Application of virus-modified microsomes electrospun scaffold by inducing fibroblast in situ reprogramming in nerve injury

Abstract

The present application relates to the technical field of biological medicine, and more particularly to the application of virus modified micro-gel electrospun scaffold in nerve injury by inducing in-situ reprogramming of fibroblasts. The present application provides a micro-gel electrospun scaffold which is modified with a polydopamine coating and grafted with a shPTB lentivirus vector; the matrix of the scaffold is prepared from brain-derived neurotrophic factor, poly-L-lactic acid and hyaluronic acid. The present application also provides a preparation method and use of the scaffold. Compared with conventional nerve injury biomaterial therapy, the micro-gel electrospun scaffold system can improve multiple characteristics such as a large number of inflammatory factors generated in the microenvironment after injury, lack of neurotrophic factors, and scar tissue proliferation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of virus-modified micro-sol-gel electrospinning scaffold in nerve injury by inducing fibroblast in situ reprogramming. BACKGROUND

[0002] Spinal cord injury (SCI) is often caused by direct or indirect injury to the spinal cord tissue due to traffic accidents, high-altitude falls, etc., resulting in sensory, motor, urinary and sexual dysfunction, and autonomic nervous dysfunction. SCI patients not only face great physical and psychological pain, but also face huge economic expenditure. Treating SCI has become a problem of concern to the whole society. At present, the main clinical treatment methods for SCI are to relieve the pressure of the vertebral plate on the spinal cord tissue through surgery, hormone shock therapy and a large number of rehabilitation therapy after surgery, but the treatment effects are poor. Due to the extremely limited self-repairing ability of the spinal cord tissue, the early inflammatory storm after SCI, the lack of neurotrophic factors, and the formation of scar tissue, neurons are difficult to grow into the lesion site, which limits the repair of the spinal cord nerve tissue. Therefore, the repair of SCI has been an international medical problem that has plagued the clinic.

[0003] In the early stage of SCI, neuronal apoptosis and necrosis, tissue vascular damage, and possible rupture of the spinal cord nerve bundle occur first; then secondary inflammatory reactions of varying severity occur, including activation of various immune cells, such as secretion of IL-1β and TNF-α by macrophages, secretion of IL-10 by neutrophils, and secretion of IL-6 by T cells, leading to inflammatory changes in the microenvironment, further exacerbating spinal cord and peripheral tissue edema, and aggravating ischemia of the damaged tissue; in the later stage of SCI, cavities, glial scars and fibrous scars form in the injury area, hindering the regeneration of neurons at the injury site and functional recovery, and affecting the repair of SCI. Axonal regeneration, orderly arrangement and effective connection are considered to be the only way for functional recovery after injury. Therefore, scar tissue is a key factor that inhibits axonal regeneration and functional recovery. Scar tissue is divided into glial scar formed by activation of reactive astrocytes and fibrous scar formed by activation of fibroblasts according to the composition of the cell matrix, and the fibrous scar is a major physical factor that hinders axonal regeneration in the pathophysiological activity after SCI due to its one-way irreversibility. The fibrous scar not only physically hinders axonal regeneration, but also secretes various axon inhibitory factors, which seriously affects the recovery of neural function. Therefore, improving the inflammatory microenvironment after SCI and inhibiting the formation of fibrous scar may be one of the keys to the recovery of neural function after SCI. SUMMARY

[0004] Therefore, the present application aims to solve the technical problem of providing application of virus-modified micro-sol-gel electrospinning scaffold in nerve injury by inducing fibroblast in situ reprogramming.

[0005] The present application provides a micro-gel electrospun scaffold which is surface modified with a polydopamine coating and grafted with a shPTB lentivirus vector; the matrix thereof is prepared from brain-derived neurotrophic factor, poly-L-lactic acid and hyaluronic acid.

[0006] The micro-gel electrospun scaffold provided by the present application comprises a BDNF-coated HA core and a PLLA shell, and the surface thereof is grafted with an LV-shPTB through a polydopamine layer. By adjusting the ratio of HA to PLLA, the controllable release of BDNF is achieved, and the scaffold has certain mechanical strength.

