CN114668836B - Application of PDIA6 in preparing medicine for treating 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 preparation of medicine for treating spinal cord injury and repairing spinal cord injury

Technical Field

The invention relates to the field of biomedicine, in particular to application of a PDIA6 protein or an active fragment thereof in preparing a medicament for treating spinal cord injury and repairing.

Background

Spinal Cord Injury (SCI) is a serious central nerve injury, resulting in dysfunction of motor and sensory functions of the limbs below the injury level, and in severe cases, can result in permanent spinal cord dysfunction, seriously affecting the quality of life of patients. The main cause of dysfunction after SCI is neuronal damage due to direct mechanical injury, followed by secondary damage including inflammatory reactions, oxidative stress and excitatory damage, which results in neuronal axonal damage and glial cell proliferation, ultimately leading to neuronal cell death. The neurological dysfunction caused by SCI is permanent because, after the spinal cord tissue is damaged, the regenerative capacity of axons 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 reconstruct nerve pathways, and thus, it is expected to improve nerve dysfunction caused by spinal cord injury.

PDIA6 is an endoplasmic reticulum protein capable of catalyzing protein folding and disulfide bond formation, possessing thioredoxin domains containing pairs of active cysteine residues that can undergo disulfide bond conversion, isomerization and reduction by conversion between disulfides and dimercapto compounds. Studies show that PDIA6 as a molecular chaperone is closely related to Endoplasmic Reticulum Stress (ERS) and can be activated into an unfolded protein reaction to reduce the accumulation of misfolded or unfolded proteins, thereby promoting the survival of cells under stress conditions. However, the role of PDIA6 in spinal cord injury is unclear.

Spastin is a microtubule-cleaving protein that regulates microtubule movement and rearrangement by cleaving long microtubules into shorter ones. Overexpression of spastin in neurons enhances branch formation of their processes, whereas silencing spastin results in decreased length of neuronal axons and inhibition of branch formation. When the Spastin plays a role, the microtubules are pulled into the central holes by combining the recognition structures with positive charges with the microtubules, so that the microtubules are cut. Spastin cuts microtubules and regulates microtubule dynamics which is essential for neuronal axonal growth. In addition, there are studies that suggest that spastin is involved in endoplasmic reticulum morphogenesis. Whether the spastin and the PDIA6 have an interaction relationship or not and whether the PDIA6 can dynamically promote nerve repair by regulating microtubules through the spastin is not clear at present.

Disclosure of Invention

The invention aims to solve the technical problems existing in the spinal cord injury repair treatment process in the prior art, thereby providing a novel spinal cord injury repair treatment target point, deeply researching the function and action mechanism of PDIA6 in the spinal cord injury repair process, disclosing the key role of PDIA6 in the spinal cord injury repair treatment, and providing 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 by the following technical solutions.

In a first aspect, the invention provides a pharmaceutical composition for treating spinal cord injury and/or repair, comprising PDIA6 protein and/or an active fragment thereof, spastin protein.

Preferably, the PDIA6 active fragment sequence is selected from SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2.

Preferably, the PDIA6 protein sequence is as set forth in SEQ ID NO: 3 is shown in the specification; the sequence of the spastin protein is shown as SEQ ID NO: 4, respectively.

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

In a second aspect, the invention provides the use of PDIA6 protein and/or active fragments thereof in the manufacture of a medicament for the treatment of spinal cord injury and/or repair.

Preferably, the PDIA6 active fragment sequence is selected from SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2.

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

In a third aspect, the invention provides a kit for detecting the severity of spinal cord injury, which comprises a primer for detecting PDIA6 protein and/or an active fragment thereof.

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

The fourth aspect of the invention provides the application of the 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 the PDIA6 comprises a primer for detecting the expression level of the 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 the PDIA6 gene is shown as SEQ ID NO: 5, and the reverse sequence is shown as SEQ ID NO: and 6.

Preferably, the reagent for detecting the protein content of PDIA6 is selected from PDIA6 monoclonal antibody and/or polyclonal antibody. The monoclonal antibody and/or the polyclonal antibody can be obtained commercially.

In a fifth aspect, the invention provides the use of the PDIA6 protein and/or active fragments thereof in the preparation of a medicament for increasing the therapeutic sensitivity of a spastin protein, in particular to spinal cord injury and/or repair.

Preferably, the PDIA6 active fragment sequence is selected from SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2.

