CN115501326A - Application of 14-3-3 protein in preparation of medicine for treating spinal cord injury repair - Google Patents

Abstract

The invention discloses the application of a 14-3-3 protein in the preparation of a medicine for treating and repairing spinal cord injury. The present invention finds that 14-3-3 protein is significantly up-regulated after spinal cord injury through immunoblotting and immunofluorescence. Through GST-Pulldown and co-immunoprecipitation experiments, it was confirmed that 14-3-3 protein and Spastin could interact in vivo and in vitro to form protein complexes. The present invention clarifies that 14-3-3 is a key protein in the repair process of spinal cord injury, and its main function is to promote the dynamics of microtubules in the damaged axon after spinal cord injury, promote the regeneration of axon to repair spinal cord injury, and provide clinical treatment for spinal cord injury. A solid scientific basis is provided.

Description

Application of 14-3-3 protein in preparation of medicine for treating spinal cord injury repair

technical field

The invention relates to the field of biomedicine, in particular to the application of a 14-3-3 protein in the preparation of medicines for treating and repairing spinal cord injuries.

Background technique

Spinal cord injury (Spinal cord injury, SCI) is the most serious complication of spinal cord injury, often leading to severe sensory and motor impairments below the level of the damaged segment. Spinal cord crush injuries are common as a result of traffic accidents, falls, or violent trauma. At present, there are nearly 30 million patients in the world, and the number of patients is increasing by 100,000 every year. Among them, there are about 3.7 million patients in China. At present, there is no effective clinical treatment to reverse the pathological process of spinal cord injury, and it often leaves various serious complications, including neuropathic pain, respiratory dysfunction, muscle atrophy, pressure sores and urinary tract infections, etc., to ensure the physical and mental health of patients. The nursing and treatment of spinal cord injury also brings huge economic burden to patients and families. Therefore, the research and development of treatment strategies for spinal cord injury has important social significance.

The pathological changes of spinal cord injury can be roughly divided into the following aspects: (1) interruption of nerve conduction bundles in the spinal cord and death of a large number of motor neurons; (2) inflammatory response; (3) toxic effects on surviving neurons after cell rupture; (4) Glial scar inhibits axon regeneration. Based on the above pathological characteristics, the current treatment strategies for spinal cord injury mainly focus on improving the microenvironment around the surviving neurons, stimulating the internal regeneration mechanism of the surviving axons, and establishing effective neural relay stations. It should be noted that exogenous treatment ultimately requires the establishment of new neural circuits through surviving neurons. The axons of differentiated mature neurons still have strong regenerative potential, so promoting the axon growth and collateral formation of surviving neurons is the first condition for the repair of spinal cord injury.

14-3-3 proteins are a class of ligand proteins that regulate the functional properties of interacting proteins by binding to specific phosphorylation sites on interacting proteins. The 14-3-3 protein is abundantly expressed in growth cones in neurons. Due to the highly similar spatial structure of each subtype of 14-3-3 proteins, although the binding substrates of 14-3-3 proteins are different, there are still certain rules to be found in the regulation of protein functions. The main effects are as follows: Affect the functional activity of the protein, change the spatial position of the substrate protein in the cell, and other related functions. However, the role of 14-3-3 protein in spinal cord injury is still unclear.

Spastin is a mutated gene found in the corticospinal tract of patients with hereditary spastic paraplegia (HSP). Spastin is a kind of microtubule-cutting protein. The loss of Spastin function leads to the degeneration of corticospinal tract in HSP patients, which makes the patients' lower limb motor dysfunction, and the walking gait is "scissors-like". Spastin is a microtubule cutting protein, which can enhance the dynamics of microtubules and promote the growth and regeneration of neuron axons by properly cutting microtubules under physiological conditions. It is very important to clarify the functional mode of Spastin in physiological state for its application to spinal cord injury. Whether 14-3-3 protein mediates spinal cord injury repair by regulating the microtubule cutting ability of Spastin is still unclear.