[0007] The micro-gel electrospun scaffold described in the present application is prepared from poly-L-lactic acid and hyaluronic acid with different proportions and molecular weights. The molecular weight of the poly-L-lactic acid is 60-110 kDa, preferably 100 kDa; the molecular weight of the hyaluronic acid is 40-100 kDa, preferably 20-40 kDa. Research shows that the molecular weight of PLLA and HA has little effect on the results, and scaffolds with good performance can be prepared from HA or PLLA with different molecular weights. The ratio of PLLA to HA affects the performance of the micro-gel electrospun scaffold, especially when the ratio of PLLA is too high, the scaffold cannot be spun, and when the ratio of PLLA is too low, the drug loading rate is insufficient. As a preferred, the mass ratio of HA to PLLA is 1:1000.

[0008] In the present application, BDNF is coated in the core-shell structure composed of PLLA, in addition to this, the core-shell structure of the micro-gel electrospun scaffold can also encapsulate other types of neurotrophic factors and / or other small molecule proteins with broad biological activity. The molecular weight of the small molecule protein is not more than 100 kDa, including but not limited to NGF, NT3.

[0009] The polydopamine described in the present application is grafted by covalent crosslinking between the ortho-phenolic group and the amino group of dopamine hydrochloride under alkaline conditions, thereby self-aggregating on the surface of the matrix, and further grafting the lentivirus vector. The lentivirus vector described in the present application can be all types of viruses, and the lentivirus vector is preferred in the present application, and further, the lentivirus vector is a shRNA carrier, i.e. a short hairpin RNA lentivirus vector. The backbone vector of the lentivirus vector is pLVTHM. In order to treat different diseases, the shRNA can be connected to the nucleic acid encoding different active proteins in the lentivirus backbone vector. In this application, in order to achieve the improvement and treatment of nerve injury, the virus vector is a lentivirus vector comprising PTBP1 short hairpin RNA.

[0010] The polydopamine modification of the surface of the micro-sol gel electrospun scaffold can significantly improve the hydrophilicity of PLLA-HA without changing the mechanical properties thereof, and adsorb excessive inflammatory cytokines in the early / acute stage of spinal cord injury. The controlled release of BDNF achieved by the core-shell structure provides long-term neurotrophic factors for the growth of neurons and the extension of fibers. Therefore, the scaffold can release LV-shPTB, infect fibroblasts, and promote the reprogramming of the fibroblasts into neurons, increase the proportion of neurons, and effectively reduce the formation of scar tissue in the late / subacute to chronic stage of spinal cord injury.

[0011] The preparation method of the micro-sol gel electrospun scaffold comprises the following steps:

[0012] Poly-L-lactic acid is mixed with dichloromethane to prepare solution A;

[0013] A brain-derived neurotrophic factor solution is mixed with a hyaluronic acid hydrogel to prepare solution B;

[0014] After the solution A, the solution B and a surfactant are mixed, the micro-sol gel electrospun solution is prepared by mixing with a spinning solution solvent;

[0015] The matrix is prepared by spinning the micro-sol gel electrospun solution;

[0016] After the dopamine hydrochloride solution is mixed with the matrix and incubated, the micro-sol gel electrospun scaffold is prepared by mixing with a shPTBP1 lentivirus vector and incubating.

[0017] The core-shell structure of the micro-sol gel electrospun scaffold prepared by the method can continuously release bioactive factors, and the polydopamine coating on the surface of the scaffold can adsorb a viral vector of short hairpin RNA (shRNA), the preparation method has mild conditions and simple steps.

[0018] In the embodiment of the present application, in the solution A, the mass ratio of poly-L-lactic acid to dichloromethane is (0.25-1):4, and preferably, the mass ratio of poly-L-lactic acid to dichloromethane is 0.5:4.