Preferably, the PDIA6 protein sequence is as set forth in SEQ ID NO: 3 is shown in the figure; the sequence of the spastin protein is shown as SEQ ID NO: 4, respectively.

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

In a sixth aspect, the present invention provides a method for screening drugs for treating spinal cord injury and/or repair, comprising the steps of:

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

(2) detecting the expression level of PDIA6 in vivo of a spinal cord injury animal model so as to obtain a target drug; when the candidate drug causes the expression level of the PDIA6 to be increased, the target drug is obtained.

Without being particularly specified, the "PDIA 6 protein" referred to in the context of the present invention is an endoplasmic reticulum protein capable of catalyzing protein folding and disulfide bond formation. Reference to an active fragment of the PDIA6 protein is to the amino acid sequence length being varied somewhat, either long or short, but maintaining the activity of the PDIA6 protein as a whole. The "spastin protein" referred to is a microtubule cleaving protein.

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

In the prior research, the rearrangement and movement of cytoskeletal microtubules are the key aspects of repair reaction after spinal cord injury, and the spastin is used as a microtubule cutting protein to regulate microtubule movement and rearrangement by cutting long microtubules into shorter microtubules. Overexpression of spastin in neurons can enhance branch formation of its processes, while silencing spastin leads to a decrease in the length of neuronal axons and inhibition of branch formation. When the Spastin plays a role, the microtubules are pulled into the central holes by combining the recognition structures with positive charges with the microtubules, so that the microtubules are cut. Spastin cuts microtubules and regulates microtubule dynamics which is essential for neuronal axonal growth.

In the invention, the protein PDIA6 is found through a great deal of research to be one of key proteins in the processes related to spinal cord injury repair. The PDIA6 protein was significantly upregulated in the spinal cord injury model and was, to some extent, positively correlated with the extent of injury. The ability of PDIA6 to interact with GST-spastin was discovered by using LC-MS method and the potential interaction between spastin and PDIA6 was verified using co-immunoprecipitation experiments and staining method. Overexpression of PDIA6 in neurons, found that overexpression of PDIA6 promotes neurite extension and collateral formation in injured neurons; if PDIA6 is overexpressed while interfering with spastin expression, the promoting function is significantly reduced. In contrast, when PDIA6 is knocked down alone, the formation of neurite extension and side branch of injured neurons is obviously weakened, and when the expression of spastin is knocked down simultaneously, the formation of neurite extension and side branch of injured neurons is weaker. In addition, it was found that the microtubule cleavage ability of spastin was significantly reduced by interfering with PDIA6 and overexpressing spastin. Therefore, the inventor creatively discovers the key role of PDIA6 in spinal cord injury and repair and the correlation between PDIA6 and spastin in the research process, and defines the important function of PDIA6 in regulating neurite growth and branching, thereby indicating that PDIA6 can be a novel target for treating spinal cord injury.

Compared with the prior art, the invention has the following technical effects:

the present invention has demonstrated through extensive studies that PDIA6 plays a key role in SCI and interacts with spastin to regulate neurite outgrowth. By identifying proteins that are differentially expressed in the upper and lower positions of SCI, to confirm that PDIA6 plays a role in this process, and to demonstrate that PDIA6 expression levels are upregulated during SCI, PDIA6 is able to 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. Since SCI is the major cause of disability, repair of structural defects in the spinal cord caused by injury or degeneration is of vital importance in the field of modern regenerative medicine. 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.

Drawings

FIG. 1 is a graph showing the results of RNA and protein levels of PDIA6 in spinal cord injury samples.

FIG. 2 is a graph showing the comparison of the PDIA6 expression level of the injury group samples in the qPCR experiment with the control group.

FIG. 3 is a graph showing the results of comparing the expression level of PDIA6 in a section of a spinal cord injury tissue with that of a control group.

FIG. 4 is a schematic diagram showing the electrophoresis results of the constructed fusion expression protein proteins of GST-Spastin and GST-PDIA 6.

FIG. 5 is a diagram showing the interaction result between PDIA6 protein and Spastin protein in vitro.

FIG. 6 is a schematic diagram showing the co-immunoprecipitation results after co-transfection of Flag-PDIA6 with GFP and GFP-Spastin plasmids, respectively.

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

FIG. 8 is a diagram showing the results of IP treatment of rat brain lysate with Spastin antibody.

