CN114522236B

CN114522236B - Pharmaceutical composition for treating and repairing spinal cord injury and application thereof

Info

Publication number: CN114522236B
Application number: CN202210432596.7A
Authority: CN
Inventors: 张国威; 陈云; 杨裕豪; 林宏生
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2022-07-22
Anticipated expiration: 2042-04-22
Also published as: CN114522236A

Abstract

The invention relates to a pharmaceutical composition for treating and repairing spinal cord injury and application thereof. The invention discovers through research that Rab3A plays a key role in SCI and interacts with Spastin to regulate neurite outgrowth. The expression level of Rab3A was shown to be down-regulated during SCI by identifying proteins that are differentially expressed in SCI. In addition, Rab3A can physically interact with Spastin, and the degradation of Spastin is regulated through a lysosome pathway, so that the function of Spastin is influenced. These findings together highlight the signal transduction pathway in which Rab3A mediates the degradation of spastin that regulates the formation and growth of neurite branches. 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. Various pharmaceutical interventions can be designed to treat SCI and assist in the associated tissue repair, and may be combined with other cellular interventions.

Description

Pharmaceutical composition for treating and repairing spinal cord injury and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a pharmaceutical composition for treating and repairing spinal cord injury and application thereof.

Background

Spinal Cord Injury (SCI) can lead to permanent spinal cord dysfunction, severely affecting the quality of life of the patient. 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.

Rab proteins are the largest subfamily of the small molecule GTP-binding proteins (small GTP-binding proteins) family, which, like other gtpases, mediate a variety of physiological activities in cells by regulating the switch between GTP and GDP. Rab plays an important role in regulating neuronal axonal transport, membrane trafficking and maintaining synaptic function. Rab is closely related to many central nervous system diseases. Upregulation of the expression of various Rab protein family members in Cholinergic Basal Forebrain (CBF) neurons may lead to cognitive dysfunction (NCI). Mutations in Rab39B elevate immature GluA2 levels, leading to the formation of AMPA receptors lacking this subunit, thereby altering neuronal synaptic function. The lack of Rab11 activity results in impaired neuronal function involved in the development of Huntington's Disease (HD). Deletion of Wdr13 upregulates Rab3A, resulting in increased density of hippocampal dendritic spines. Whether Rab3A can influence the spinal cord injury repair process is not clear at present, and the action mechanism of the Rab3A is blank.

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, deeply researching the function and action mechanism of Rab3A in the spinal cord injury repair process, disclosing the key function of Rab3A in the spinal cord injury repair treatment, and providing practical theoretical basis and direction for the clinical treatment of spinal cord injury.

In order to solve the above-mentioned technical problems, the present invention is achieved by the following technical means.

In a first aspect of the invention there is provided a pharmaceutical composition for use in the treatment and repair of spinal cord injury comprising an inhibitor of Rab3A and a spastin protein or an active fragment thereof.

Preferably, the Rab3A inhibitor is selected from siRNA designed based on Rab3A gene (siRab 3A).

Preferably, the siRNA sequence designed based on Rab3A gene is shown in SEQ ID NO: 1 is shown.

Preferably, the sequence of the spastin protein is as shown in SEQ ID NO: 2, respectively.

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

In a second aspect, the invention provides the use of an Rab3A inhibitor in the manufacture of a medicament for the treatment and repair of spinal cord injury.

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

In a third aspect, the invention provides a kit for detecting spinal cord injury and repair, which comprises a first specific primer for detecting Rab3A protein or an active fragment thereof and/or a second specific primer for detecting spastin protein or an active fragment thereof.

Preferably, the forward sequence of the first specific primer is shown in SEQ ID NO: 3, the reverse sequence is shown as SEQ ID NO: 4, respectively.

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

In a fourth aspect, the present invention provides a method for screening drugs for treating spinal cord injury and 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 Rab3A and/or spastin in a spinal cord injury animal model body; when the candidate drug causes the expression level of Rab3A to be reduced and/or causes the expression level of spastin to be increased, the target drug is obtained.