[0019] In the embodiment of the present application, in the solution B, the concentration of the brain-derived neurotrophic factor solution is 100-1000 μg / ml; the mass fraction of HA in the hyaluronic acid hydrogel is 00.5%-2%; and the volume ratio of the brain-derived neurotrophic factor solution to the hyaluronic acid hydrogel is 1:(2-8). Preferably, the concentration of the brain-derived neurotrophic factor solution is 500 μg / ml; the mass fraction of HA in the hyaluronic acid hydrogel is 1%; and the volume ratio of the brain-derived neurotrophic factor solution to the hyaluronic acid hydrogel is 1:5.

[0020] In the embodiment of the present application, the surfactant is Span-80;

[0021] The spinning solution solvent is N,N-dimethylformamide.

[0022] In the embodiment of the present application, the ratio of the solution A, the solution B, the surfactant, and the spinning solution solvent is (4g-6g):(40μL-80μL):(0.01g-0.02g):(2g-3g), and preferably, the ratio of the solution A, the solution B, the surfactant, and the spinning solution solvent is 4.5g:60μL:0.01g:2g.

[0023] In the embodiment of the present application, the electrospinning condition is 10-15kV voltage, 10-15mL / h flow rate, 10-15cm needle-target distance, and 16-20G needle size. Preferably, the electrospinning condition is 10-15kV voltage, 10mL / h flow rate, 12cm needle-target distance, and 20G needle size.

[0024] In the embodiment of the present application, the dopamine hydrochloride solution is 10mM Tris buffer containing 2mg / ml dopamine hydrochloride.

[0025] The condition for mixing and incubating the dopamine hydrochloride solution with the matrix is 12-36h incubation at room temperature in the dark, and preferably 24h.

[0026] In the embodiment of the present application, the condition for mixing and incubating with the shPTBPl lentivirus vector is 15-30min standing at 4℃, and preferably 15min.

[0027] More specifically, the preparation method of the micro-sol gel electrospun scaffold of the present application comprises:

[0028] Preparation of polydopamine coating: the matrix of the micro-sol gel electrospun scaffold is immersed in 10mM Tris buffer containing 2mg / ml dopamine hydrochloride at room temperature in the dark for 24h, and then cleaned with sterilized double-distilled water to remove free polydopamine; the room temperature is preferably 18-30℃.

[0029] Virus grafting: the virus is directly added on the surface of the micro-sol gel electrospun scaffold, and then standing at 4℃ for 15min, and then washing off the unabsorbed virus with sterile PBS.

[0030] In some specific embodiments, the preparation of the matrix of the micro-sol gel electrospun scaffold comprises:

[0031] The PLLA is mixed with dichloromethane and stirred for 1h.

[0032] The bioactive factor (500μg / ml) is uniformly mixed with 5 times the volume of 1wt% HA hydrogel;

[0033] The HA hydrocolloid and Span-80 encapsulating bioactive factors are added into the dichloromethane solution of PLLA, and a uniform and stable water-in-oil emulsion is obtained by stirring at room temperature for 30 minutes;

[0034] The water-in-oil emulsion is dissolved in N,N-dimethylformamide to obtain a micro-gel electrospinning solution. The electrospinning conditions are 10-15 kV voltage, 10 mL / h flow rate, 12 cm needle-target distance and 20G needle size.

[0035] The preparation method provided by the present application has simple process parameters and strong reproducibility, and can be stably implemented within the above parameter range. Compared with other conditions, the optimal effect can be achieved within the preferred range described in the examples of the present application.

[0036] Further, the present application also provides the use of the micro-gel electrospinning scaffold as described above or prepared by the preparation method as described above in the preparation of a product for treating nerve injury.

[0037] Further, the present application also provides a product for treating nerve injury, which comprises the micro-gel electrospinning scaffold as described above or prepared by the preparation method as described above. The product for treating nerve injury described in the present application also comprises other drugs having activity in treating nerve injury.

[0038] Further, the present application also provides a method for treating nerve injury, which comprises implanting the product described in the present application into the injury site.