FIG. 9 is a graph showing the effect of PDIA6 and Spastin on the growth of hippocampal neurites, wherein 9A is a status diagram of the growth of hippocampal neurites in a tag plasmid (Vector) group; 9B is a protrusion growth state diagram of Flag-PDIA6 plasmid (PDIA 6) Hippocampus neurons; 9C is a protruding growth state diagram of Flag-Spastin plasmid (Spastin) group hippocampal neurons; 9D is the protuberant growth state diagram of Flag-PDIA6+ GFP-Spastin plasmid (Spastin + PDIA 6) hippocampal neurons.

FIG. 10 is a graph showing the quantitative results of the effects of PDIA6 and Spastin on the growth of hippocampal neurites, wherein 10A is a graph showing the results of the total number of hippocampal branches in each group; 10B is a schematic diagram of the total length result of each group of hippocampal neuron branches; 10C is a schematic diagram of the result of the number of hippocampal neurites in each group; 10D is a graphical representation of the results for each group of hippocampal neurite outgrowth.

FIG. 11 is a graph showing the effect of PDIA6 and sispasatin on growth of hippocampal neurites.

FIG. 12 is a graph showing the quantitative results of the effects of PDIA6 and sispasatin on the growth of hippocampal neurites.

FIG. 13 is a graph showing the effect of siPDIA6 and siSpastin on the growth of hippocampal neurites.

FIG. 14 is a graph showing the quantitative results of the effects of siPDIA6 and siSpastin on the growth of hippocampal neurites.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is described in further detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Of the reagents used in the context of the present invention, they are commercially available. The related proteins such as PDIA6 and spastin and the gene sequences thereof mentioned in the invention can be obtained by conventional means (such as Pubmed and Uniprot), and the experimental methods used in the invention, such as DNA extraction, gene sequencing, primer design, vector construction, Western blot, cell experiments, animal experiments and the like, are all conventional methods and technologies in the field. For animal experiments, the related 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.

Representative results from selection of biological experimental replicates are presented in the context figure, and data are presented as mean ± SD and mean ± SEM as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 22.0 software. And comparing the difference of the average values of two or more groups by adopting conventional medical statistical methods such as t test, chi-square test, variance analysis and the like.p< 0.05 was considered to be a significant difference.

In the description of the specification, reference to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

EXAMPLE 1 preparation of plasmids and constructs

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

(2) pull-down assay with glutathione S-transferase (GST): spinal cord tissue sections were ground and lysed, followed by rinsing of GST-agarose beads (Invitrogen, CA, USA), mixing with the lysate, incubation at 4 ℃ for 1h, and centrifugation at 1000g for 10 min at 4 ℃. The supernatant was then collected and the above steps repeated once. The appropriate bead fusion protein was then added to spinal cord tissue and the sample was incubated overnight at 4 ℃. After centrifugation at 1000g for 5 minutes at 4 ℃, unbound protein is washed with 1mL of wash buffer, and the sample is centrifuged at 1000g for 1 minute at 4 ℃. This washing step was repeated six times, and then the proteins that had been pulled down were analyzed by western blotting and Mass Spectrometry (MS).

The mass spectrum detection method comprises the following steps:

the proteins were first separated using NuPAGE 4-12% gels (Life Technologies) and then MS analysis of the splastin-GST pull-down assay was performed using the colloidal blue staining kit (Life Technologies). The excised protein bands were trypsinized and then used with nanoscale reverse phase liquid chromatography-tandem mass spectrometry together with HPLC Ultimate 3000 (DIONEX, usa) coupled to a linear ion trap (LTQ, thermo electron, usa) to analyze the peptides in data-dependent acquisition mode.

IPA analysis was also performed on the differentially expressed proteins (http:// www.ingenuity.com). IPA analysis utilizes a curated database to identify overlapping functions and relationships between these proteins, assigning scores to specific biological function networks such that a score of > 2 generally indicates significant enrichment of a particular biological function in a given set of protein protons. These scores correspond to the log probability of detecting a given network due to random chance only.

Through the screening process, PDIA6 is found to be one of the key proteins related to spinal cord injury and also a potential protein interacting with spastin.

