CN114668836B - Application of PDIA6 in the preparation of drugs for spinal cord injury and repair - Google Patents

Abstract

The invention relates to application of PDIA6 protein or its active fragment in preparing medicine for treating spinal cord injury and repair. A large number of researches prove that PDIA6 plays a key role in SCI and interacts with spastin to regulate neurite outgrowth, and the invention proves that the expression level of PDIA6 is up-regulated in the SCI process, and PDIA6 can physically interact with spastin in vivo and in vitro. Meanwhile, the effect of PDIA6 and Spastin is found to promote not only the growth of neuron protuberance, but also the repair of injured neurons, and the promotion effect of co-transferring PDIA6 and Spastin is more obvious than that of single-transferring Spastin. The invention defines that PDIA6 is a key target point in the process of spinal cord injury and repair, plays a role in regulating neurite growth and branching, and provides practical experimental evidence and scientific basis for clinical treatment of spinal cord injury.

Description

Application of PDIA6 in the preparation of drugs for spinal cord injury and repair

technical field

The present invention relates to the field of biomedicine, in particular, the present invention relates to the application of PDIA6 protein or its active fragment in preparing medicine for treating spinal cord injury and repairing.

Background technique

Spinal cord injury (SCI) is a serious injury to the central nervous system, which leads to motor and sensory dysfunction of the limbs below the level of injury. In severe cases, it can lead to permanent spinal cord dysfunction and seriously affect the quality of life of patients. The main cause of dysfunction after SCI is the direct mechanical damage leading to neuronal damage, and subsequent secondary damage, including inflammatory response, oxidative stress response and excitatory damage, etc., resulting in neuronal axonal damage and glial damage. Plasma cells proliferate, eventually leading to neuronal cell death. Neurological dysfunction caused by SCI is permanent because after spinal cord tissue damage, the ability of axons to regenerate is inhibited and it is difficult to pass through the damaged area, thereby irreversibly impairing the transmission of motor and sensory information. Therefore, promoting axonal regeneration of damaged neurons can rebuild neural pathways, which is expected to improve neurological dysfunction caused by spinal cord injury.

PDIA6 is an endoplasmic reticulum protein that catalyzes protein folding and disulfide bond formation. It possesses thioredoxin domains that contain pairs of active cysteine residues that can be activated by disulfide bond formation. The conversion between the compound and the dimercapto compound realizes the conversion, isomerization and reduction of the disulfide bond. Studies have shown that PDIA6, as a molecular chaperone, is closely related to endoplasmic reticulum stress (ERS) and can be activated to respond to unfolded proteins, reducing the accumulation of misfolded proteins or unfolded proteins, thereby promoting cell survival under stress. However, the role of PDIA6 in spinal cord injury is unclear.

Spastin is a microtubule cleavage protein that regulates microtubule movement and rearrangement by cutting long microtubules into shorter ones. Overexpression of spastin in neurons enhanced the branching of their neurites, whereas silencing spastin reduced the length of neuronal axons and inhibited branching. When Spastin functions, it binds to the microtubule through a positively charged recognition structure, pulling the microtubule into the central pore to cut the microtubule. Spastin cleaves microtubules to regulate microtubule dynamics and is required for neuronal axonal growth. In addition, studies have shown that spastin is involved in endoplasmic reticulum morphogenesis. So whether spastin and PDIA6 have an interaction relationship, and whether PDIA6 can promote neural repair through spastin's regulation of microtubule dynamics, is still unclear.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the technical problems existing in the process of spinal cord injury repair and treatment in the prior art, thereby providing a new spinal cord injury repair treatment target, and the function and mechanism of PDIA6 in the process of spinal cord injury repair were carried out. In-depth research revealed the key role of PDIA6 in the repair and treatment of spinal cord injury, and provided a practical theoretical basis and direction for the clinical treatment of spinal cord injury.

In order to solve the above technical problems, the present invention is achieved through the following technical solutions.

A first aspect of the present invention provides a pharmaceutical composition for treating and/or repairing spinal cord injury, comprising PDIA6 protein and/or its active fragment, spastin protein.

Preferably, the PDIA6 active fragment sequence is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2.

Preferably, the PDIA6 protein sequence is shown in SEQ ID NO: 3; the spastin protein sequence is shown in SEQ ID NO: 4.