[0039] The treatment of nerve injury described in the present application comprises inducing fibroblasts to be reprogrammed into neurons in situ and to be distributed in a targeted manner, reducing scar tissue and / or increasing the number of neurons at the injury site. The nerve injury comprises central nerve injury or lesion, peripheral nerve injury or lesion. Further, the nerve injury is sciatic nerve injury or spinal cord injury.

[0040] The present application provides a micro-gel electrospinning scaffold which is surface-modified with a polydopamine coating and grafted with a shPTB lentivirus vector; the matrix thereof is prepared from brain-derived neurotrophic factor, poly-L-lactic acid and hyaluronic acid. The present application also provides a preparation method and use of the scaffold. Compared with traditional nerve injury biomaterial therapy, the micro-gel electrospinning scaffold system can improve multiple characteristics of the microenvironment after injury, such as a large amount of inflammatory factors generated, lack of neurotrophic factors and hyperplasia of scar tissue. The LV-shPTB induces fibroblasts to be reprogrammed into neurons in situ and to be distributed in a targeted manner, reduces scar tissue and increases the number of neurons at the injury site. At the same time, the core-shell structure releases BDNF and the polydopamine coating adsorbs inflammatory factors, which are beneficial to the growth and development of reprogrammed neurons while improving the microenvironment of nerve injury. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The schematic diagram of the polydopamine modified micro-gel electrospun scaffold system of the application;

[0042] Figure 2 The surface scanning electron microscope image of the polydopamine modified micro-gel electrospun scaffold of the application;

[0043] Figure 3 The fiber diameter measurement of the polydopamine modified micro-gel electrospun scaffold of the application;

[0044] Figure 4 The water contact angle measurement of the micro-gel electrospun scaffold of the application before and after polydopamine modification;

[0045] Figure 5 The projection electron microscope image of the core-shell structure of the micro-gel electrospun scaffold of the application;

[0046] Figure 6 The tensile test and Young's modulus of the micro-gel electrospun scaffold of the application before and after polydopamine modification;

[0047] Figure 7 The core-shell structure of the micro-gel electrospun scaffold of the application encapsulating the release curve of neurotrophic factor BDNF;

[0048] Figure 8 The shPTB lentiviral vector sequence of the application

[0049] Figure 9 The shPTB lentiviral vector constructed by the application induces the reprogramming of rat embryonic fibroblasts to neurons;

[0050] Figure 10 The schematic diagram of implanting the animal experiment material before and after implantation;

[0051] Figure 11 BBB score of each experimental group (**P<0.01, n=3);

[0052] Figure 12 HE staining of spinal cord tissue in the injury area of each experimental group;

[0053] Figure 13 Fibronectin immunofluorescence staining of spinal cord tissue in the injury area of each experimental group;

[0054] Figure 14 Glial fibrillary acidic protein immunofluorescence staining of spinal cord tissue in the injury area of each experimental group;

[0055] Figure 15 Class III beta-tubulin immunofluorescence staining of spinal cord tissue in the injury area of each experimental group;

[0056] Figure 16The nerve filament protein 200 immunofluorescence staining of the spinal cord tissue in the damaged area of each experimental group. DETAILED DESCRIPTION

[0057] The application provides application of a virus-modified micro-sol gel electrospun scaffold in nerve injury by inducing in-situ reprogramming of fibroblasts, and those skilled in the art can refer to the content herein and appropriately improve process parameters to achieve the application. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the application. The method and application of the application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the application to achieve and apply the technical solution of the application.