To date, cytoskeletal remodeling, including microtubule and microfilament reorganization, is critical for axonal regeneration after SCI. In spinal cord lesion sites, the retracting balls of severed axons require dynamic cytoskeletal remodeling to successfully regenerate, and this process is closely related to microtubule invasion. Mechanistically, microtubules at the distal end of axons not only provide mechanical force, but also guide intracellular transport to regulate the distribution of specific molecules, including mitochondria, peroxisomes, growth factors, and other molecules that mediate axon extension. Thus, timely invasion of microtubules can drive the formation of growth cones with growth capabilities from retracting bulbs. Spastin is a microtubule cutting protein that can enhance microtubule cutting and promote microtubule dynamics, thereby promoting axon regeneration after spinal cord injury.

Contents of the invention

The purpose of the present invention is to propose the application of a 14-3-3 protein in the preparation of medicines for the treatment of spinal cord injury repair, to solve the problem of difficult regeneration of axons in the treatment process of spinal cord injury repair in the prior art, and at the same time provide a A new therapeutic target for spinal cord injury repair.

The purpose of the present invention will be achieved through the following technical solutions:

One aspect of the present invention provides the application of a 14-3-3 protein in the preparation of medicine or pharmaceutical composition for treating spinal cord injury or promoting the repair of spinal cord injury.

Another aspect of the present invention provides the application of a 14-3-3 protein in the preparation of a drug or a pharmaceutical composition for promoting neuron axon regeneration.

Another aspect of the present invention provides an application of a 14-3-3 protein in the preparation of a drug or a pharmaceutical composition for improving the microtubule cutting ability of the Spastin protein.

Another aspect of the present invention provides the application of a 14-3-3 protein in the preparation of a drug for improving the therapeutic sensitivity of Spastin protein, and the therapeutic sensitivity is the therapeutic sensitivity to spinal cord injury repair.

Further, the 14-3-3 protein is a protein sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 One or more of those shown.

Further, the spinal cord injury is acute spinal cord injury.

Further, the Spastin protein is M87 Spastin.

Another aspect of the present invention provides a detection reagent or detection kit for detecting spinal cord injury or evaluating the severity of spinal cord injury, including a reagent for detecting the expression level of 14-3-3 protein.

Further, the reagents for detecting the expression level of 14-3-3 protein include reagents for detecting the content of 14-3-3 protein, and the reagents for detecting the content of 14-3-3 protein are selected from 14-3-3 monoclonal antibody and/or polyclonal antibodies. The monoclonal antibodies and/or polyclonal antibodies are commercially available.

Another aspect of the present invention provides a method for screening drugs for the treatment of spinal cord injury repair, comprising the steps of:

(1) Apply the candidate drug to the animal model of spinal cord injury;

(2) Obtain the target drug by detecting the expression level of 14-3-3 protein in the animal model of spinal cord injury; when the candidate drug increases the expression level of 14-3-3 protein, it is the target drug.

Unless otherwise specified, the "14-3-3 protein" mentioned in the context of the present invention is a protein that can recognize the phosphorylation site of a target substrate and change the function of the substrate protein. The "Spastin protein" referred to is a microtubule cutting protein.

The outstanding effects of the present invention are:

The present invention finds that 14-3-3 is an important protein in the process of repairing spinal cord injury through extensive research. Firstly, by immunoblotting and immunofluorescence, it was found that 14-3-3 protein was significantly up-regulated after spinal cord injury. GST-Pulldown and co-immunoprecipitation experiments confirmed that 14-3-3 protein and Spastin can interact in vivo and in vitro to form protein complexes. 14-3-3 protein can enhance the microtubule cutting ability of Spastin, and promote the growth and regeneration of neuronal processes. Adding CHX to inhibit protein synthesis after overexpressing 14-3-3 protein and Spastin found that 14-3-3 protein can inhibit the degradation of Spastin protein. 14-3-3 was found to upregulate the microtubule cutting ability of Spastin in COS7 cells. The application of 14-3-3 protein agonist in neurons can promote the growth of hippocampal neuron neurites, and at this time, interfering with Spastin will make the promoting effect disappear; add 14-3-3 agonist to the cultured cortical neurons in the scratch experiment Agents and Spastin inhibitors found that 14-3-3 promotes the regeneration of neuronal processes by interacting with Spastin. In the animal model of spinal cord injury, 14-3-3 protein was used to mediate the recovery of motor function after spinal cord injury by interacting with Spastin, and immunostaining experiments showed that 5-HT axons regenerated in large numbers after spinal cord injury. The present invention clarifies that 14-3-3 is a key protein in the repair process of spinal cord injury, and its main function is to promote the dynamics of microtubules in the damaged axon after spinal cord injury, promote the regeneration of axon to repair spinal cord injury, and provide clinical treatment for spinal cord injury. Provides a solid scientific basis.