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

The second aspect of the present invention provides the use of PDIA6 protein and/or its active fragment in the preparation of a medicament for treating spinal cord injury and/or repair.

Preferably, the PDIA6 active fragment sequence is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2.

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

A third aspect of the present invention provides a kit for detecting the severity of spinal cord injury, comprising primers for detecting PDIA6 protein and/or its active fragments.

Preferably, the forward sequence of the primer is shown in SEQ ID NO:5, and the reverse sequence is shown in SEQ ID NO:6.

The fourth aspect of the present invention provides the use of a reagent for detecting the expression level of PDIA6 in the preparation of a kit for evaluating the severity of spinal cord injury.

Preferably, the reagent for detecting the expression level of PDIA6 includes a primer for detecting the expression level of PDIA6 gene and/or a reagent for detecting the content of PDIA6 protein.

Preferably, the forward sequence of the primer for detecting the expression level of PDIA6 gene is shown in SEQ ID NO:5, and the reverse sequence is shown in SEQ ID NO:6.

Preferably, the reagent for detecting PDIA6 protein content is selected from PDIA6 monoclonal antibody and/or polyclonal antibody. The monoclonal and/or polyclonal antibodies are commercially available.

The fifth aspect of the present invention provides the use of PDIA6 protein and/or its active fragment in the preparation of a drug for improving the therapeutic sensitivity of spastin protein, specifically the therapeutic sensitivity to spinal cord injury and/or repair.

Preferably, the PDIA6 active fragment sequence is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2.

Preferably, the PDIA6 protein sequence is shown in SEQ ID NO: 3; the spastin protein sequence is shown in SEQ ID NO: 4.

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

A sixth aspect of the present invention provides a method for screening a drug for the treatment of spinal cord injury and/or repair, comprising the steps of:

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

(2) The target drug is obtained by detecting the expression level of PDIA6 in an animal model of spinal cord injury; when the candidate drug increases the expression level of PDIA6, it is the target drug.

Unless otherwise specified, the "PDIA6 protein" mentioned in the context of the present invention is an endoplasmic reticulum protein capable of catalyzing protein folding and disulfide bond formation. The mentioned PDIA6 protein active fragment means that the amino acid sequence has some changes in length, longer or shorter, but the overall activity of the PDIA6 protein is maintained. The "spastin protein" referred to is a microtubule cleavage protein.

The terms "first" and "second" are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature delimited with "first", "second" may expressly or implicitly include at least one of that feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.

Previous studies have found that the rearrangement and movement of cytoskeletal microtubules are key aspects of the repair response after spinal cord injury. Tube movement and rearrangement. Overexpression of spastin in neurons enhanced the branching of their neurites, whereas silencing spastin reduced the length of neuronal axons and inhibited branching. When Spastin functions, it binds to the microtubule through a positively charged recognition structure, pulling the microtubule into the central pore to cut the microtubule. Spastin cleaves microtubules to regulate microtubule dynamics and is required for neuronal axonal growth.

In the present invention, the inventors have found that PDIA6 protein is one of the key proteins in the process of spinal cord injury repair through extensive research. PDIA6 protein was significantly up-regulated in spinal cord injury models, and to a certain extent, it was positively correlated with the degree of injury. It was found that PDIA6 could interact with GST-spastin by LC-MS method, and the potential interaction between spastin and PDIA6 was verified using co-immunoprecipitation experiments and staining methods. PDIA6 was overexpressed in neurons, and it was found that overexpression of PDIA6 could promote the extension of neurites and the formation of collaterals in injured neurons; if overexpression of PDIA6 interfered with the expression of spastin at the same time, the above promoting functions were significantly weakened. On the contrary, knockdown of PDIA6 alone significantly weakened the neurite extension and collateral formation of injured neurons. In addition, by interfering with PDIA6 and overexpressing spastin, it was found that the microtubule cleavage ability of spastin was significantly reduced. Therefore, the inventors creatively discovered the key role of PDIA6 in spinal cord injury and repair and the relationship with spastin during the research process, and clarified its important function in regulating neurite growth and branching, indicating that PDIA6 may be a treatment for spinal cord. A novel target of injury.