[0058] The application provides an implantable poly-L-lactic acid (PLLA)-hyaluronic acid (HA) micro-sol gel electrospun scaffold loaded with exogenous recombinant brain-derived neurotrophic factor (BDNF), which can stably release BDNF through a core-shell structure; a polydopamine coating on the surface of the scaffold can adsorb adverse factors in the microenvironment of spinal cord injury, and regulate the inhibitory microenvironment caused by the injury; a lentiviral vector (LV-shPTB) of shPTB grafted on the polydopamine coating on the surface of the micro-sol gel electrospun scaffold reduces the expression of PTBP1 in the damaged area, induces in-situ reprogramming of fibroblasts to neurons, thereby reducing the area of fibrous scar, providing an excellent platform for the growth of axons and the migration of stem cells, and being beneficial to the regeneration and repair of spinal cord injury; and the PLLA-HA micro-sol gel scaffold prepared in the application has a directional structure, can reprogram the directional growth of nerve cells, and promotes the survival of neurons. In addition, the sol gel electrospun scaffold has a certain mechanical strength, and can reduce secondary damage to spinal cord tissue caused by activity.

[0059] The test materials used in the application are ordinary commercially available products and can be purchased in the market.

[0060] The BDNF is purchased from Peprotech 450-02, and the molecular weight is 27.0 kDa, and see https: / / www.peprotech.com / zh / recombinant-humanmurinerat-bdnf.

[0061] The lentiviral vector of shPTB is purchased from Suzhou Dongling Biotechnology Co., Ltd.

[0062] The application will be further described below in combination with examples:

[0063] Example 1: Preparation of a micro-sol gel electrospun scaffold grafted with a lentivirus and slowly releasing a cell factor

[0064] The application will be further described below in combination with examples:Figure 1 Schematic diagram of the micro-microgel electrospun scaffold virus delivery tool constructed according to the present application.

[0065] (1) Preparation of micro-microgel electrospun scaffold: 0.5 g of PLLA was mixed with 4 g of dichloromethane and stirred for 1 hour. To encapsulate BDNF, 10 ul of BDNF (500 pg / ml) was uniformly mixed with 50 ul of 1 wt% HA hydrogel. Then the BDNF-encapsulated HA hydrogel and 0.01 g of Span-80 were added to the dichloromethane solution of PLLA. A uniform and stable water-in-oil emulsion was obtained by stirring at room temperature for 30 minutes. Finally, the water-in-oil emulsion was dissolved in 2 g of N,N-dimethylformamide to obtain a micro-microgel electrospun solution. The electrospinning conditions were 10-15 kV voltage, 10 mL / h flow rate, 12 cm needle-target distance and 20G needle size.

[0066] (2) Preparation of polydopamine coating: the micro-microgel electrospun scaffold was immersed in a dopamine hydrochloride solution (2 mg / ml dopamine hydrochloride solution prepared with 10 mM Tris buffer, pH = 8.5) in the dark at room temperature for 24 hours, then washed with sterilized double distilled water to remove free polydopamine.

[0067] (3) Virus grafting: the shPTB lentiviral vector was directly added dropwise (20 pL / mm 3 ) on the surface of the micro-microgel electrospun scaffold, and then incubated at 4°C for 15 min, and then washed with sterile PBS to remove unabsorbed viruses.

[0068] (4) Application of micro-microgel electrospun scaffold system: the micro-microgel electrospun scaffold was implanted locally in situ at the site of spinal cord injury in rats.

[0069] Example 2: Characterization of materials related to the micro-microgel electrospun scaffold system

[0070] (1) Scanning electron microscopy (SEM) image of the polydopamine coating modified micro-microgel electrospun scaffold

[0071] To observe the microstructure and orientation of the electrospun fibers, the fibers were coated with gold and photographed with SEM at 3 kV ( Figure 2 ). 50 fibers were randomly selected and the diameter was measured using ImageJ software to obtain the average fiber diameter ( Figure 3 ). The average diameter of the polydopamine coating modified micro-microgel electrospun fiber scaffold was 0.59 ± 0.15 pm. Observation of the SEM image showed that the fibers were roughly parallelly distributed. After soaking the fibers in the PDA solution and grafting with lentivirus, uniform spherical particles (orange arrows) were observed on the fibers.