The "Rab 3A protein" referred to in the context of the present invention is a small molecule GTP-binding protein, unless otherwise specified; the active fragment of the Rab3A protein refers to the amino acid sequence with length changed slightly, or longer or shorter, but the activity of the Rab3A protein is maintained as a whole. The "spastin protein" referred to is a microtubule cleaving protein; reference to an active fragment of a spastin protein is a reference to a change in the length of the amino acid sequence, either long or short, but which overall retains the activity of the spastin 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 implicitly indicating 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 previous studies, spastin has been found to regulate microtubule movement and rearrangement by cleaving long microtubules into shorter microtubules. 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 the invention, the inventor finds that Rab3A protein is spinal cord injury related protein through a large number of researches, the expression level of the protein in a SCI model is remarkably reduced, and Rab3A and spastin can directly interact with each other. In injured spinal cord tissue, Rab3A protein and spastin protein co-localize with each other. These interactions suggest that Rab3A and spastin may both play important roles in microtubule dynamics regulation. The interaction between Rab3A and spastin can inhibit SCI damage and initiate the associated repair process. Thus, through the interaction of these proteins, drug intervention can be used to achieve SCI repair and treatment.

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

(1) in the present invention, proteomics methods are used to identify key proteins involved in the repair process of spinal cord injury. Proteins capable of interacting with GST-Spastin were identified by using LC-MS method and potential interaction between Rab3A and Spastin was verified using co-immunoprecipitation experiments and staining methods.

(2) The invention discovers that the over-expression of Rab3A in neurons can inhibit protrusion extension and collateral formation; in contrast, several perturbed Rab3A expressions may promote neurite outgrowth and collateral formation. By co-transfecting COS7 cells with Rab3A and spastin, Rab3A was found to reduce the microtubule cleavage ability of spastin, while Rab3A was found to influence its function by inducing degradation of spastin through the lysosomal pathway.

(3) In the invention, Rab3A and spastin are co-transfected into hippocampal neurons, and Rab3A is found to inhibit the ability of spastin to promote the growth of hippocampal neurites. And the specific mechanism of Rab3A participating in the spinal cord injury and repair process is determined by constructing a spinal cord injury animal model, utilizing immunofluorescence staining, protein mass spectrometry, high-throughput sequencing and the like. The invention defines Rab3A as 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 the clinical treatment of spinal cord injury.

Drawings

FIG. 1 is a schematic diagram showing the immunofluorescence staining results of primary cultured hippocampal neurons transfected with GFP and GFP-Rab3A for 48h by the calcium phosphate method.

FIG. 2 is a graph showing the quantitative statistics of the effect of Rab3A on the axon and process length of hippocampal neurons.

FIG. 3 is a graph showing the quantitative statistics of the effect of Rab3A on the number of axons and processes in hippocampal neurons.

FIG. 4 is a diagram showing the WB detection results of siRab3A on Rab3A expression.

FIG. 5 is a graph showing the immunofluorescence staining results 48h after transfection of primary cultured hippocampal neurons with GFP and GFP-siRab 3A.

FIG. 6 is a diagram showing the statistical results of the Rab3A expression amount in two samples with typical Rab3A staining as a positive standard.

FIG. 7 is a graphical representation of the quantitative statistics of the effect of SiRab3A on hippocampal cell axon and process length.

Fig. 8 is a graphical representation of the quantitative statistics of the effects of siRab3A on the number of axons and processes in hippocampal neurons.

FIG. 9 is a schematic representation of the results of Rab3A gene transcription levels in spinal cord injury and sham surgery groups.

FIG. 10 is a graph showing the results of gene transcription levels of spastin in spinal cord injury and sham groups.

Fig. 11 is a graph showing the results of protein expression levels of Rab3A in spinal cord injury and sham operation groups.

FIG. 12 shows the protein expression level of spastin in the group of spinal cord injury and sham operation.

Fig. 13 is a graph showing the correlation results of Rab3A transcription level and protein expression level after spinal cord injury.

FIG. 14 is a schematic diagram showing the results of immunofluorescence assay of spastin protein expression levels after the lysis of spinal cord tissues of rats in the group of spinal cord injury and sham operation.

FIG. 15 is a schematic diagram showing the results of detecting the expression level of spastin protein by immunoblotting after the spinal cord tissues of rats in the spinal cord injury and sham operation groups are lysed.

FIG. 16 is a schematic diagram showing the results of western blot detection performed after the GST and GST-spasitn recombinant proteins are purified and run.

FIG. 17 is a schematic representation of the electrophoretic staining results of purified GST and GST-spasitn proteins after interaction with lysates and rat brain lysates, respectively.

FIG. 18 shows Rab3A peptide fragment interacting with spastin, its sequence is MSESLDTADPAVTGAK.

FIG. 19 shows the Rab3A peptide fragment with LQIWDTAGQER sequence interacting with spastin.

FIG. 20 shows Rab3A peptide fragment interacting with spastin, with sequence LQIWETAGQER.