The present invention has the following technical effects with respect to the prior art:

The present invention confirms that PDIA6 plays a key role in SCI through a large number of studies, and interacts with spastin to regulate neurite outgrowth. By identifying the differentially expressed proteins in the upper and lower positions of SCI, to clarify the role of PDIA6 in this process, and to demonstrate that the expression level of PDIA6 is up-regulated during SCI, PDIA6 can physically interact with spastin in vivo and in vitro. At the same time, it was also found that the interaction of PDIA6 and spastin can not only promote the growth of neuron neurites, but also promote the repair of damaged neurons. Since SCI is a major cause of disability, repairing structural defects in the spinal cord caused by injury or degeneration is critical in the modern field of regenerative medicine. The invention clarifies that PDIA6 is a key target in the process of spinal cord injury and repair, plays a role in regulating neurite growth and branching, and provides practical experimental evidence and scientific basis for clinical treatment of spinal cord injury.

Description of drawings

Figure 1 is a schematic diagram of the results of the RNA and protein levels of PDIA6 in spinal cord injury samples.

Figure 2 is a schematic diagram showing the results of the comparison of the PDIA6 expression levels of the samples in the injury group and the control group in the qPCR experiment.

Figure 3 is a schematic diagram showing the results of the comparison of PDIA6 expression in spinal cord injury tissue sections with the control group.

Figure 4 is a schematic diagram showing the results of protein electrophoresis of the constructed fusion expression protein of GST-Spastin and GST-PDIA6.

Figure 5 is a schematic diagram of the interaction results between PDIA6 protein and Spastin protein in vitro.

Figure 6 is a schematic diagram of the co-immunoprecipitation results after Flag-PDIA6 was co-transfected with GFP and GFP-Spastin plasmids, respectively.

Figure 7 is a schematic diagram of the co-immunoprecipitation results after co-transfection of GFP-Spastin with Flag and Flag-PDIA6 plasmids, respectively.

Figure 8 is a schematic diagram showing the results of IP of rat brain lysate using Spastin antibody.

Figure 9 is a schematic diagram of the effect of PDIA6 and Spastin on the growth of hippocampal neurons, in which 9A is the state diagram of the growth of hippocampal neurons in the tag plasmid (Vector) group; 9B is the growth of hippocampal neurons in the Flag-PDIA6 plasmid (PDIA6) group. State diagram; 9C is the neurite outgrowth status diagram of the hippocampal neurons in the Flag-Spastin plasmid (Spastin) group; 9D is the neurite outgrowth status diagram of the hippocampal neurons in the Flag-PDIA6+GFP-Spastin plasmid (Spastin+PDIA6) group.

Figure 10 is a schematic diagram of the quantitative results of the effects of PDIA6 and Spastin on the growth of hippocampal neurons, wherein 10A is a schematic diagram of the total number of hippocampal neuron branches in each group; 10B is a schematic diagram of the total length of hippocampal neuron branches in each group; 10C is a schematic diagram of the hippocampal neurons in each group Schematic diagram of the results of the number of neurites; 10D is the schematic diagram of the results of the length of the hippocampal neurons in each group.

Figure 11 is a schematic diagram showing the effect of PDIA6 and siSpastin on the growth of hippocampal neuron neurites.

Figure 12 is a schematic diagram showing the quantitative results of the effects of PDIA6 and siSpastin on the growth of hippocampal neurons.

Figure 13 is a schematic diagram showing the effect of siPDIA6 and siSpastin on the growth of hippocampal neuron neurites.

Figure 14 is a schematic diagram showing the quantitative results of the effects of siPDIA6 and siSpastin on the growth of hippocampal neuron neurites.

Detailed ways

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

The reagents used in the context of the present invention are all commercially available. The related proteins such as PDIA6 and spastin mentioned in the present invention and their gene sequences can be obtained by conventional means (such as Pubmed, Uniprot). The experimental methods used in the present invention, such as DNA extraction, gene sequencing, primer design, vector construction , Western blot, cell experiments, animal experiments, etc. are all conventional methods and techniques in the field. For animal experiments, the relevant procedures and methods meet the requirements of medical ethics. The experimental methods used in the present invention are all conventional methods and techniques in the art.

