Application of lamprey transcription factor MKX in preparation of medicine for promoting spinal cord injury repair

Abstract

The invention discloses application of a lamprey transcription factor MKX in preparation of a medicine for promoting spinal cord injury repair, and belongs to the technical field of biology. The lamprey MKX gene is found to have a spinal cord injury repairing function by taking a mouse as a model for the first time, and can promote nerve cell proliferation and axon growth. The invention provides a new application of the lamprey MKX gene in spinal cord injury repair treatment, and provides a new perspective for research and clinical treatment of human spinal cord injury.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of lamprey transcription factor MKX in preparing a drug for promoting spinal cord injury repair. Background Art

[0002] Spinal cord injury (SCI) is usually caused by traumatic events, common causes include traffic accidents, falls from heights, falls, tumbles and injuries. In addition, sports injuries occurring in competitive sports are also one of the common factors leading to spinal cord injury. Spinal cord injury may cause temporary or permanent changes in spinal cord function, which can cause a series of symptoms, such as loss of sensation in the body parts below the injury site, weakened muscle function or autonomic dysfunction. Depending on the severity and specific symptoms of the spinal cord injury, patients may suffer from different effects ranging from mild body numbness to complete paralysis. In addition, spinal cord injury may also cause a series of complications, such as urinary incontinence. In the process of treating spinal cord injury, traditional rehabilitation training and drug therapy often have limited effects and cannot effectively promote the regeneration of damaged nerve tissue. Therefore, it is particularly important to find new treatment strategies.

[0003] Transcription factors are a class of key proteins that play a vital role in the regulation of gene expression and signal transduction networks. In the process of nervous system pathology, transcription factors mediate the proliferation, development and regeneration of nerve cells. Transcription factors play a key role in the process of nervous system injury and repair by regulating the expression of a series of genes, affecting the differentiation, migration and synapse formation of nerve cells. Therefore, in-depth research on the function of transcription factors in spinal cord injury can provide us with valuable information to understand the molecular mechanism of axon regeneration after nervous system injury. Summary of the invention

[0004] The present invention provides an application of lamprey transcription factor MKX in preparing a drug for promoting spinal cord injury repair, so as to solve the above problems.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] The present invention provides application of lamprey transcription factor MKX in preparing a medicine for promoting the repair of spinal cord injury.

[0007] Furthermore, the nucleotide sequence of the transcription factor MKX is shown in SEQ ID NO.1.

[0008] Furthermore, the drug for promoting spinal cord injury repair includes a viral vector and a transcription factor MKX loaded on the viral vector.

[0009] Furthermore, the viral vector is adeno-associated virus AAV.

[0010] Furthermore, the concentration of the adeno-associated virus in the drug for promoting spinal cord injury repair is 1×10 13 PFU / ml.

[0011] Furthermore, the drug is administered by fixed-point injection.

[0012] The beneficial effects of the present invention are:

[0013] The present invention discloses an application of the lamprey transcription factor MKX in the preparation of a drug for promoting spinal cord injury repair. Based on the results of bioinformatics analysis, transcription factors with potential effects on the repair of lamprey spinal cord injury are screened out, and the efficacy of the transcription factor MKX in promoting the repair of spinal cord injury is evaluated by using a variety of methods such as 5-ethynyl-2'-deoxyuridine (EdU) experiment, methylthiazolium tetrazolyl (MTT) test, immunofluorescence staining, protein spectrum analysis, and cell scratch experiment. Finally, the characteristic of the lamprey transcription factor MKX in promoting the repair of spinal cord injury is used to construct a mouse spinal cord injury model, and the function of the transcription factor MKX is verified by experiments such as BBB scoring, immunofluorescence, and protein blotting, which provides practical experimental evidence and scientific basis for the clinical treatment of spinal cord injury, lays a foundation for revealing the molecular mechanism of regeneration of lamprey spinal cord injury, and provides a new perspective for the research and clinical treatment of human spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 Screening of differentially expressed genes after Baclofen and GABA treatment, where A, E: heat map of differentially expressed genes, red indicates up-regulated genes, blue indicates down-regulated genes; B, F: volcano map showing differentially expressed genes; C, G: KEGG pathway of differentially expressed genes; D, H: GO analysis;

[0016] Figure 2 Screening of key modules of spinal cord injury for WGCNA and GO analysis, including: A: topological network analysis under various soft threshold powers; B: clustering dendrogram of co-expressed genes in spinal cord injury; C: heat map of gene network; D: network diagram of modules and characteristic genes; E: scatter plot between green-yellow module membership and gene significance at spinal cord injury stage; F: GO analysis; G: hub gene network diagram; H: module feature association;

[0017] Figure 3 Western Blot was used to detect the expression of various proteins in PC-12 cells;

[0018] Figure 4 MTT assay was used to evaluate the effects of transcription factors on PC-12 cell proliferation;