The specific implementation of the present invention will be described in further detail below in conjunction with the examples, so as to make the technical solution of the present invention easier to understand and grasp.

Description of drawings

Fig. 1 is the protein level of 14-3-3 total protein in different periods after spinal cord injury in Example 1 of the present invention;

Fig. 2 shows that the part with increased expression of 14-3-3 protein in the tissue section after spinal cord injury in Example 1 of the present invention is mainly located in neurons;

Figure 3 is a schematic diagram of the results of protein electrophoresis after the expression and purification of the GST-14-3-3 protein constructed in Example 2 of the present invention;

Figure 4 is a schematic diagram of the Pulldown results of the GST-14-3-3 protein in Example 2 of the present invention;

Fig. 5 is a schematic diagram of the co-immunoprecipitation results after co-transfection of Flag-14-3-3 protein and GFP-Spastin plasmid in Example 2 of the present invention;

Fig. 6 is a schematic diagram of the co-immunoprecipitation results of rat brain lysate using Spastin antibody in Example 2 of the present invention;

Figure 7 is a schematic diagram of the GFP-Spastin protein stability results after co-transfection of Flag-14-3-3 and GFP-Spastin with the plasmid in Example 3 of the present invention;

Fig. 8 is a schematic diagram of the results of changes in microtubule cutting ability after Flag-14-3-3 and GFP-Spastin plasmid co-transfection in Example 4 of the present invention;

Figure 9 is a quantitative analysis of changes in microtubule cutting ability after Flag-14-3-3 and GFP-Spastin plasmid co-transfection in Example 4 of the present invention;

Figure 10 is a schematic diagram of the results of 14-3-3 promoting the growth of hippocampal neuron neurites through Spastin in Example 5 of the present invention;

Figure 11 is the quantitative analysis of 14-3-3 promoting the growth of hippocampal neuron neurites through Spastin in Example 5 of the present invention;

Figure 12 is a schematic diagram of the results of 14-3-3 promoting regeneration of neuron processes after injury through Spastin in Example 5 of the present invention;

Fig. 13 is a schematic diagram of the results of 14-3-3 promoting the recovery of spinal cord injury motor function through Spastin in Example 6 of the present invention;

Figure 14 is a quantitative analysis of the increase in the step length and the reduction in the error rate of the affected limb after 14-3-3 mediates the spinal cord injury through Spastin in Example 6 of the present invention;

Fig. 15 is a schematic diagram of the results of 14-3-3 promoting axon regeneration after spinal cord injury through Spastin in Example 6 of the present invention.

detailed description

In order to make the object, technical solution and effect of the present invention more clear and definite, the present invention will be further described in detail below with reference to the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The reagents used in the context of the present invention are all commercially available. The 14-3-3 protein mentioned in the present invention and related proteins such as Spastin and their gene sequences can be obtained by conventional means (such as NCBI, Uniprot). The experimental methods used in the present invention, such as molecular cloning, Western blot, cell experiments and animal experiments, are all conventional methods and techniques in the art. For animal experiments, the relevant procedures and methods meet the requirements of medical ethics. The experimental methods used in the present invention are conventional methods and techniques in the art.

Selected representative results from biological experiment replicates are presented in contextual figures, and data are presented as mean ± standard deviation as specified in the figure. All experiments were repeated at least three times. The experimental data were statistically analyzed using Graphpad Prism 8.4. Chi-square test, analysis of variance and other routine medical statistical methods were used to compare the mean differences of each group. The test value P<0.05 indicated that the difference was statistically significant.