Selected representative results from replicates of biological experiments are presented in the contextual figures, and data are presented as mean ± SD and mean ± SEM as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 22.0 software. The mean differences of two or more groups were compared by conventional medical statistical methods such as t-test, chi-square test and analysis of variance. p < 0.05 was considered a significant difference.

In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples, without conflicting each other.

Example 1 Preparation of plasmids and constructs

(1) PDIA6 and spastin sequences (available through commonly used databases such as Pub, ed or Uniprot) were obtained for cDNA preparation, respectively, which were then cloned into pEGFP-C1 (Clontech, CA, USA), pGEX-5x-3 (Amer - shamPharmacia Biotech, NJ, USA) and pCMV-Tag2 (Stratagene, CA, USA) vectors, and the constructed constructs were confirmed by DNA sequencing;

(2) Pull-down analysis using glutathione S-transferase (GST): Spinal cord tissue sections were ground and lysed, then GST-agarose beads (Invitrogen, CA, USA) were rinsed and mixed with lysis buffer at 4 After incubation for 1 h at 4°C, centrifugation was performed at 1000 g for 10 minutes. The supernatant was then collected and the above steps were repeated once. The appropriate bead fusion protein was then added to the spinal cord tissue and the samples were incubated overnight at 4°C. After centrifugation at 1000 g for 5 minutes at 4°C, unbound protein was washed with 1 mL of wash buffer, and then the samples were centrifuged at 1000 g for 1 minute at 4°C. This washing step was repeated six times before the proteins that had been pulled down were analyzed by Western blotting and mass spectrometry (MS).

The mass spectrometry detection method is as follows:

These proteins were first separated using NuPAGE 4–12% gels (Life Technologies), followed by MS analysis of the spastin-GST pull-down assay using a colloidal blue staining kit (Life Technologies). The excised protein bands were trypsinized and then analyzed by nanoscale reversed-phase liquid chromatography-tandem mass spectrometry using an HPLC Ultimate 3000 (DIONEX, USA) connected to a linear ion trap (LTQ, ThermoElectron, USA) These peptides are in data-dependent acquisition mode.

The differentially expressed proteins were also subjected to IPA analysis (http://www.ingenuity.com). IPA analysis utilizes curated databases to identify overlapping functions and relationships between these proteins, assigning scores to specific biological function networks such that scores > 2 generally indicate significant significance for a specific biological function within a given subset of proteins enrichment. These scores correspond to the log-probabilities of detecting a given network due to random chance alone.

Through the above screening process, it was found that PDIA6 may be one of the key proteins related to spinal cord injury, and it is also a potential protein that interacts with spastin.

Example 2 Study on the role of PDIA6 in the repair of spinal cord injury

First, the construction of the rat SCI model includes the following steps:

The Sprague-Dawley (SD) rats used in the experiment were purchased from the Laboratory Animal Center of Sun Yat-Sen University. The rats were housed individually in a facility at 25 ± 3 °C and provided with regular food and water. Specifically, the following steps were included:

(1) SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.35 mL/100 g body weight), after which a 2.5 cm longitudinal dorsal incision was made to expose the T9-11 spinous process and lamina;

(2) The entire T10 lamina is removed, and the exposed area of the spine is about 2.5mm × 3mm;

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

(4) Adjust the collision depth to different damage levels. The collision depth is set to 0.6mm, 1.0mm or 1.8mm, representing the damage levels of Light, Medium and Severe respectively, 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 injury site was assessed, and bleeding was suppressed, and finally the muscle and skin of the rat were sutured with 3-0 silk suture.

The successful spinal cord injury model was screened by observing the activities of the lower limbs of the rat. The evaluation criteria included: paralytic paralysis, tail wagging reflex, body and leg flicking, spinal cord ischemia and edema around the wound site. Sham-operated animals underwent T10 total laminectomy, but the spinal cord was not injured. All rats were treated with gentamicin at 2000 U/day, and each rat's bladder was manually squeezed every 8 hours to assist voiding until spontaneous voiding was observed. Using the SCI rat model constructed above, the rats were perfused with paraformaldehyde 72 hours after injury, and eluted samples were collected.

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 transcriptomic high-throughput side aspiration to analyze the changes in RNA and protein levels of PDIA6 in spinal cord injury samples .

