold for repairing spinal cord injury according to an embodiment of the present invention;

[0031] Figure 4 This is a diagram showing the results of cell differentiation and MuSK expression detection after 14 days of in vitro culture of an engineering scaffold in the method for constructing an engineering scaffold in an embodiment of the present invention; DETAILED DESCRIPTION

[0032] In order to more concisely and clearly demonstrate the technical solutions, purposes and advantages of the present invention, the technical solutions of the present invention are described in detail below in conjunction with specific embodiments. Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0033] Example 1 A method for constructing an engineering scaffold for repairing spinal cord injury comprises the following steps:

[0034] (1) Construction of the main body of the stent

[0035] The main body of the columnar stent with a 2 mm cylindrical structure was prepared using porous gelatin sponge (see the actual picture). Figure 1 ), and a tubular membrane structure was prepared with decellularized porcine optic nerve epimembrane, which was sterilized and used separately. The main body of the scaffold was cryosectioned and the porous structure was observed under a scanning electron microscope. The scanning electron microscope image is shown in Figure 2 , where 2-B is an enlarged view of the porous area of ​​2-A. Gel sponge was purchased from Jiangxi Xiangen Medical Technology Development Co., Ltd., and the porcine optic nerve was sponsored by the College of Animal Science and Technology of Northwest Agriculture and Forestry University;

[0036] (2) Seed cell culture

[0037] To facilitate the implementation of the experiment, the neural stem cells (hereinafter referred to as NSCs) of this example were obtained from 1-3 day old GFP transgenic suckling mice; Schwann cells (hereinafter referred to as SCs) were obtained from 3-5 day old GFP suckling mice;

[0038] NSCs were obtained from 1-3 day old GFP transgenic suckling mice. After alcohol disinfection, the whole hippocampus was removed under sterile conditions, the epineurium was removed, and the tissue was separated in D-Hank's solution. Transfer to a 15 mL centrifuge tube, blow several times until there are no obvious lumps and bubbles, centrifuge at 1000 rpm for 5 minutes, and then discard the supernatant. Use NSCs culture medium to blow the cell pellet again to suspend it, transfer it to a culture bottle and culture it in a 37°C, 5% CO2 incubator. When a large number of cell clones are observed to begin to form, use a fine-headed glass pipette to mechanically blow and separate the NSCs clones for passaging every other day. Select P2 generation NSCs with good growth status, and the transfection steps are as follows: before virus infection, NSCs are first washed with D-Hank's solution for 3 times, and then fresh culture medium of AdV-GFP-TrkC with a certain MOI value of 200 is added. After culturing for 48 hours, the old supernatant is discarded, and fresh culture medium is added after washing with D-Hank's solution, and then cultured for another 24 hours to obtain TrkC gene modified neural stem cell suspension;

[0039] SCs were obtained from GFP suckling mice 3-5 days after birth. After alcohol disinfection, the sciatic nerve and brachial plexus were separated and placed in a culture dish containing frozen D-Hank's solution. The epineurium was peeled off under a dissecting microscope, and the nerve was cut into pieces of about 0.5-1 mm, transferred to a centrifuge tube, and 0.125% trypsin was added to digest for 3-5 minutes at 37°C, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and washed three times with D-Hank's solution. Then a small amount (about 1 mL) of DMEM / F12 culture medium containing 10% FBS was used to resuspend the cells, and the tissue suspension was transferred to a culture bottle paved with poly-lysine, and cultured in a 37°C, 5% CO2 incubator. After 40 minutes of culture, the culture medium was added to a sufficient amount. The culture medium of cells was changed every 3 days. The exponential growth period of fibroblasts is within 1 to 2 days after implantation. Therefore, the mitotic inhibitor Ara-C (10 mol / L) can be used within 48 to 72 hours after implantation to inhibit fibroblasts and purify SCs. When the cells grow to more than 90%, the cells are passaged, and 0.125% trypsin is used for differential digestion, and differential adhesion is used according to the specific situation to further purify SCs. The purified SCs are inoculated into a 6-well culture plate with poly-lysine at 2×104 cells / well, and cultured overnight in a 37°C, 5% CO2 incubator. AdV-GFP-NT-3 with an MOI value of 100 is added to infect SCs. After 48 hours of culture, the old supernatant is discarded, and fresh culture medium is added after washing with D-Hank's solution, and culture is continued for 24 hours to obtain Schwann cell suspension overexpressing NT-3.