Example 1: The role of 14-3-3 protein in the repair of spinal cord injury

Firstly, the mouse spinal cord injury model was established, which specifically included the following steps:

(1) C57 experimental mice were anesthetized with 1.25% avertin (2,2,2-tribromoethanol), and fixed in prone position after intraperitoneal anesthesia at a dose of 0.2mL/10g body weight;

(2) Hair was cut with the T10 spinous process as the center, the skin was incised with a routine disinfection drape, the muscles were separated, and the T9-11 spinous process and lamina were exposed. Under a stereomicroscope, bite off the T10 spinous process and lamina to expose the segment of the spinal cord;

(3) Use a stabilizer to fix both sides of the T10 cut surface, and set the nitrogen tank to control the impact head to 18psi or 124kPa. Load the U-shaped stabilizer with the rat onto the platform of the Louisville Injury Systems Apparatus (LISA) and adjust the dura/spinal height directly below the impactor while monitoring via the laser beam;

(4) Adjust the collision depth to different damage levels. The collision depth is set to 0.6mm, 1.0mm or 1.8mm, respectively representing the damage degree of Light, Medium and Severe, and the time is set to 0.5s;

(5) After the injury was induced, the stabilizer was removed from the platform, the rat was removed from the stabilizer, the injured site was assessed, and bleeding was suppressed. Finally, the muscle and skin of the rat were sutured with 3.0 silk suture.

The successful spinal cord injury models were screened by observing the activities of the lower limbs of the rats. The evaluation criteria included: paralytic paralysis, tail wagging reflex, flicking of the body and legs, spinal cord ischemia and edema around the wound site, etc. Sham-operated animals underwent T10 total laminectomy, but the spinal cord was not injured. Using the SCI rat model constructed above, the rats were perfused with paraformaldehyde 72 hours after injury, and the eluted samples were collected for immunofluorescence staining.

Using the rat T10 spinal cord injury model constructed above, the spinal canal was opened again 72 hours after the injury, and the injured segment was taken out for Western blot to analyze the total protein of 14-3-3 (including: SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) protein level changes after spinal cord injury. The analysis results are shown in Figure 1. Figure 1 shows the change and quantitative analysis of the 14-3-3 protein level, and the results show that the expression level of the 14-3-3 protein in the tissue after spinal cord injury is up-regulated compared with the control group.

Further, the obtained spinal cord samples were gradually dehydrated to make paraffin sections, and 14-3-3 total protein (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5. Incubation with SEQ ID NO: 6) antibody and βIII-tubulin antibody, then stained with fluorescent secondary antibody and DAPI, and then placed under confocal laser for observation, the detection results are shown in Figure 2. The experimental results further illustrate that the protein expression level of 14-3-3 protein increases after spinal cord injury.

Example 2: Interaction of 14-3-3 protein with Spastin

First, verify the interaction of 14-3-3 protein with Spastin in vitro:

(1) Construction of GST-14-3-3β(SEQ ID NO:1), GST-14-3-3γ(SEQ ID NO:2), GST-14-3-3ε(SEQ ID NO:3), GST -14-3-3ζ (SEQ ID NO: 4), GST-14-3-3η (SEQ ID NO: 5), GST-14-3-3θ (SEQ ID NO: 6) plasmids, the plasmids that will be constructed Transformed into competent BL21 bacteria;

(2) After overnight culture, select monoclonal positive colonies and confirm that they are correct, take 1 mL of the bacterial liquid and culture it in 250 mL of LB medium containing ampicillin resistance, and culture it in a shaker at 37°C at 180 rpm until the OD value of the bacterial liquid is 0.4 -0.8 (log phase of growth);

(3) Add 0.3mM IPTG and carry out another 8 hours at 30°C;

(4) In a centrifuge at 4°C, centrifuge the bacterial liquid at 12000rpm, discard the supernatant, take the precipitate, add an appropriate amount of lysate, and use an ultrasonic breaker to sonicate after 1h in ice bath, intensity: 30%, time: 5X5s, total 1min;

(5) Add 150 μL beads to the centrifuge tube, add 1 mL lysate, remove the supernatant after centrifugation, add the supernatant obtained in step (4), and incubate overnight at 4°C;

(6) Centrifuge at 4°C, 1000rpm for 5min to remove the supernatant, add 1mL of lysate for 3 repeated washings, and obtain purified protein;

(7) Perform SDS-PAGE electrophoresis on the purified protein, and finally perform Coomassie brilliant blue staining. The result is shown in Figure 3.