Example 2 study of the role of PDIA6 in spinal cord injury repair

Firstly, constructing a rat SCI model, which specifically comprises the following steps:

Sprague-Dawley (SD) rats used in the experiment were purchased from the Experimental animals center of Zhongshan university, and the rats were individually housed in a facility at 25 + -3 deg.C with a supply of conventional food and water, and specifically included the following steps:

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

(2) the entire T10 vertebral plate was removed and the exposed area of the spine was approximately 2.5mm by 3 mm;

(3) the T10 section was secured on both sides with a stabilizer and the nitrogen tank controlling the impact head was set at 18psi or 124 kPa. Loading a U-shaped stabilizer with a rat onto a platform of a Lewis vascular injury system device (LISA), and adjusting the dura mater/spinal cord height directly below an impactor, while monitoring by a laser beam;

(4) adjusting the collision depth to different damage levels, setting the collision depth to be 0.6mm, 1.0mm or 1.8mm respectively representing the Light, Medium and Severe damage degrees, and setting the time to be 0.5 s;

(5) after induction of injury, the stabilizer was removed from the platform, the rat was removed from the stabilizer, the injured site was evaluated and bleeding was inhibited, and finally the muscle and skin of the rat were sutured using 3-0 silk.

Screening a spinal cord injury model successfully modeled by observing the activity condition of two lower limbs of a rat, wherein the evaluation standard comprises the following steps: paralytic paralysis, tail sway reflex, body and leg flicks, spinal cord ischemia, edema around wound sites, and the like. Sham operated animals received a T10 total vertebrectomy, but the spinal cord was not injured. All rats received 2000U/day gentamicin treatment and the bladder of each rat was manually squeezed every 8 hours to aid in urination until spontaneous urination was observed. Using the SCI rat model constructed above, rats were perfused 72 hours after injury with paraformaldehyde and elution samples were collected.

The rat T10 spinal cord injury model constructed in the above way is used, the vertebral canal is opened again after 72h of injury, the injured segment is taken out for transcriptomic high-throughput lateral suction, and the change condition of the RNA level and the protein level of PDIA6 in a spinal cord injury sample is analyzed.

The results of the analysis are shown in FIG. 1. Wherein FIGS. 1A and 1B are a heat map and a line graph of changes in PDIA6 RNA and protein levels, the results indicate that PDIA6 protein expression levels and RNA levels in the injured spinal cord samples are upregulated compared to the control group, and both increase with the severity of the spinal cord injury; FIG. 1C is a correlation analysis of the PDIA6 protein level and RNA level in samples, indicating that the two are in positive correlation.

To clarify the expression of PDIA6 in spinal cord injury samples, RNA was extracted from the removed spinal cord samples, followed by reverse transcription, followed by detection of the RNA level of PDIA6 in the samples using qPCR apparatus with primers designed in advance, the forward sequences of which are shown in SEQ ID NO: 5, the reverse sequence is shown as SEQ ID NO: FIG. 6 shows the results of the detection in FIG. 2. The results show that the RNA level of PDIA6 in all spinal cord injury samples is up-regulated compared with the control group, and the Severe group with the most serious injury in the experimental design is up-regulated more obviously compared with other experimental groups; the experimental result shows that the expression level of PDIA6 is up-regulated in the injured spinal cord injury, and the expression level is in positive correlation with the spinal cord injury degree to some extent.

Further, the obtained spinal cord samples were gradually dehydrated to prepare paraffin sections, and the PDIA6 antibody, fluorescent secondary antibody and DAPI stained nuclei were incubated dropwise and then observed under a fluorescent microscope, and the detection results are shown in fig. 3. The results show that the experimental group has significantly more PDIA6 with green fluorescence label than the control group, and increases with the degree of spinal cord injury, which is consistent with the expression of PDIA6 in the high-throughput sequencing results. The results of this experiment further demonstrate that PDIA6 is up-regulated in expression in injured spinal cord injury.

Example 3 interaction study of PDIA6 with spastin

(1) Constructing prokaryotic expression vector plasmids of GST-Spastin and GST-PDIA6, and transforming the prokaryotic expression vector plasmids into BL21 competence;

(2) selecting the single clone to be cultured in LB culture solution until OD value is measured to be 0.4-0.6, adding IPTG, and inducing in a shaker at 25 ℃ overnight;

(3) after induction, the bacterial solution was centrifuged and lysed, followed by purification of the fusion expression protein using GST beads, electrophoresis and staining with coomassie brilliant blue.

The results of the detection are shown in FIG. 4. The results show that the molecular weight of the purified fusion expressed protein is consistent with the expectation.