[0040] (3) Planting and culture of seed cells on the scaffold

[0041] The TrkC gene modified neural stem cell suspension was injected into the scaffold; the NT-3 overexpressing Schwann cells were implanted around the neural stem cells and differentiated into oligodendrocytes, which were then cultured in vitro so that the oligodendrocytes surrounding the neurons continued to differentiate into spinal nerve fiber structures; the TrkC gene modified neural stem cell suspension and the NT-3 overexpressing Schwann cell suspension were added in a volume ratio of 3:1; the scaffolds were cultured for 14 days, during which the cell distribution of the scaffolds was detected by electron microscopy, i.e., part of the scaffolds were frozen and sectioned, and then observed by scanning electron microscopy. Figure 3 ,in Figure 3 -A is the non-color-developed state SEM observation picture, Figure 3 -B is a fluorescence display state observation image, which shows that neural stem cells and Schwann cells have filled the porous inner cavity of the scaffold.

[0042] (4) Overexpression of MuSK in the scaffold

[0043] TrkC gene-modified NSCs and NT-3 gene-modified SCs were co-cultured in the main body of the scaffold, with a total cell volume of 5×10 5 , the TrkC gene-modified NSCs suspension was injected into the scaffold at a ratio of 3:1; the NT-3 gene-modified SCs were planted around the neural stem cells, and then the scaffold was placed in a 24-well plate containing 300μl of 10% FBS DMEM / F12, with one scaffold material per well. Cultured in a 37°C, 5% CO2 incubator for 14 days, the culture medium was replaced every other day, and AAV-CMV-MuSK-GFP (DJ type) with an MOI value of 100 was added to the serum-free culture medium to infect the neurons in the neural network tissue on the 10th day of culture. After 24 hours of culture, the new culture medium was replaced, and the engineered scaffold became a spinal cord neural scaffold overexpressing MuSK;

[0044] (5) the scaffold body is immersed in rAAV-hSyn-agrin-mCherry adeno-associated virus culture medium;

[0045] Add rAAV-hSyn-agrin-mCherry adeno-associated virus culture medium to the culture medium of the spinal cord nerve scaffold overexpressing MuSK, the concentration of rAAV-hSyn-agrin-mCherry adeno-associated virus is MOI=100, and the tubular mantle is coated with the columnar scaffold body to form a scaffold, which is cultured for implantation.

[0046] In the process of engineering scaffold construction, in particular, the TrkC gene-modified NSCs and the NT-3 gene-modified SCs were co-cultured in the main body of the prose scaffold, and after 14 days of induction culture, the engineering scaffold was subjected to immunofluorescence staining to identify the differentiation of NSCs. NSCs highly expressed the mature neuron marker Map2, and a small part expressed the astrocyte marker GFAP and the oligodendrocyte marker MBP; the experimental results showed that the recombinant adeno-associated virus of the MuSK gene constructed in this example can successfully infect neurons differentiated from NSCs and make the neurons express MuSK (see Figure 4 ).

[0047] Example 2

[0048] To facilitate the transplantation effect test of tissue engineering scaffolds, adult female SD rats were selected as a complete spinal cord transection model. In this example, 6 adult female SD rats were selected, of which 2 were used as experimental group 1, 2 were used as experimental group 2, 1 was used as a blank control group, and 1 was used as a positive control group.

[0049] All adult female SD rats were injected with cyclosporine A at a dose of 1 mg / kg body weight 3 days before modeling. Sodium pentobarbital (25-35 mg / kg) was injected intraperitoneally for anesthesia during the complete spinal cord transection surgery. After skin preparation and disinfection, the body position was fixed, and the skin and superficial fascia were incised under sterile conditions. The surgical area was fixed with a homemade retractor, and the muscles and ligaments were bluntly separated to clearly expose the T9 spinous process and vertebral arch. The T9 spinous process was gently lifted with toothed forceps, and the pedicles were gently bitten open along the T9-T10 vertebral arch space with ophthalmic needle forceps, and the T9 vertebral arch was gradually bitten off to expose the T10 spinal cord. After cutting the dura mater with straight-tipped trabecular scissors, the spinal cord was quickly and completely transected again. The spinal cord was quickly and completely transected again 2 mm away from this incision, and the spinal cord between the two knives was tweezed out with pointed nuclear forceps. After sufficient hemostasis, the cut site of the spinal cord of the rats in experimental group 1 was filled with about 2 mm of spinal cord neural scaffolds overexpressing MuSK soaked in rAAV-hSyn-agrin-mCherry adeno-associated virus culture medium; the cut site of the spinal cord of the rats in experimental group 2 was filled with about 2 mm of spinal cord neural scaffolds overexpressing MuSK that were not soaked in rAAV-hSyn-agrin-mCherry adeno-associated virus culture medium; the rats in the blank control group did not receive transplanted materials; the rats in the positive control group received transplants back into their autologous spinal cord segments.