Then the purified GST-14-3-3 protein (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) was combined with large The mouse brain lysate was mixed and incubated overnight, and then the GST-Pulldown experiment was performed. The specific steps are as follows:

(1) Weigh 100 mg of rat brain tissue;

(2) Extract an appropriate amount of brain lysate and add PMSF (500 μL cell lysate per 100 mg of mouse brain tissue);

(3) grind the mouse brain tissue on ice with a grinding rod, so that the mouse brain tissue is fully lysed;

(4) Centrifuge at 4°C and 12,000 rpm for 15 minutes, retain the supernatant, add another 20 μL of the supernatant to the loading buffer, heat at 95°C for 6 minutes in a dry heat apparatus, and use it as a positive control (Input);

(5) Add 50 μL beads and 1 mL cell lysate to the EP tube, wash the beads by centrifugation, add 500 μL mouse brain lysate, invert at 4°C for 1 hour, and centrifuge to get the supernatant;

(6) Take 50 μg of the purified GST-14-3-3 protein, add it to the mouse brain lysate, invert overnight at 4°C, centrifuge to remove the supernatant, and keep the beads;

(7) Wash the beads obtained in step (6) with Washing Buffer, add an appropriate amount of lysate, and then add loading buffer, heat with dry heat apparatus for 10 minutes, and perform Western Blot detection.

The result is shown in Figure 4. The results showed that GST-14-3-3β(SEQ ID NO:1), GST-14-3-3γ(SEQ IDNO:2), GST-14-3-3ε(SEQ ID NO:3), GST-14 -3-3ζ (SEQ ID NO: 4), GST-14-3-3η (SEQ ID NO: 5), GST-14-3-3θ (SEQ ID NO: 6) can all be combined with rat brain lysate Spastin binds to each other.

Subsequently, we constructed eukaryotic expression plasmids of 14-3-3 and Spastin, and transfected them into HEK293T cells for co-immunoprecipitation experiments to verify the intracellular interaction between 14-3-3 and Spastin. The specific implementation steps are as follows:

(1) Construct the eukaryotic expression plasmids of Flag-14-3-3 and GFP-Spastin;

(2) 293T cells were plated one day in advance, and Flag-14-3-3β (SEQ ID NO: 1), Flag-14-3-3γ (SEQ ID NO: 2), Flag-14-3-3ε (SEQ ID NO:3), Flag-14-3-3ζ (SEQ ID NO:4), Flag-14-3-3η (SEQ ID NO:5), Flag-14-3-3θ (SEQ ID NO:6) The plasmids were co-transfected with GFP and GFP-Spastin plasmids into 293T cells;

(3) After 24 hours of transfection, the green fluorescence of the GFP tag can be observed through a fluorescence microscope, indicating that the transfection is successful. Collect and lyse the cells, take an appropriate amount of samples as a positive control, and use Agrose-proteinA/G to remove non-specific proteins;

(4) Add the GFP tag antibody to the cell lysate and incubate overnight at 4°C;

(5) Add new Agrose-protein A/G to the sample the next day, incubate at 4°C, remove the supernatant and wash with cell lysate, and finally perform Western blot detection, and detect the target bands with GFP and Flag antibodies.

The test results are shown in Figure 5. The results showed that each subtype protein of Flag-14-3-3 could interact with GFP-Spastin in cells. And by detecting the expression of GFP-Spastin protein, it was found that GFP-Spastin was well expressed in each sample, and Agrose-proteinA/G beads could recruit GFP-Spastin to the beads.

Subsequently, the spastin antibody was used in the rat brain lysate to carry out co-immunoprecipitation experiments, and the lysate was prepared as described above, with 14-3-3 total protein (including: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2, ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) antibodies were detected, and the results are shown in Figure 6. The results showed that Spastin combined with beads could successfully pull down the 14-3-3 protein in the brain, while IgG in the control group could not, indicating that the brain tissue contains natural 14-3-3 protein (including: SEQ ID NO: 1, Protein complexes of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) and Spastin.

Embodiment 3: The influence of 14-3-3 protein on the stability of Spastin protein

(1) After the HEK293T cells are overgrown, transfer the cells to a 6-well plate the day before;

(2) GFP-Spastin is transferred alone or together with Flag-14-3-3 (SEQ ID NO: 6) into HEK293 cells;

(3) After incubation for 12 hours, cycloheximide (CHX) was added to inhibit protein synthesis;

(4) Western blot detection with GFP antibody.

The results are shown in Figure 7, GFP-Spastin degrades over time after 9h, and cotransfection of Flag14-3-3 (SEQ IDNO: 6) can inhibit the degradation of GFP-Spastin, proving that 14-3-3 enhances Spastin protein stability.