FIG. 21 is a schematic diagram showing the results of the experiment of the CoIP using the spastin antibody as an IP antibody and the rat brain lysis.

FIG. 22 is a diagram showing the results of a CoIP experiment using Rab3A antibody as an IP antibody and rat brain lysis.

FIG. 23 is a diagram showing the results of CoIP experiments using Flag as an IP antibody after HEK293 cells overexpress Flag-spastin and GFP-Rab 3A.

FIG. 24 is a diagram showing the result of CoIP experiment using GFP as IP antibody after HEK293 cells overexpress Flag-spastin and GFP-Rab 3A.

FIG. 25 is a diagram showing the results of detecting the distribution of COS7 cells endogenous Spastin and Rab3A using antibodies to Spastin and Rab 3A.

FIG. 26 is a diagram showing the results of detecting the distribution of endogenous Spastin and Rab3A in neuronal cells using antibodies to Spastin and Rab 3A.

FIG. 27 is a graph showing the results of fluorescence of target protein expression and microtubules.

FIG. 28 is a schematic diagram showing the results of fluorescence intensity of each group of GFP + mCherry, GFP + mCherry-Rab3A, GFP-Spastin + mCherry and GFP-Spastin + mCherry-Rab3A relative to microtubules.

FIG. 29 is a graph showing the fluorescence intensity results of each group of GFP-Spastin + mCherry and GFP-Spastin + mCherry-Rab3A relative to microtubules.

FIG. 30 is a graph showing the effect of Rab3A transfection on the expression level of spastin protein in COS7 cells.

FIG. 31 is a schematic diagram showing the effect of transfection of Rab3A and spastin on the expression level of spastin protein in COS7 cells.

FIG. 32 is a graph showing the effect of the expression level of spastin protein in COS7 of different groups after Rab3A transfection.

FIG. 33 is a graph showing the effect of expression levels of spastin protein in different groups of COS7 after transfection of spastin.

FIG. 34 is a graph showing the effect of expression level of spastin protein in COS7 of different groups after transfection of Rab3A and spastin.

FIG. 35 is a diagram showing the effect of the expression level of spastin protein in COS7 of different groups on the quantitative results after Rab3A transfection.

FIG. 36 is a diagram showing the effect of the expression level of spastin protein in COS7 of different groups on the quantitative results after transfection of spastin.

FIG. 37 is a diagram showing the effect of the expression level of spastin protein in COS7 of different groups on the quantitative results after Rab3A and spastin are transfected.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in 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.

Among the reagents used in the context of the present invention, those commercially available are not specifically mentioned. 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 herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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) Rab3A and spastin sequences were obtained for cDNA preparation, which was 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;

(2) pull down assay 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 the lysate, incubated at 4 ℃ for 1h, and then centrifuged at 1000g for 10 min. 4 deg.C. The supernatant was then collected and these steps were repeated once more. The appropriate bead fusion protein was then added to spinal cord tissue, and the sample was incubated overnight at 4 ℃. After 5 minutes of spinning at 1000g at 4 ℃, unbound protein is washed with 1mL of wash buffer, and then the sample is spun 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 that a given network is detected due to random chance only.

Example 2 construction of rat SCI model

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

(1) 10% chloral hydrate (0.35 mL/100g body weight) was intraperitoneally injected for anesthetizing SD rats, after which a 2.5 cm longitudinal dorsal incision was made exposing the T9-11 spinal process and the 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 fixed 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 and setting the time to be 0.5s for mild, moderate and severe injuries;

(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.

The target SCI model animals met the following injury criteria: paralytic paralysis, tail sway reflex, body and leg flicks, spinal cord ischemia and edema around the wound site. 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.

Rats were perfused with paraformaldehyde 72 hours after injury using the SCI rat model constructed above, and elution samples were collected.

Example 3 Rab3A regulates neuronal growth and development

Firstly, researching the growth and development conditions of the neurons by overexpression of Rab3A, and specifically comprising the following steps:

(1) transfection into primary cultured hippocampal neurons for 72h with GFP-Rab3A and GFP (blank control), respectively;

(2) after a further 48h, hippocampal neuronal cells were fixed and photographed at 100 μm scale.

The results are shown in FIGS. 1-3, wherein FIG. 1 is a graph showing immunofluorescence staining results of hippocampal neuron cells, FIG. 2 is a graph showing quantitative statistics of lengths of axons and processes of hippocampal neuron cells, FIG. 3 is a graph showing quantitative statistics of the number of axons and processes of hippocampal neuron cells, n = 20/group, and the results are expressed as Mean + -SDpIs less than 0.05. The scale bar is 100. mu.m. According to the results, the excessive expression of Rab3A in the cells can obviously inhibit the growth of neurons (P < 0.05).