[0050] After transplantation, the animals were sutured layer by layer in the order of muscle layer, subcutaneous tissue, and skin. Each animal was injected intramuscularly with 1 mL of penicillin 160,000 units for 3 consecutive days after surgery, and artificial urination was performed twice a day. To prevent the unhealed wound from being bitten open, the animals were fed in a single cage for at least 5 days after surgery. The animals were raised for a total of 60 days, during which time they were given cyclosporine A to suppress immune rejection, heat preservation, natural light exposure time, and adequate diet.

[0051] The physical characteristics of rats in experimental group 1, experimental group 2, blank control group, and positive control group were compared respectively:

[0052] (1) Evaluation of the recovery of hind limb motor function in rats:

[0053] Rats were subjected to the Basso-Beattie-Bresnahan (BBB) ​​test and the inclined grid climbing test weekly before surgery and after SCI. That is, during the BBB test, rats were allowed to freely explore the open area for 5 minutes to assess the frequency of hindlimb movement, range of joint motion, weight bearing, and coordination. The BBB test followed a double-blind process. A 45° inclined grid climbing test was performed in the last week to qualitatively assess the accuracy of foot placement and coordination. The test results showed that the hindlimb motor function of rats in experimental group 1 and the positive control group recovered well, while the hindlimb motor function of rats in the blank control group had no motor function, and the hindlimb motor function of rats in experimental group 2 recovered poorly.

[0054] (2) Electrophysiological testing

[0055] Eight weeks after the transplantation, the TMSMEP data acquisition and analysis system was used to record cortical motor evoked potentials to evaluate the conductivity of the cortical motor pathway. Specifically, the rats in each group were anesthetized (using 1% sodium pentobarbital, 30 mg / kg), and the recording electrodes were inserted into the Achilles tendon and tibialis anterior muscle of the rats. The rats were electrically stimulated according to the standard protocol and the cortical motor evoked potential (CMEP) was recorded. Each rat was given 10 single pulse stimulations (lasting 50 milliseconds), and 20 stable waveforms were collected for each test of each rat, and the average was taken. By comparing the amplitude and latency of CMEP, we found that the conductivity of the cortical motor pathway of rats in experimental group 1 was similar to that of the positive control group, while the conductivity of the cortical motor pathway of the blank control group was 0, and the conductivity of the cortical motor pathway of rats in experimental group 2 was much lower than that of the positive control group.

[0056] (3) Immunofluorescence detection

[0057] The rats in each group were sampled and subjected to immunofluorescence histochemistry to detect the expression of synaptophysin (SYP) and postsynaptic density 95 (PSD95). The regenerated axons of CST entered the injury area and came into close contact with the transplanted cells, and SYP and PSD95 were expressed in the area where they were in close contact. Compared with experimental group 2, the expression of SYP and PSD95 in experimental group 1 was increased.

[0058] The above three test results clearly show that the overexpressed MuSK spinal cord nerve scaffold immersed in rAAV-hSyn-agrin-mCherry adeno-associated virus culture medium can activate the formation of acetylcholine receptor-low-density lipoprotein receptor-related protein complex on the postsynaptic membrane, cause tyrosine phosphorylation, activate the target neuron mitogen-activated protein kinase cyclic adenosine monophosphate effector element binding protein signaling pathway, promote the transmission of synaptic signals between neurons, and thus promote the repair of spinal cord injury. The repair effect of spinal cord injury is close to that of the autologous spinal cord segment.

[0059] It can be seen that the engineering scaffold of the present invention enables neural stem cells to overexpress tyrosine receptor kinase (MuSK) through AAV-CMV-MuSK-GFP (DJ type) adeno-associated virus, and transfects neuronal cells in the injured area through rAAV-hSyn-agrin-mCherry adeno-associated virus with a sustained release effect in the inner columnar porous structure, thereby promoting the overexpression of agrin, and then the agrin of the presynaptic membrane activates the formation of acetylcholine receptor low-density lipoprotein receptor-related protein complex on the postsynaptic membrane, causing tyrosine phosphorylation, activating the target neuron mitogen-activated protein kinase cyclic adenosine monophosphate response element binding protein signaling pathway, promoting the transmission of synaptic signals between neurons, and thus promoting the repair of spinal cord injury.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An engineering scaffold for repairing spinal cord injury, characterized in that: The engineering scaffold includes a columnar scaffold body and a tubular mantle wrapped around the outer wall of the columnar scaffold body, wherein the columnar scaffold body is a porous structure made of porous gelatin sponge, and the material of the tubular mantle is a biodegradable material; neural stem cells and Schwann cells are distributed in the porous structure of the columnar scaffold body, wherein the neural stem cells are transfected with AdV-TrkC, and the Schwann cells are transfected with AdV-NT-3; AAV-CMV-MuSK-GFP (DJ type) adeno-associated vector virus and rAAV-hSyn-agrin-mCherry adeno-associated vector virus are also diffusely distributed in the porous structure of the columnar scaffold body.