[0072] (2) Water contact angle measurement of micro-gel electrospun scaffold modified by polydopamine coating

[0073] To evaluate the surface hydrophilicity of micro-gel electrospun scaffold modified by polydopamine coating, the micro-gel electrospun scaffold before and after modification was subjected to 10 seconds of handle-free drop test using WCA measurement instrument. Figure 4 The results showed that the water contact angle of micro-gel electrospun scaffold was reduced from 114.57 ± 1.80° before modification to 0°, which indicated that the micro-gel electrospun scaffold modified by polydopamine had good hydrophilicity.

[0074] (3) Transmission electron microscopy (TEM) images of micro-gel electrospun scaffold modified by polydopamine coating

[0075] To confirm that we successfully constructed the core-shell structure of micro-gel electrospun scaffold, we observed the internal structure of micro-gel electrospun scaffold by TEM, and the results showed that the core-shell structure of micro-gel electrospun scaffold was successfully constructed. Figure 5

[0076] (4) Tensile test of micro-gel electrospun scaffold modified by polydopamine coating

[0077] Sometimes, the surface modification of fibers can affect their physicochemical properties. Therefore, we conducted a tensile test on the surface-modified electrospun fibers. Figure 6 The results showed that after the micro-gel electrospun fibers were immersed in PDA solution, their mechanical properties did not change significantly. After modification by polydopamine coating, the micro-gel electrospun scaffold still maintained its original tensile properties.

[0078] (5) BDNF release curve of micro-gel electrospun scaffold modified by polydopamine coating

[0079] As one of the most commonly used neurotrophic factors in the treatment of spinal cord injury, BDNF can promote myelination and maintain the survival of damaged axon neurons. To achieve the sustained release of BDNF in the area of spinal cord injury, we constructed P / H-BNDF-PDA fibers based on core-shell structure. ELISA results showed that BDNF could be continuously released for more than two weeks. Figure 7

[0080] Example 3: Study on micro-gel electrospun scaffold modified by polydopamine coating, grafted with shPTB lentivirus and loaded with BDNF for the treatment of spinal cord injury

[0081] (1) Experimental animals

[0082] ​​50 female rats were randomly divided into 5 groups and housed in the animal experimental center of Suzhou University. The treatment method was in line with the Guidelines for the Treatment of Laboratory Animals and was approved by the Ethics Committee of Suzhou University.

[0083] (2) shPTB lentivirus vector (LV-shPTB) induced reprogramming of rat embryonic fibroblasts (REFs) to neurons

[0084] As lentivirus has the ability of sustained transgene expression, it is widely used as a gene vector. After the successful extraction of rat embryonic fibroblasts, we infected REFs with LV-shPTB. 48 hours after infection, we detected the infection efficiency of LV-shPTB and control lentivirus vector (LV-shCTR) in REFs by the expression of green fluorescent protein (GFP). Representative IF images showed that the GFP expressed in LV-shPTB infected cells overlapped with the pan-neuronal marker Tuj1, while no such colocalization was observed in LV-shCTR infected cells. In addition, only DAPI positive cells were observed in uninfected cells (Empty). Figure 8

[0085] (3) Construction of spinal cord injury rat model and implantation of materials

[0086] Twenty-five female rats were anesthetized with 2% sodium pentobarbital solution (50 mg / kg). The paraspinal muscles, lamina and spinal cord were exposed in turn. The entire spinal cord was excised 4 mm from the T8 position. The same operation was performed on the sham group, but the spinal cord was not excised. In the first week after surgery, all rats were injected intramuscularly with penicillin every day, and the bladder of the spinal cord injury rats was manually emptied twice a day. The transected spinal cord injury rats were modeled and divided into 4 groups according to the implanted scaffold (axial length: 4 mm, radius: 1.5 mm, n = 10 / group) at the injury site, named P / H-PDA group, P / H-BDNF-PDA group, P / H-PDA-shPTB group, P / H-BDNF-PDA-shPTB group, respectively. At the same time, spinal cord injury rats without implanted scaffold were set as control group (sham, n = 10). Figure 9 Schematic diagram of implantation of materials before and after.