And then, using a prepared rat brain lysate in advance, reserving a proper amount of Input as a positive control, using GST beads to remove non-specific protein, adding the successfully purified fusion expression protein of GST-Spastin and GST-PDIA6 into the brain lysate, turning and incubating at 4 ℃ overnight, and simultaneously using GST protein as a negative control test. And finally, carrying out Western blot detection on the finished Pull down sample, wherein the detection result is shown in FIG. 5. The results indicate that the PDIA6 protein and the Spastin protein can interact in vitro.

Further, the relation between PDIA6 and spastin is researched through eukaryotic expression vector plasmids, and the method specifically comprises the following steps:

(1) the 293T cells are plated one day in advance, and Flag-PDIA6 plasmid and GFP-Spastin plasmid are respectively co-transferred to the 293T cells;

(2) after 24h of transfection, the green fluorescence of the GFP label can be observed through a fluorescence microscope, which indicates that the transfection is successful, cells are collected and lysed, a proper amount of sample is reserved as a positive control, and Agrose-protein A/G is used for removing non-specific protein;

(3) adding the GFP tag antibody into cell lysate, turning and incubating at 4 ℃ overnight, and combining Flag-PDIA6 fusion expression protein;

(4) adding new Agrose-protein A/G into the sample the next day, turning over and incubating at 4 ℃, removing supernatant, washing with cell lysate, performing Western blot detection, and detecting a target band by using GFP and Flag antibodies.

The results of the detection are shown in FIG. 6. The results show that in the samples co-transferred by the Flag-PDIA6 and the GFP-Spastin plasmid, the GFP-Spastin protein can be detected by the GFP antibody, and the Flag-PDIA6 can be detected by the Flag antibody, which indicates that the Agrose-protein A/G successfully pulls down the GFP-Spastin fusion expression protein, and simultaneously the GFP-Spastin protein successfully pulls down the Flag-PDIA6 protein combined with the GFP-PDIA fusion expression protein. The use of GFP antibody in the Flag-PDIA6 and GFP samples detected GFP, while the failure to detect Flag-PDIA6 by the Flag antibody indicated that Agrose-proteINA/G successfully pulled down the GFP-Spastin fusion expression protein, but the failure of GFP to pull down Flag-PDIA 6. Meanwhile, the supernatant of the cell lysate is used as a positive control, which indicates that each transfected plasmid is successfully transfected and expressed.

GFP-Spastin was transfected with Flag and Flag-PDIA6, respectively, in the same manner, and subjected to CO-IP experiments, the results of which are shown in FIG. 7. The results show that the Flag-PDIA6 protein successfully pulls down the GFP-Spastin protein, while the Flag protein as a negative control cannot pull down the GFP-Spastin protein, and meanwhile, the positive control indicates that each transfected plasmid is successfully transfected and expressed.

Subsequently, IP experiments were performed in rat brain lysates using Spastin antibodies, and detected using PDIA6 and Spastin antibodies, respectively, with the results shown in fig. 8. The results show that the protein of Spastin bound to beads successfully pulls down the protein PDIA6, meanwhile IgG can not pull down the protein, and the positive control shows that the brain lysate contains abundant protein of Spastin and PDIA 6.

The results are combined to confirm that the PDIA6 protein and the Spastin protein can interact in vivo and in vitro.

Example 4 Effect of PDIA6 on neurite outgrowth and neuronal repair

Firstly, the research on the growth and development conditions of the neurons by the overexpression of PDIA6 specifically comprises the following steps:

(1) respectively transfecting tag plasmids, Flag-PDIA6 plasmids, GFP-Spastin plasmids and Flag-PDIA6+ GFP-Spastin plasmids into primary cultured hippocampal neuron cells for 72h by a calcium-phosphorus precipitation transfection method;

(2) and after continuously culturing for 24h, fixing and photographing the hippocampal neuron cells, performing immunofluorescence staining, and finally observing and photographing in a laser confocal microscope to count the number and the length of the processes.

The results of the experiments are shown in fig. 9-10, with n = 20/group, and the results are expressed as Mean ± SDp< 0.05, on a scale of 100 μm. The results show that the transfection of PDIA6 or spastin can promote the growth of the hippocampal neuron processes and the increase of the branch number; the co-transformation of PDIA6 and Spastin can obviously promote the growth of total hippocampal neurite, and the promotion effect is more obvious than that of single transformation of Spastin.