Example 4: 14-3-3 protein enhances the microtubule cutting ability of Spastin

(1) Use PDL to coat slides, incubate for 30 minutes, wash off residual PDL with double distilled water, and irradiate with ultraviolet light for 30 minutes in an ultra-clean bench;

(2) Inoculate the cultured COS7 cells on a glass slide and culture them for 12 hours;

(3) Transfect GFP-Spastin alone or with Flag-14-3-3 (SEQ ID NO: 6) into COS7 cells, and incubate for 12 hours;

(4) After COS7 cells were fixed with 4% PFA, they were washed with pre-cooled PBS;

(5) Punch holes with TBST for 10 minutes at room temperature;

(6) Transfer the slides to the antibody incubation box, with the slides of Korean cells facing up, wash with TBST, add 3% BSA blocking solution, and block at room temperature for 1 hour;

(7) After washing 3 times with TBST, incubate with Tubulin antibody dilution, overnight at 4°C;

(8) Wash off residual antibody with TBST, 5 min, 3 times; add fluorescent secondary antibody diluent, incubate at room temperature for 1 h in the dark;

(9) wash with TBST, 5min, 3 times, then cover the cells on the glass slide with mounting medium upside down;

(10) Observation and taking pictures with a confocal fluorescence microscope.

The results are shown in Figure 8, and Figure 9 is the quantitative analysis of the microtubule cutting ability, where * represents P<0.05, and the difference is statistically significant. Compared with the control group, Spastin can cut microtubules, the middle of the microtubules is fragmented, and the fluorescence intensity is significantly decreased; while overexpressing Flag-14-3-3 (SEQ ID NO:6) at the same time, it is found that the content of Spastin microtubules With further reduction, the microtubule cutting ability of Spastin is further enhanced.

Example 5: The effect of the interaction between 14-3-3 and Spastin on the neurite outgrowth of hippocampal neurons

(1) After separating the hippocampal tissue under a microscope, digest it with 2mg/ml papain;

(2) Add 10% FBS to the DMEM-F12 medium to stop the digestion, turn over gently 5 times, and let stand for 1 min;

(3) Add 2 mL of DMEM-F12 medium with 10% FBS, shake the hippocampal tissue 10 times, then let it stand for 1 min and collect the supernatant;

(4) Repeat the operation 2-3 times to fully collect neurons;

(5) Inoculate neurons onto coated glass slides and incubate at 37°C for 30 min in an incubator;

(6) Slowly add 500 uL of DMEM-F12 medium containing 10% FBS;

(7) Wash off the medium after 4 hours, and add NB27 neuron culture medium;

(8) Transfection and drug treatment are carried out when the cells grow to 2 days;

(9) Transfer the original medium to auxiliary wells, and add 500 μL Neurobasal medium to each well for starvation;

(10) Using a calcium phosphate transfection kit, add 1 μg of GFP, GFP-Spastin plasmid and 25 μL of CaCl2 solution to the EP tube, and add BBS solution to another EP tube;

(11) Mix the solutions in step (10), and let stand in a dark box for 15 minutes;

(12) Add 50 μL of the mixed solution dropwise to each well. The mixed solution was added dropwise in different positions in the well, and gently shaken several times. After 40 min, the calcium and phosphorus particles were washed with 1×SA twice, and then the original medium or 14- 3-3 agonist medium FC-A;

(13) 24 hours after transfection, the cells were fixed with 4% PFA, stained with immunofluorescence, and observed under confocal laser light.

The results are shown in Figure 10, and Figure 11 is the quantitative analysis of the length and branching of neuron processes, where * means p<0.05, which is statistically significant, and the scale bar is μm. The results showed that, relative to the blank control group GFP, 14-3-3 protein (including: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6) agonist significantly promoted the growth of neuron neurite, while the effect of 14-3-3 agonist on promoting the growth of neuron neurite was significantly decreased after simultaneous transfection of Spastin interference fragment.