The analysis results are shown in Figure 1. Figure 1A and Figure 1B are heat maps and line graphs of the changes in PDIA6 RNA and protein levels. The results show that the PDIA6 protein expression and RNA levels in the injured spinal cord samples were up-regulated compared with the control group, and both increased with the severity of the spinal cord injury. Then increased; Figure 1C is the correlation analysis of PDIA6 protein level and RNA level in the sample, indicating that the two are positively correlated.

In order to clarify the expression of PDIA6 in spinal cord injury samples, RNA was extracted from the spinal cord samples taken out, and then reverse transcribed, and then the RNA level of PDIA6 in the samples was detected by qPCR using primers designed in advance. The forward sequence of the primers is shown in SEQ ID NO: 5, the reverse sequence is shown in SEQ ID NO: 6, and the detection result is shown in Figure 2. The results showed that the RNA levels of PDIA6 in all spinal cord injury samples were up-regulated compared with the control group, and the Severe group with the most severe injury in the experimental design was more significantly up-regulated than other experimental groups; the experimental results indicated that the expression level of PDIA6 in injured spinal cord injury It was up-regulated, and the expression level was positively correlated with the degree of spinal cord injury to a certain extent.

Further, the obtained spinal cord samples were gradually dehydrated and made into paraffin sections, and PDIA6 antibody, fluorescent secondary antibody and DAPI were added dropwise to stain the nucleus, and then placed under a fluorescence microscope for observation. The detection results are shown in Figure 3. The results showed that the PDIA6 with green fluorescent label in the experimental group was significantly higher than that in the control group, and it increased with the degree of spinal cord injury, which was consistent with the expression of PDIA6 in the high-throughput sequencing results. The experimental results further indicate that the expression of PDIA6 is up-regulated in injured spinal cord.

Example 3 Study on the interaction between PDIA6 and spastin

(1) Construct prokaryotic expression vector plasmids of GST-Spastin and GST-PDIA6 and transform them into BL21 competent cells;

(2) Pick a single clone and culture it in LB medium until the measured OD value is 0.4-0.6, add IPTG, and induce overnight in a shaker at 25°C;

(3) After the induction, the bacterial solution was centrifuged and lysed, and then the fusion protein was purified by GST beads, electrophoresed and stained with Coomassie brilliant blue.

The detection results are shown in Figure 4. The results showed that the molecular weight of the purified fusion protein was in line with expectations.

Then, using the prepared rat brain lysate, set aside an appropriate amount of Input as a positive control, and use GSTbeads to remove non-specific proteins, and add the successfully purified fusion expression protein of GST-Spastin and GST-PDIA6 to the brain lysate for 4 Incubate overnight at °C with inversion, while GST protein was also used as a negative control. Finally, the completed Pull down samples were subjected to Western blot detection, and the detection results were shown in Figure 5. The results indicated that PDIA6 protein and Spastin protein could interact in vitro.

Further, the relationship between PDIA6 and spastin was studied through the eukaryotic expression vector plasmid, which specifically included the following steps:

(1) The 293T cells were plated one day in advance, and the Flag-PDIA6 plasmid and the GFP and GFP-Spastin plasmids were co-transfected into the 293T cells;

(2) 24 hours after transfection, the green fluorescence of the GFP label can be observed by fluorescence microscope, indicating that the transfection is successful, harvest and lyse the cells, take an appropriate amount of samples as a positive control, and use Agrose-proteinA/G to remove non-specific proteins;

(3) Add the GFP-tagged antibody to the cell lysate and incubate at 4°C overnight to bind to the Flag-PDIA6 fusion expression protein;

(4) The next day, new Agrose-proteinA/G was added to the samples, incubated at 4°C by inversion, the supernatant was removed and washed with cell lysate. Finally, Western blot was performed and the target bands were detected with GFP and Flag antibodies.

The test results are shown in Figure 6. The results showed that in samples co-transfected with Flag-PDIA6 and GFP-Spastin plasmids, GFP-Spastin protein could be detected by GFP antibody, while Flag-PDIA6 could be detected by Flag antibody, indicating that Agrose-proteinA/G successfully fused GFP-Spastin The expressed protein was pulled down, and the GFP-Spastin protein successfully pulled down the Flag-PDIA6 protein bound to it. GFP can be detected by GFP antibody in Flag-PDIA6 and GFP samples, but Flag-PDIA6 cannot be detected by Flag antibody, indicating that Agrose-proteinA/G successfully pulled down the GFP-Spastin fusion protein, but GFP could not detect Flag-PDIA6. PDIA6 pulled down. At the same time, the cell lysate supernatant was used as a positive control, indicating that the transfected plasmids were successfully transfected and expressed.