2. The engineering scaffold for repairing spinal cord injury according to claim 1, characterized in that: The vector of the AAV-CMV-MuSK-GFP (DJ type) adeno-associated vector virus can also be adenovirus and lentivirus.

3. The engineering scaffold for repairing spinal cord injury according to claim 1, characterized in that: The rAAV-hSyn-agrin-mCherry adeno-associated vector virus carries the DNA sequence of the agrin gene as shown in SEQ ID NO:

1.

4. The engineering scaffold for repairing spinal cord injury according to claim 3, characterized in that: The vector of the rAAV-hSyn-agrin-mCherry adeno-associated vector virus can also be adenovirus and lentivirus.

5. The engineering scaffold for repairing spinal cord injury according to claim 1, characterized in that: The inner columnar porous structure is loaded with NT-3.

6. The engineering scaffold for repairing spinal cord injury according to claim 1, characterized in that: The material of the tubular mantle is acellular optic nerve epimembrane.

7. The method for constructing an engineering scaffold for repairing spinal cord injury according to any one of claims 1 to 6, characterized in that: The steps include: (1) Construction of the main columnar support A porous gelatin sponge is used to prepare a columnar stent body with a porous structure, and a biodegradable material is used to prepare a tubular mantle membrane. The tubular mantle membrane and the columnar stent body are sterilized and then used for standby; (2) Seed cell culture A recombinant adenovirus carrying the tyrosine kinase C gene, AdV-TrkC, was constructed, neural stem cells were cultured in vitro, and the neural stem cells were transfected with AdV-TrkC to prepare a TrkC gene-modified neural stem cell suspension; a recombinant adenovirus carrying the neurotrophin-3 gene, AdV-NT-3, was constructed, Schwann cells were cultured in vitro, and Schwann cells were transfected with AdV-NT-3 to prepare a Schwann cell suspension overexpressing NT-3; (3) Scaffold implantation and culture of seed cells Injecting a suspension of TrkC gene-modified neural stem cells into the scaffold; injecting a suspension of NT-3-overexpressing Schwann cells around the TrkC gene-modified neural stem cells, culturing the scaffolds on which the seed cells have been planted, allowing the TrkC gene-modified neural stem cells to differentiate into functional neurons, allowing the NT-3-overexpressing Schwann cells to differentiate into oligodendrocytes, and further culturing the scaffolds in vitro, allowing the oligodendrocytes surrounding the neurons to continue to differentiate into spinal cord nerve fiber structures; (4) Overexpression of MuSK in the scaffold A recombinant adenovirus AAV-CMV-MuSK-GFP (DJ type) expressing a tyrosine receptor kinase gene was constructed, and AAV-CMV-MuSK-GFP (DJ type) was added to the culture medium of the scaffold that differentiated into the spinal nerve fiber structure. After culturing for 24 hours to form the spinal nerve scaffold overexpressing MuSK, the culture medium was replaced with serum-free medium. (5) Constructing a recombinant adenovirus rAAV-hSyn-agrin-mCherry carrying the aggregation protein gene, adding the rAAV-hSyn-agrin-mCherry adeno-associated virus culture medium to the culture medium of the spinal cord nerve scaffold overexpressing MuSK, and then wrapping the tubular mantle around the columnar scaffold body to form a scaffold for culture and standby use.

8. The method for constructing an engineering scaffold for repairing spinal cord injury according to claim 7, characterized in that: The drop volume ratio of the TrkC gene modified neural stem cell suspension to the NT-3 overexpressing Schwann cell suspension is 3:

1.

9. The method for constructing an engineering scaffold for repairing spinal cord injury according to claim 7, characterized in that: The concentration of the AAV-CMV-MuSK-GFP (DJ type) adeno-associated virus was MOI=100.

10. The method for constructing an engineering scaffold for repairing spinal cord injury according to claim 6, characterized in that: The virus concentration of the rAAV-hSyn-agrin-mCherry adeno-associated virus culture solution is MOI=100.

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