[0087] (4) Experimental sampling

[0088] Eight weeks after spinal cord injury surgery, rats were perfused with 4% paraformaldehyde through the heart, and then the entire spinal cord tissue was taken out. After 4 hours of fixation, the spinal cord tissue was dehydrated with 20% and 30% sucrose solution gradient in turn. The Tissue-Tek OCT embedded spinal cord specimen was sectioned in the sagittal plane, with a thickness of 6 μm.

[0089] ​(5) Motor function and histological evaluation of spinal cord injured rats after implantation of scaffolds

[0090] a. BBB score per week after implantation of scaffolds.

[0091] Basso, Beattie and Bresnahan (BBB) score was performed weekly to assess the motor function of the hindlimbs of rats. In all groups, there was a different degree of recovery of the motor function of the hindlimbs over time ( Figure 10 ). However, the P / H-BDNF-PDA-shPTB group achieved the best recovery of motor function.

[0092] b. HE staining of spinal cord tissue

[0093] By hematoxylin-eosin (H&E) staining, cavities and scar tissue were observed near the injured area of spinal cord injured rats without implantation of scaffolds ( Figure 11 ). However, after implantation of scaffolds, the area of cavities was significantly reduced and many host cells were found to enter the scaffolds, especially in the P / H-BDNF-PDA-shPTB and P / H-PDA-shPTB groups. In addition, newly formed tissue and degenerated fibers coexisted in the injured area. These data well demonstrated that the implanted scaffolds coexisted well with the host tissue, filled the defect area, and the P / H-BDNF-PDA-shPTB electrospun scaffold could better promote the recovery of motor function of spinal cord injured rats than the other groups.

[0094] (6) Evaluation of scar tissue in the spinal cord injury area

[0095] a. Immunofluorescence staining of fibronectin in the spinal cord injury area

[0096] Fibrotic scar gradually formed after spinal cord injury, and fibronectin is an important component of fibrotic scar. Different degrees of fibrotic scar were observed in the injured spinal cord by the expression of fibronectin. As expected, immunofluorescence staining showed that GFP-positive cells were observed in the fibrotic scar in the P / H-BDNF-PDA-shPTB and P / H-PDA-shPTB groups ( Figure 12 ). In addition, the area of fibronectin in these two groups was significantly reduced, while the other three groups still had a high expression of fibronectin. This indicates that LV-shPTB grafted on the surface of the scaffold successfully infected the cells within the fibrotic scar, reducing the formation of fibrotic scar.

[0097] b. Immunofluorescence staining of glial fibrillary acidic protein (GFAP) in the spinal cord injury area

[0098] It is well known that glial scar is also an important component of scar tissue after spinal cord injury. Previous studies have shown that PTB expression affects the activity of astrocytes. Glial fibrillary acidic protein (GFAP) is often used to represent reactive astrocytes, which play a crucial role in the formation of glial scar. Therefore, we used GFAP antibody staining to evaluate the changes of reactive astrocytes after scaffold implantation. Surprisingly, the frequency of reactive astrocytes in the injured area decreased with the implantation of P / H-BDNF-PDA-shPTB and P / H-PDA-shPTB scaffolds, with the P / H-BDNF-PDA-shPTB group decreasing the most Figure 13 ) In summary, P / H-BDNF-PDA-shPTB scaffolds can effectively reduce PTB expression, reduce fibrosis and glial scar area around the injured site after spinal cord injury, and facilitate axon growth and migration.

[0099] (7) Evaluation of nerve fibers in the spinal cord injury area

[0100] a. Immunofluorescence staining of nerve class III β-tubulin (Tuj1) in the spinal cord injury area

[0101] Reducing scar tissue and reprogramming fibroblasts should encourage more neurons to cross the injury site. We then evaluated the number of rostral and caudal neurons in the injured area of each group. As a pan-neuronal marker, Tuj1 expression roughly reflects the overall morphology of neurons in the injured area. The area of Tuj1-positive cells in the P / H-BDNF-PDA-shPTB group and the P / H-PDA-shPTB group was significantly higher than that in the other groups, although there was no difference between the two groups Figure 14 ).