Subsequently, the research on the growth and development of the neurons after the Rab3A expression inhibition specifically comprises the following steps:

(1) siRNA (siRab 3A) is designed based on Rab3A gene, and the sequence of the siRNA is shown in SEQ ID NO: 1, GCCAUGGGCUUCAUUCUAATT; transfecting the cells into primary cultured hippocampal neuron cells for 72h, and simultaneously transfecting blank vectors into the hippocampal neuron cells as a control group under the same experimental conditions;

(2) and after continuing culturing for 48 hours, collecting cell lysate for Western blot detection.

The results are shown in FIGS. 4-5. The results show that the endogenous Rab3A expression level of hippocampal neurons transfected with siRab3A interference fragments is remarkably reduced (p＜0.05）。

The Rab3A interference fragment and the blank vector were subsequently transfected in hippocampal neurons under the same experimental conditions, respectively, and after 48h the cells were harvested and the length and total number of branch neurites quantified. The results are shown in fig. 6-8, wherein fig. 6 is a graph showing statistics of Rab3A expression in two samples with typical Rab3A staining as positive standard, fig. 7 is a graph showing quantitative statistics of the lengths of axons and processes of hippocampal neurons, fig. 8 is a graph showing quantitative statistics of the numbers of axons and processes of hippocampal neurons, n = 20/group, and the results are expressed as Mean ± SD, and are expressed as ×pIs less than 0.05. The scale bar is 100. mu.m. The results show that significantly more branches were observed in Rab 3A-perturbed neurons than in the blank control group (c)p< 0.05). The above experimental results clearly indicate that overexpression and interference of Rab3A can affect branch formation and neurite outgrowth.

Example 4 Rab3A correlation with spastin

High throughput sequencing and protein mass spectrometry of spinal cord tissue samples from rats obtained according to example 2 was performed to determine the relationship of spastin and Rab3A to SCI.

High throughput sequencing analysis was first performed on samples from the sham and SCI groups, and the results are shown in fig. 9-12, where fig. 9 and 10 show the gene transcription levels of Rab3A and spastin in the spinal cord injury and sham groups, and fig. 11 and 12 show the protein expression levels of Rab3A and spastin in the spinal cord injury and sham groups. The results show that Rab3A was down-regulated at both protein and RNA levels in the SCI group samples and that spastin was up-regulated at protein levels in the SCI group samples. Further analysis revealed a clear correlation between Rab3A transcript levels and protein levels after spinal cord injury (see figure 13).

Then, the SCI group samples were analyzed by western blot and immunofluorescence staining, and the results are shown in fig. 14-15, where fig. 14 shows that after the spinal cord tissue of rats in the spinal cord injury and sham operation groups was lysed, the immunofluorescence detects the expression level of spastin protein, the arrow points to the expressed spastin, and the magnification is 200 times; FIG. 15 shows the level of spastin protein expression detected by immunoblotting after the spinal cord tissue of rats in the group of spinal cord injury and sham operation was lysed, with GAPDH as an internal control. The results show that the protein expression of the spastin in SCI group samples is increased, and further verify the key role of the spastin in the spinal cord injury repair process.

The above results show that Rab3A expression is down-regulated and spastin expression is up-regulated during SCI, providing strong evidence that Rab3A is associated with spastin and is related to SCI.

In order to further determine which peptide fragments in Rab3A can interact with the spastin, GST-spastin plasmid is transformed into BL21 escherichia coli competence, a large amount of GST-spastin is obtained by inducing BL21 expression, GST-spastin is purified by GST specific magnetic beads, and western blot detection is carried out at the same time. The detection results are shown in FIGS. 16-20, in which FIG. 16 shows the results of western blot detection after running purified GST and GST-spasitn recombinant proteins, and the GST antibody is used for incubation; FIG. 17 shows the Coomassie blue staining results of purified GST and GST-spasitn proteins interacting with lysates and rat brain lysates, respectively, after running electrophoresis gel on the interacting products; FIGS. 18-20 show three peptides of Rab3A interacting with spastin, MSESLDTADPAVTGAK, LQIWDTAGQER and LQIWETAGQER, respectively. The above results further confirm the ability of Rab3A to interact with GST-spastin and the specific peptide stretch acting.