[0019] Figure 5 The EdU method was used to evaluate the effect of transcription factors on PC-12 cell proliferation, where A: EdU method was used to detect PC-12 cell proliferation; B: Histogram showing the percentage of proliferating cells in the total number of cells;

[0020] Figure 6 The effects of transcription factors on the migration ability of PC-12 cells in an in vitro wound healing assay were demonstrated;

[0021] Figure 7 This is a graph showing the effects of different transcription factors on neurite growth in PC-12 cells;

[0022] Figure 8 The results of Western Blot detection of MKX protein expression in spinal cord injury tissues of mice in each group;

[0023] Fig. 9 Figure 2 is the BBB score result of mice in each group;

[0024] Fig.10 This is the result of immunofluorescence detection of spinal cord neuron survival in each group of mice. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Screening of differentially expressed genes

[0027] The data used in this example were downloaded from the GEO database, and the GSE number is GSE137858.

[0028] Screening of differentially expressed genes was performed as follows:

[0029] a. Differential expression analysis was performed using the WGCNA R software package (version 3.5.1);

[0030] b. Transcripts with |log2FC|>1 and adjusted p-value<0.05 were considered differentially expressed genes;

[0031] c. All differentially expressed genes were mapped to corresponding terms in the GO and KEGG databases; GO annotation analysis mainly focused on biological process (BP), molecular function (MF) and cellular component (CC);

[0032] d. The adjusted p-value < 0.05 indicates significant enrichment; the volcano plot is used to visually observe the relationship between qValue and |log2FC|; cluster heat maps are used to perform cluster analysis on genes with similar expression patterns to explore the functions of unknown genes or unknown functions of known genes;

[0033] Figure 1 To screen for differentially expressed genes after Baclofen and GABA treatment, KEGG analysis and GO analysis revealed that the genes were mainly enriched in pathways related to disease and protein synthesis.

[0034] Example 2: Analysis and screening of key modules for spinal cord injury

[0035] The data used in this example were downloaded from the GEO database, and the GSE number is GSE95686.

[0036] Weighted gene co-expression network analysis (WGCNA) R software package (version 3.5.1) was used to perform WGCNA analysis on all transcription factors in GSE95686 data as follows:

[0037] a. Remove outliers from normalized gene expression data using hierarchical clustering;

[0038] b. The optimal soft threshold is determined based on the R2 value and slope, and the scale-free network is verified;

[0039] c. A gene-level clustering tree matrix was constructed using hierarchical clustering and topological overlap;

[0040] d. Module membership (MM) and gene significance (GS) were selected to verify the association between genes and clinical information, thereby identifying key modules and genes.

[0041] Figure 2Screening of key modules for spinal cord injury for WGCNA and GO analysis, including: A: topological network analysis under various soft threshold powers; B: clustering dendrogram of co-expressed genes in spinal cord injury; C: heat map of gene network; D: network diagram of modules and characteristic genes; E: scatter plot between green and yellow module membership and gene significance at spinal cord injury stage; F: GO analysis; G: hub gene network diagram; H: module feature association. After hub gene screening, HOXD8, HOXC9, ETV7, MKX, RUNX1, PRDM8, SMAD1, SOX9 and NFIA genes are hub genes and are most relevant to spinal cord injury repair. The nucleotide sequence of MKX gene is shown in SEQ ID NO.1, the nucleotide sequence of ETV7 is shown in SEQ ID NO.2, the nucleotide sequence of HOXD8 gene is shown in SEQ ID NO.3, the nucleotide sequence of NFIA gene is shown in SEQ ID NO.4, the nucleotide sequence of RUNX1 gene is shown in SEQ ID NO.5, the nucleotide sequence of SMAD1 gene is shown in SEQ ID NO.6, the nucleotide sequence of SOX9 gene is shown in SEQ ID NO.7, the nucleotide sequence of HOXC9 gene is shown in SEQ ID NO.8, and the nucleotide sequence of PRDM8 gene is shown in SEQ ID NO.9.

[0042] Example 3: Western Blot detection of the expression of various gene proteins in PC-12 cells

[0043] The cells used in this example are PC-12 cells purchased from Procell Life Science Technology Co., Ltd. HOXD8, HOXC9, ETV7, MKX, RUNX1, PRDM8, SMAD1, SOX9 and NFIA screened out in Example 2 were fused with the Flag tag sequence and then integrated into the plasmid pcDNA3.1(+), which was synthesized by GenScript Biotech.