Using the same method, GFP-Spastin was transfected with Flag and Flag-PDIA6, respectively, and CO-IP experiments were performed. The experimental results are shown in Figure 7. The results showed that the Flag-PDIA6 protein successfully pulled down the GFP-Spastin protein, while the Flag protein as a negative control could not pull down the GFP-Spastin protein. At the same time, the positive control indicated that all the transfected plasmids were successfully transfected and expressed.

Subsequently, IP experiments were performed using Spastin antibody in rat brain lysate, and detected with PDIA6 and Spastin antibodies, respectively. The results are shown in Figure 8. The results showed that the Spastin protein bound to the beads successfully pulled down the PDIA6 protein, while IgG could not pull down the protein, while the positive control showed that the brain lysate was rich in Spastin and PDIA6 proteins.

Based on the above results, it is clear that PDIA6 protein and Spastin protein can interact in vivo and in vitro.

Example 4 Study on the effect of PDIA6 on neuronal process growth and neuron repair

First, the effect of PDIA6 overexpression on neuron growth and development was studied, including the following steps:

(1) The tag plasmid, Flag-PDIA6 plasmid, GFP-Spastin plasmid and Flag-PDIA6+GFP-Spastin plasmid were respectively transfected into hippocampal neurons of primary culture for 72h by calcium-phosphorus precipitation transfection method;

(2) After culturing for 24 hours, the hippocampal neuron cells were fixed and photographed, and immunofluorescence staining was performed. Finally, the cells were observed and photographed in a laser confocal microscope, and the number and length of neurites were counted.

The experimental results are shown in Figure 9-10, n=20/group, the results are expressed as Mean±SD, * means p < 0.05, and the scale is 100 μm. The results showed that transfection of PDIA6 or spastin could promote the growth of hippocampal neurons and increase the number of branches; while co-transfection of PDIA6 and spastin could significantly promote the growth of total neurites in hippocampal neurons, and the effect was more obvious than that of single transfection of Spastin. .

The above results show that overexpression of PDIA6 and spastin can both promote the growth of hippocampal neurons, and the effect of overexpression of spastin is more significant. On the basis of overexpression of spastin, increasing the expression level of PDIA6 in cells at the same time can more effectively increase the length and number of neuronal neurites, that is, PDIA6 can further enhance the effect of spastin on the growth of neuronal neurites.

Further, using hippocampal neuron cells to study the effect of PDIA6 on hippocampal neuron repair, the specific steps include the following:

(1) 120 μM glutamate was added to the hippocampal neurons of SD rats cultured for 1 day for injury;

(2) After culturing for 48 hours, the cells were transferred into the tag plasmid, Flag-PDIA6 plasmid, and Flag-PDIA6+siSpastin plasmid by calcium-phosphorus precipitation transfection method, wherein the siSpastin sequence is shown in SEQ ID NO: 7;

(3) After 24 hours, the cells were fixed, and immunofluorescence staining was performed. Finally, the cells were observed and photographed in a laser confocal microscope, and the number and length of protrusions were counted.

The experimental results are shown in Figure 11-12, n=20/group, the results are expressed as Mean±SD, * means p < 0.05, and the scale is 100 μm. The results showed that by increasing the expression level of PDIA6 in cells, the repair effect of neurons after injury could be effectively increased; however, after the use of siSpastin to inhibit the expression of spastin, the repair effect of PDIA6 was greatly reduced.

Subsequently, the above experiments were repeated using the siPDIA6 plasmid and the siPDIA6+siSpastin plasmid, wherein the siPDIA6 sequence is shown in SEQ ID NO: 8, and the siSpastin sequence is shown in SEQ ID NO: 7. It was found that when siPDIA6 was used to inhibit the expression of intracellular PDIA6, the repair effect of spastin on neurons was also significantly reduced (see Figures 13-14), indicating that siPDIA6 can inhibit spastin's ability to cut microtubules. The above experiment was further repeated with siPDIA6 having the sequence of SEQ ID NO: 9, showing the same results as above (results not shown).