To assess whether Rab3A specifically interacts with spastin, further validation was performed using Co-immunoprecipitation (Co-IP) analysis of rat brain extracts. Specifically, using a spastin-specific antibody to pull down spastin in rat brain lysates, and using a detector Rab3A via western blot, it was found that spastin can co-precipitate Rab3A (see fig. 21). Also, spastin can be co-precipitated using the same method with Rab3A specific antibodies (see fig. 22). In addition, Rab3A with a tag and spastin were co-expressed in HEK293 cells using the same assay and detected by tag antibodies, which further demonstrated that Rab3A interacts with spastin (see fig. 23-24). Subsequently, the distribution of endogenous Rab3A and spastin in neurons and COS7 cells was examined by Rab3A and spastin antibodies, and the results are shown in FIGS. 25-26, where the co-localization of Rab3A and spastin is shown in the merged image, and Enlargement is an enlarged view of the corresponding box with a scale of 100 or 25 μm. The immunofluorescence analysis results revealed co-localization of Rab3A and spastin in neurons.

Example 5 Effect of Rab3A on the cleavage function of spastin and its mechanism

COS7 cells are respectively transfected by GFP + mCherry, GFP + mCherry-Rab3A, GFP-Spastin + mCherry or GFP-Spastin + mCherry-Rab3A, the cells are harvested after 12h transfection, and the Tubulin antibody and the corresponding fluorescent secondary antibody are used for detecting the condition of the microtubules.

The results are shown in fig. 27-29, where fig. 27 is a graph showing the results of target protein expression and microtubule status fluorescence, fig. 28 and 29 are graphs showing the results of comparing the relative microtubule fluorescence intensity between groups and performing statistics, n = 30/group, results are expressed as mean ± SD and ×, or # indicates P < 0.05, and scales are 100 μm and 20 μm. The result shows that the fluorescence intensity of microtubules can be obviously weakened by transfecting the spastin, which indicates that the overexpression of the spastin can cut microtubules in a cell body; the strength of the microtubules of the co-transfected Rab3A and spastin group is obviously stronger than that of the spastin group, which shows that Rab3A can inhibit the ability of the spastin to cut the microtubules.

In order to search a mechanism that Rab3A influences the function of the Spastin, COS7 cells are transfected with GFP, GFP-Rab3A or co-transfected with GFP-Rab3A and Flag-Spastin, cell lysates are collected, Westen blot is carried out, and the endogenous and exogenous expression levels of the Spastin of the cells are detected by using Spastin and Flag antibodies respectively. The results of the tests are shown in FIGS. 30 to 31. The results show that Rab3A can obviously reduce the expression amount of endogenous and exogenous spastin.

GFP-Rab3A, Flag-Spastin or GFP-Rab3A and Flag-Spastin were then transfected into COS7 cells, respectively, and divided into blank, Leupeptin and MG-132 treatment groups, with the results shown in FIGS. 32-37. The results show that both Leupeptin and MG-132 can reverse Rab3A mediated degradation of spastin, indicating that Rab3A mediates degradation of spastin through lysosome pathway.

The above results indicate that Rab3A plays a key role in SCI and interacts with spastin to regulate neurite outgrowth. By identifying proteins that are differentially expressed in SCI, it can be determined that Rab3A plays a role in this process, and it was demonstrated that Rab3A expression levels are down-regulated during SCI. In addition, Rab3A can physically interact with spastin in vivo and in vitro, and Rab3A regulates the degradation of spastin through a lysosome pathway, so that the function of spastin is influenced. These findings collectively highlight the signal transduction pathway in which Rab3A mediates the degradation of spastin to regulate the formation and growth of neurite branches. 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. Various pharmaceutical interventions can be designed to treat SCI and assist in the associated tissue repair, and may 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 limitation of the related contents. The present invention can 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 should also fall within the protection scope of the present invention.

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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> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcctcag ccacagactc t 21

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcagcaggcg caatcctg 18

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggccgcca agaggagc 18

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttaaacagtg gtgtctccaa agtcc 25

Claims

The application of Rab3A inhibitor in preparing medicine for treating spinal cord injury and repair is characterized in that the Rab3A inhibitor is selected from siRNA designed based on Rab3A gene, and the siRNA sequence designed based on Rab3A gene is shown in SEQ ID NO: 1 is shown.
The application of the Rab3A inhibitor in preparing the medicine for improving the therapeutic action of the spastin protein is characterized in that the therapeutic action is specifically the therapeutic action on spinal cord injury and repair, the Rab3A inhibitor is selected from siRNA designed based on Rab3A gene, the siRNA sequence designed based on Rab3A gene is shown as SEQ ID NO: 1 is shown.