Furthermore, we used the same method to obtain primary cortical neurons for in vitro culture. After the cells grew to 7 days, the processes were fully fused, and then scratched the center of the glass slide with a pipette tip. After scratching, the medium was replaced. 14-3-3 protein (including: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) agonist added to the base FC-A or Spastin inhibitor spastazoline were used to fix the cells after 1 day, and immunofluorescent staining was performed with βIII-tubulin to observe the regeneration of neuronal processes. The results are shown in Figure 12, 14-3-3 protein (including: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 ) agonist FC-A can significantly promote neurite regeneration, while spastazoline can significantly inhibit 14-3-3 agonist-mediated neurite regeneration.

Example 6: Study on the role of 14-3-3 in promoting the repair of spinal cord injury through Spastin

(1) The preparation of the mouse spinal cord injury model is consistent with Example 1;

(2) 1 day after the mouse spinal cord was injured, the mice were intraperitoneally injected with 14-3-3 protein (including: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO:5, SEQ ID NO:6) agonists and Spastin inhibitors;

(3) BMS scores were performed on 1d, 3d, 5d, 7d, 9d, 12d and 14d after modeling to evaluate the motor function of the hindlimbs of mice in each group. The experiment was double-blind, and the animals with spinal cord injury were scored by two testers. The scoring rules are as follows: no ankle movement is 0 points, slight ankle movement is 1 point, a wide range of ankle joint movement is 2 points, hind limbs can be well homed, regardless of whether they can support weight, 3 points; occasional solid footing Score 4; footsteps often or always firm, but uncoordinated 5 points; footsteps often or always firm, sometimes coordinated, paws parallel at first contact with floor, whether parallel or rotated when leaving floor 6 points; footsteps often or always firm, coordinated most of the time, paws parallel at initial contact with floor, and rotated at exit 7 points; footsteps often or always firm, coordinated most of the time, paws parallel at initial contact with floor, torso stable , tail drooping or sometimes hanging off the ground is an 8 points; footsteps are often or always firm, coordinated most of the time, paws are parallel at the time of initial contact and leaving the floor, the body is stable, and the tail is always off the ground is a 9 points;

(4) 15 days after the mouse spinal cord injury, the stepping test was carried out, and the mice were placed in a parallel rod device and allowed to walk freely. Video recording for 3 minutes, recording the total number of steps and the number of falls of the right lower limb of the mouse;

(5) After the mouse spinal cord injury, 16 days on the narrow board of the training animal, lay a white paper with the same length and width, and record the animal's number and test date on the paper; after the animal gets used to it, put the black ink Apply to the soles of the feet; immediately place the animal on one side of the white paper and make it walk towards the cassette; after the animal enters the cassette, put the white paper away and let the gap dry; continue the above steps until all animals are analyzed.

The results are shown in Figure 13-14, 14-3-3 protein (including: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) The agonist significantly improved the motor function score of the mice and reduced the drop frequency of the affected limb during walking, while the Spastin inhibitor decreased the score of the mouse motor function and increased the drop frequency of the affected limb in the mouse The gait analysis experiment of mice showed that 14-3-3 agonists can significantly increase the step length of the affected limbs of mice, and the difference is statistically significant. The promotion effect is significantly reduced. The above experiments show that giving you 14-3-3 can promote the recovery of motor function after spinal cord injury through Spastin.

Further, immunohistochemical staining was performed on the animal spinal cord tissue 17 days after the mouse spinal cord injury, and the specific steps were as follows:

(1) Mice were anesthetized with 1.25% avertin (2,2,2-tribromoethanol), fixed in the prone position after intraperitoneal anesthesia at a dose of 0.2 mL/10 g body weight, and perfused with 4% PFA.

(2) The spinal cord was cut at 1 cm above and below the spinal cord injury, and placed in 4% PFA fixative solution overnight, and then dehydrated with 30% sucrose solution for 2 days.

(3) Place the tissue block flat in a soft plastic bottle cap or a special small box (about 2cm in diameter). Add an appropriate amount of OCT embedding agent to immerse the tissue, and then slowly and flatly place the special small box into a small cup filled with liquid nitrogen. After making the frozen block, put it into the cryostat slicer to freeze and slice. Apply a layer of OCT embedding glue on the sample holder, place the quick-frozen tissue on it, and pre-cool in a 4°C refrigerator for 5-10 minutes to allow the OCT glue to soak into the tissue.