According to the above results, it is clear that PDIA6 plays a key role in SCI and interacts with spastin to regulate neurite outgrowth. By identifying the differentially expressed proteins in the upper and lower positions of SCI, to clarify the role of PDIA6 in this process, and to demonstrate that the expression level of PDIA6 is up-regulated during SCI, PDIA6 can physically interact with spastin in vivo and in vitro. At the same time, it was also found that the interaction of PDIA6 and spastin can not only promote the growth of neuron neurites, but also promote the repair of damaged neurons. Since SCI is a major cause of disability, repairing the structural defects of the spinal cord caused by injury or degeneration is crucial in the modern field of regenerative medicine. Thus, studies based on the present invention can design a variety of drug interventions to treat SCI and assist related tissue repair, and can be combined with other cellular interventions.

The above-mentioned section of the detailed description has made a specific introduction to the analysis method involved in the present invention. It should be noted that the above description is only for helping those skilled in the art to better understand the method and idea of the present invention, rather than limiting the related content. Without departing from the principles of the present invention, those skilled in the art can also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications should also belong to the protection scope of the present invention.

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Thr Arg Ser Asp Ile Val Ser Arg Ala Leu Asp Leu Phe Ser Asp Asn

260 265 270

Ala Pro Pro Pro Glu Leu Leu Glu Ile Ile Asn Glu Asp Ile Ala Lys

275 280 285

Lys Thr Cys Glu Glu His Gln Leu Cys Val Val Ala Val Leu Pro His

290 295 300

Ile Leu Asp Thr Gly Ala Thr Gly Arg Asn Ser Tyr Leu Glu Val Leu

305 310 315 320

Leu Lys Leu Ala Asp Lys Tyr Lys Lys Lys Met Trp Gly Trp Leu Trp

325 330 335

Thr Glu Ala Gly Ala Gln Tyr Glu Leu Glu Asn Ala Leu Gly Ile Gly

340 345 350

Gly Phe Gly Tyr Pro Ala Met Ala Ala Ile Asn Ala Arg Lys Met Lys

355 360 365

Phe Ala Leu Leu Lys Gly Ser Phe Ser Glu Gln Gly Ile Asn Glu Phe

370 375 380

Leu Arg Glu Leu Ser Phe Gly Arg Gly Ser Thr Ala Pro Val Gly Gly

385 390 395 400

Gly Ser Phe Pro Asn Ile Thr Pro Arg Glu Pro Trp Asp Gly Lys Asp

405 410 415

Gly Glu Leu Pro Val Glu Asp Asp Ile Asp Leu Ser Asp Val Glu Leu

420 425 430

Asp Asp Leu Glu Lys Asp Glu Leu

435 440

<210> 4

<211> 581

<212> PRT

<213> Artificial Sequence

<400> 4

Met Ser Ser Pro Ala Gly Arg Arg Lys Lys Lys Gly Ser Gly Gly Ala

1 5 10 15

Ser Pro Ala Pro Ala Arg Pro Pro Pro Pro Ala Ala Val Pro Ala Pro

20 25 30

Ala Ala Gly Pro Ala Pro Ala Pro Gly Ser Pro His Lys Arg Asn Leu

35 40 45

Tyr Tyr Phe Ser Tyr Pro Leu Val Val Gly Phe Ala Leu Leu Arg Leu

50 55 60

Leu Ala Cys His Leu Gly Leu Leu Phe Val Trp Leu Cys Gln Arg Phe

65 70 75 80

Ser Arg Ala Leu Met Ala Ala Lys Arg Ser Ser Gly Thr Ala Pro Ala

85 90 95

Pro Ala Ser Pro Ser Thr Pro Ala Pro Gly Pro Gly Gly Glu Ala Glu

100 105 110

Ser Val Arg Val Phe His Lys Gln Ala Phe Glu Tyr Ile Ser Ile Ala