[0102] b. Immunofluorescence staining of glial neurofilament protein 200 (neurofilament-H, NF-200) in the spinal cord injury area

[0103] After spinal cord injury, the main cause of disrupted neural signaling is the breakage and loss of nerve fibers. NF-200 is a marker for mature neurons, and we applied it to evaluate the nerve fiber remodeling of spinal cord injury rats. Many NF-200-positive cells were present in the P / H-BDNF-PDA-shPTB group and the P / H-PDA-shPTB group, and the shape of the nerve fibers was significantly Figure 15 ). At the same time, the percentage area of NF-200 in the P / H-BDNF-PDA-shPTB group was higher than that in the P / H-PDA-shPTB group, which may be due to the sustained release of BDNF promoting the maturation of reprogrammed neurons.

[0104] The above merely is the preferred embodiment of the present application, it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1.A micro-gel electrospun scaffold surface modified with a polydopamine coating and grafted with a shPTB lentivirus vector, wherein the matrix is prepared from brain-derived neurotrophic factor, poly-L-lactic acid and hyaluronic acid; the mass ratio of HA to PLLA is 1:1000; the backbone vector of the lentivirus vector is pLVTHM, and the vector comprises a PTBP1 short hairpin RNA. 2.A method for preparing the micro-gel electrospun scaffold of claim 1, comprising: mixing poly-L-lactic acid with dichloromethane to prepare a solution A; mixing a brain-derived neurotrophic factor solution with a hyaluronic acid hydrogel to prepare a solution B; mixing the solution A, the solution B and a surfactant, and then mixing with a spinning solution solvent to prepare a micro-gel electrospun solution; spinning the micro-gel electrospun solution to prepare a matrix; mixing a dopamine hydrochloride solution with the matrix and incubating, and then mixing with a shPTBP1 lentivirus vector and incubating to prepare the micro-gel electrospun scaffold. 3.The method of claim 2, wherein: in the solution A, the mass ratio of poly-L-lactic acid to dichloromethane is (0.25-1) : 4; in the solution B, the concentration of the brain-derived neurotrophic factor solution is 100-1000 μg / ml, the mass fraction of HA in the hyaluronic acid hydrogel is 0.5%-2%, and the volume ratio of the brain-derived neurotrophic factor solution to the hyaluronic acid hydrogel is 1:(2-8). 4.The method of claim 2, wherein: the surfactant is Span-80; the spinning solution solvent is N,N-dimethylformamide; the ratio of the solution A, the solution B, the surfactant and the spinning solution solvent is (4-6) g:(40-80) μL:(0.01-0.02) g:(2-3) g; the electrospinning conditions are 10-15 kV voltage, 10-15 mL / h flow rate, 10-15 cm needle-target distance, and needle size 16-20 G; the dopamine hydrochloride solution is a 10 mM Tris buffer containing 2 mg / ml dopamine hydrochloride; the incubation conditions of the dopamine hydrochloride solution with the matrix are incubation in the dark at room temperature for 12-36 h; and the incubation conditions with the shPTBP1 lentivirus vector are standing at 4 ℃ for 15-30 min. 9.Use of the micro-gel electrospun scaffold of claim 1 or the micro-gel electrospun scaffold prepared by the method of any one of claims 2-8 in the preparation of a product for treating nerve injury. 10.A product for treating nerve injury, comprising the micro-gel electrospun scaffold of claim 1 or the micro-gel electrospun scaffold prepared by the method of any one of claims 2-8. ​ ​ ​ ​ ​ ​ ​ ​ ​ 5. The preparation method according to claim 2, characterized in that, ​ 6. The preparation method according to claim 2, characterized in that, ​ 7. The preparation method according to claim 2, characterized in that, ​ ​ 8. The preparation method according to claim 2, characterized in that, ​ ​ ​

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