(4) Remove the tissue and put it on a glass slide, and freeze the sample holder quickly. Place the tissue on the sample holder, add a layer of OCT glue on it, it is advisable to completely cover it, and place it on the quick-freezing rack for 30 minutes. Thin slices were subsequently cut serially to 50 μm.

(5) After cutting, place at room temperature for 30 minutes;

(6) Add ice-acetone fixative solution to fix for 5-10 minutes, and dry in oven for 20 minutes.

(7) Rinse with PBS, 5min×3.

(8) Incubate with 3% H2O2 for 5-10min to eliminate the activity of endogenous peroxidase.

(9) Add the diluted 5-HT antibody diluent and incubate overnight at 4°C;

(10) wash with PBS/Triton three times, each time for 5 minutes;

(11) Incubate with the corresponding secondary antibody mixture at room temperature in the dark for 1 h according to the source of the primary antibody; wash twice with PBS/Triton, 10 min each time; then add Mounting Medium dropwise and seal the slide with a cover slip.

(12) Collect pictures under a fluorescence microscope.

The results are shown in Figure 15: in the case of spinal cord injury, the 5-HT axon immunogenicity increased to a certain extent compared with the control group, and the application of 14-3-3 protein (including: SEQ ID NO: 1, SEQ ID NO: 2. After using SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) agonists, the immunogenicity of 5-HT in the spinal cord injury was significantly increased, and on this basis, adding Spastin Inhibitors significantly reduced the immunogenicity of 5-HT. The above results indicated that 14-3-3 promoted the regeneration of 5-HT axons through Spastin.

According to the above results, it can be clarified that the 14-3-3 protein (including: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) It plays an important role after spinal cord injury, and is coupled with Spastin to control the growth and regeneration of neuron processes and promote the repair of spinal cord injury. Through immunoblotting and immunofluorescence experiments, the 14-3-3 protein (including: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: ID NO:6) in the repairing process of spinal cord injury; the 14-3-3 protein (including: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6) interact with Spastin to function; 14-3-3 (SEQ ID NO: 6) enhances the protein stability of Spastin, up-regulates the microtubule cutting function of Spastin, and promotes neural and 14-3-3 proteins (including: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6) Promote axon regeneration and recovery of motor function after spinal cord injury through Spastin. As spinal cord injury is the leading cause of disability, repairing neural tissue defects caused by spinal cord injury is of paramount importance to modern regenerative medicine. Therefore, based on the research of the present invention, various drugs and intervention measures can be designed to assist the repair of spinal cord injury.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, any person familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solution of the present invention Any equivalent replacement or change of the inventive concepts thereof shall fall within the protection scope of the present invention.

Claims (10)

1. An application of 14-3-3 protein in preparing the medicine or medicine composition for treating or repairing the injury of spinal cord.

2. An application of 14-3-3 protein in preparing the medicines or medicine composition for promoting the regeneration of the axon of neuron.

3. An application of 14-3-3 protein in preparing a medicine or a medicine composition for improving the microtubule cutting ability of Spastin protein.

4. An application of 14-3-3 protein in preparing a medicament for improving the sensitivity of Spastin protein treatment, which is characterized in that: the treatment sensitivity is a treatment sensitivity to spinal cord injury repair.

5. Use according to any one of claims 1 to 4, characterized in that: the 14-3-3 protein is one or more of the protein sequences shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6.

6. Use according to claim 1 or 4, characterized in that: the spinal cord injury is acute spinal cord injury.

7. Use according to claim 3 or 4, characterized in that: the Spastin protein is M87 Spastin.

8. A test reagent or test kit for detecting spinal cord injury or assessing the severity of spinal cord injury, comprising: comprises a reagent for detecting the expression level of the 14-3-3 protein.

9. Use according to claim 8, characterized in that: the reagent for detecting the expression level of the 14-3-3 protein comprises a reagent for detecting the content of the 14-3-3 protein, and the reagent for detecting the content of the 14-3-3 protein is selected from a 14-3-3 monoclonal antibody and/or a polyclonal antibody.

10. A method of screening for a drug for use in the treatment of spinal cord injury repair comprising the steps of:

(1) Acting the candidate drug on the spinal cord injury animal model;

(2) Obtaining a target drug by detecting the expression level of 14-3-3 protein in a spinal cord injury animal model body; when the candidate drug increases the expression level of the 14-3-3 protein, the drug is the target drug.

Images