115 120 125

Leu Arg Ile Asp Glu Glu Glu Lys Gly Gln Lys Glu Gln Ala Val Glu

130 135 140

Trp Tyr Lys Lys Gly Ile Glu Glu Leu Glu Lys Gly Ile Ala Val Ile

145 150 155 160

Val Thr Gly Gln Gly Glu Gln Tyr Glu Arg Ala Arg Arg Leu Gln Ala

165 170 175

Lys Met Met Thr Asn Leu Val Met Ala Lys Asp Arg Leu Gln Leu Leu

180 185 190

Glu Ser Gly Ala Val Pro Lys Lys Lys Asp Pro Leu Thr His Ala Ser

195 200 205

Asn Ser Leu Pro Arg Ser Lys Thr Val Met Lys Ser Gly Ser Thr Gly

210 215 220

Leu Ser Gly His His Arg Ala Pro Ser Cys Ser Gly Leu Ser Met Val

225 230 235 240

Ser Gly Ala Arg Pro Gly Ser Gly Pro Ala Ala Thr Thr His Lys Gly

245 250 255

Thr Ser Lys Pro Asn Arg Thr Asn Lys Pro Ser Thr Pro Thr Thr Ala

260 265 270

Val Arg Lys Lys Lys Asp Leu Lys Asn Phe Arg Asn Val Asp Ser Asn

275 280 285

Leu Ala Asn Leu Ile Met Asn Glu Ile Val Asp Asn Gly Thr Ala Val

290 295 300

Lys Phe Asp Asp Ile Ala Gly Gln Glu Leu Ala Lys Gln Ala Leu Gln

305 310 315 320

Glu Ile Val Ile Leu Pro Ser Leu Arg Pro Glu Leu Phe Thr Gly Leu

325 330 335

Arg Ala Pro Ala Arg Gly Leu Leu Leu Phe Gly Pro Pro Gly Asn Gly

340 345 350

Lys Thr Met Leu Ala Lys Ala Val Ala Ala Glu Ser Asn Ala Thr Phe

355 360 365

Phe Asn Ile Ser Ala Ala Ser Leu Thr Ser Lys Tyr Val Gly Glu Gly

370 375 380

Glu Lys Leu Val Arg Ala Leu Phe Ala Val Ala Arg Glu Leu Gln Pro

385 390 395 400

Ser Ile Ile Phe Ile Asp Glu Val Asp Ser Leu Leu Cys Glu Arg Arg

405 410 415

Glu Gly Glu His Asp Ala Ser Arg Arg Leu Lys Thr Glu Phe Leu Ile

420 425 430

Glu Phe Asp Gly Val Gln Ser Ala Gly Asp Asp Arg Val Leu Val Met

435 440 445

Gly Ala Thr Asn Arg Pro Gln Glu Leu Asp Glu Ala Val Leu Arg Arg

450 455 460

Phe Ile Lys Arg Val Tyr Val Ser Leu Pro Asn Glu Glu Thr Arg Leu

465 470 475 480

Leu Leu Leu Lys Asn Leu Leu Cys Lys Gln Gly Ser Pro Leu Thr Gln

485 490 495

Lys Glu Leu Ala Gln Leu Ala Arg Met Thr Asp Gly Tyr Ser Gly Ser

500 505 510

Asp Leu Thr Ala Leu Ala Lys Asp Ala Ala Leu Gly Pro Ile Arg Glu

515 520 525

Leu Lys Pro Glu Gln Val Lys Asn Met Ser Ala Ser Glu Met Arg Asn

530 535 540

Ile Arg Leu Ser Asp Phe Thr Glu Ser Leu Lys Lys Ile Lys Arg Ser

545 550 555 560

Val Ser Pro Gln Thr Leu Glu Ala Tyr Ile Arg Trp Asn Lys Asp Phe

565 570 575

Gly Asp Thr Thr Val

580

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 5

atgcgggtga tcagcatgg 19

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 6

tcacaactcg tccttctcta ggtca 25

<210> 7

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 7

ccagucagau gagaaauau 19

<210> 8

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 8

gcacugaaag auguuguua 19

<210> 9

<211> 19

<212> RNA

<213> Artificial Sequence

<400> 9

uaacaacauc uuucagugc 19

Claims (2)

1. Use of the PDIA6 protein in the preparation of a medicament for the treatment and/or repair of acute spinal cord injury.
2. The application of the PDIA6 protein in preparing a medicament for improving the therapeutic sensitivity of the spastin protein, wherein the therapeutic sensitivity is particularly the therapeutic sensitivity to acute spinal cord injury and/or repair.

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