The results show that the over-expression of PDIA6 and the over-expression of spastin can promote the growth of hippocampal neurite, and the over-expression of spastin has more obvious effect. On the basis of overexpression of the spastin, the expression level of PDIA6 in the cells is improved, so that the increase of the length and the number of the neuron cell processes can be effectively improved, namely the PDIA6 can further improve the promoting effect of the spastin on the growth of the neuron processes.

Further, the method for researching the influence of PDIA6 on repair of the hippocampal neurons by using the hippocampal neuron cells specifically comprises the following steps:

(1) adding 120 mu M glutamic acid into the cultured SD rat hippocampal neuron cells for injury;

(2) after continuous culture for 48h, the plasmids are respectively transferred into a tag plasmid, a Flag-PDIA6 plasmid and a Flag-PDIA6+ plasmid of the sispasatin by a calcium-phosphorus precipitation transfection method, wherein the sequences of the sispasatin are shown as SEQ ID NO: 7 is shown in the specification;

(3) and fixing cells after 24 hours, performing immunofluorescence staining, and finally observing and photographing in a laser confocal microscope to count the number and the length of the protrusions.

The results of the experiments are shown in fig. 11-12, with n = 20/group, and the results are expressed as Mean ± SDp< 0.05, on a scale of 100 μm. The results show that the repair effect of the damaged neurons can be effectively increased by improving the expression level of the PDIA6 in the cells; after the inhibition of the expression of spastin by the sispasatin, the repair effect of PDIA6 is greatly reduced.

The experiment was then repeated using the siPDIA6 plasmid, siPDIA6+ siSpastin plasmid, where the siPDIA6 sequence is as set forth in SEQ ID NO: 8, the sequence of the siSpastin is shown as SEQ ID NO: shown at 7. As a result, when the expression of intracellular PDIA6 is inhibited by utilizing siPDIA6, the repair effect of the spastin on neurons is also remarkably reduced (see figures 13-14), and the fact that the siPDIA6 can inhibit the microtubule cleavage capability of the spastin is suggested. Further using a polypeptide having a sequence of SEQ ID NO: 9 siPDIA6 the above experiment was repeated showing the same results as above (results not shown).

From the above results it is clear that PDIA6 plays a key role in SCI and interacts with spastin to regulate neurite outgrowth. By identifying proteins that are differentially expressed in the upper and lower levels of SCI, to clarify that PDIA6 plays a role in this process, and to demonstrate that PDIA6 expression levels are up-regulated during SCI, PDIA6 is capable of physically interacting 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. Since SCI is the leading cause of disability, repair of structural defects in the spinal cord caused by injury or degeneration is of paramount importance in the field of modern regenerative medicine. Thus, studies based on the present invention can design a variety of pharmaceutical interventions to treat SCI and assist in the associated tissue repair, and can be combined with other cellular interventions.

The above description of the embodiments specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the purpose of limiting the relevant contents. The present invention may be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification also fall within the scope of the present invention.

Sequence listing

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Gly Phe Pro Thr Ile Lys Ile Phe Gly Ala Asn Lys Asn Lys Pro Glu

100 105 110

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

115 120 125

Ser Ala Leu Arg Gln Leu Val Lys Asp Arg Leu Gly Gly Arg Ser Gly

130 135 140

Gly Tyr Ser Ser Gly Lys Gln Gly Arg Gly Asp Ser Ser Ser Lys Lys

145 150 155 160

Asp Val Val Glu Leu Thr Asp Asp Thr Phe Asp Lys Asn Val Leu Asp

165 170 175

Ser Glu Asp Val Trp Met Val Glu Phe Tyr Ala Pro Trp Cys Gly His

180 185 190

Cys Lys Asn Leu Glu Pro Glu Trp Ala Ala Ala Ala Thr Glu Val Lys

195 200 205

Glu Gln Thr Lys Gly Lys Val Lys Leu Ala Ala Val Asp Ala Thr Val

210 215 220

Asn Gln Val Leu Ala Ser Arg Tyr Gly Ile Lys Gly Phe Pro Thr Ile

225 230 235 240

Lys Ile Phe Gln Lys Gly Glu Ser Pro Val Asp Tyr Asp Gly Gly Arg

245 250 255

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 (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 (Artificial Sequence)

<400> 5

atgcgggtga tcagcatgg 19

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcacaactcg tccttctcta ggtca 25

<210> 7

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccagucagau gagaaauau 19

<210> 8

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcacugaaag auguuguua 19

<210> 9

<211> 19

<212> RNA

<213> Artificial Sequence (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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