[0044] (A) Plasmids containing nine different transcription genes were transfected into PC-12 cells:

[0045] Transfection was performed when PC-12 cells reached 60-80% confluence. Transfection was performed using Lipofectamine 3000 reagent and following the manufacturer's instructions to successfully transfect plasmids into PC-12 cells;

[0046] (B) Western Blot detection of target protein expression in PC-12 cells

[0047] a. Wash the successfully transfected PC-12 cells with PBS, then add RIPA lysis buffer and PMSF to fully lyse the cells, heat them in a 100°C water bath for 10 min, centrifuge them at 12,000 rpm for 10 min, and transfer the supernatant into a test tube for later use;

[0048] b. Take an equal amount of the protein sample obtained in the above steps, separate the protein by SDS-PAGE electrophoresis, transfer to PVDF membrane at 30V for 70min, block the PVDF membrane in blocking solution at room temperature, add primary antibody and secondary antibody for binding, wash 3 times with PBST, 10min each time, drain, add ECL Plus reagent (supersensitive ECL luminescent liquid), and develop in an automatic developer.

[0049] Figure 3 Western Blot was used to detect the expression of various proteins in PC-12 cells. It can be seen from the figure that various genes were successfully transfected into the cells and their expression in the cells was almost the same.

[0050] Example 4: Detection of cell proliferation using diphenyltetrazolium bromide (MTT) method and 5-ethyl-2′-deoxyuridine (EdU) method

[0051] (A) Cell proliferation was detected by MTT method. The steps are as follows:

[0052] a. 1×10 per well 4 PC-12 cells were cultured at 37°C and 5% CO 2 Cultured in 96-well culture plates;

[0053] b. Using the transfection method of Example 3, PC-12 cells were transfected with plasmids inserted with different transcription factors, and 10 μl of MTT was added to each well at 0, 24, 48 and 72 hours after transfection, and incubated for another 4 hours;

[0054] c. Remove the cell supernatant and add 100 μl DMSO to dissolve the formazan crystals;

[0055] d. Finally, the absorbance (OD value) was measured at a wavelength of 570 nm using an enzyme reader.

[0056] Figure 4 This is the result of evaluating the effect of transcription factors on PC-12 cell proliferation using the MTT method. As can be seen from the figure, all nine genes have the phenomenon of promoting cell proliferation, among which the MKX gene has the most significant effect.

[0057] (B) 5-ethyl-2′-deoxyuridine (EdU) method was used to detect cell proliferation. The steps are as follows:

[0058] a. PC-12 cells were collected at 1×10 4The cells were seeded at a density of 10 cells / well in a 96-well plate;

[0059] b. Using the transfection method in Example 3, PC-12 cells were transfected with plasmids having different transcription factors inserted therein, and 48 hours after transfection, the cells were incubated with EdU reagent for 2 hours;

[0060] c. Wash the cells with PBS;

[0061] d. Fix in 4% paraformaldehyde for 30 minutes;

[0062] e. Add 2 mg / ml glycine to neutralize paraformaldehyde;

[0063] f. Add penetrant and treat for 10 minutes;

[0064] g. Strictly follow the instructions of the kit for EdU staining and observe the proliferating cells under a fluorescence microscope. All measurements were repeated three times (n=3).

[0065] Figure 5 EdU method was used to evaluate the effect of transcription factors on PC-12 cell proliferation. Figure 5 A and Figure 5 B shows that all nine genes promote cell proliferation, among which the MKX gene has the most significant effect.

[0066] Example 5: In vitro wound healing assay (cell scratch assay) verifies the effect of transcription factors on PC-12 cell migration ability

[0067] The experiment was carried out as follows:

[0068] a. PC-12 cells were plated at 2.5×10 6 The cells were seeded at a density of 10 cells / mL in a six-well plate;

[0069] b. When 90% confluence is reached, scrape evenly using the tip of a sterile pipette;

[0070] c. Each well was washed with PBS, and then the plasmids inserted with transcription genes were transfected into PC-12 cells according to the transfection method of Example 3;

[0071] d. Observe the cell confluence under a microscope and take pictures at 0, 24, 48, and 72 hours after transfection.

[0072] Figure 6 The effect of transcription factors on the migration ability of PC-12 cells in an in vitro wound healing experiment is shown. As can be seen from the figure, all nine genes promote cell migration, among which MKX and HOXD8 genes have the most significant effects.

[0073] Example 6: Effects of different transcription factors on neurite outgrowth in PC-12 cells

[0074] The cell immunofluorescence experiment was performed as follows:

[0075] a. Incubate PC-12 cells at 37°C, 5% CO 2 The cells were cultured in air, the culture medium was changed every 3-4 days, and the cells were subcultured after 90% confluence at 1x10 4 The cells were plated at a density of cells / well in six-well plates;

[0076] b. In the absence of serum, 100ng / ml Fgf2 (fibroblast growth factor 2) was added to each well as the positive control group, and 2.5ug transcription factor DNA was added to the test group to induce cell differentiation for 3 days. PC-12 cells were used as negative controls. After the induction culture, the cells were fixed and stained with mouse anti-tubulin antibody and secondary anti-mouse CoraLite488-conjugated antibody for quantitative measurement of neurite growth.

[0077] Figure 7 The effects of different transcription factors on neurite outgrowth in PC-12 cells. Figure 7 A and Figure 7 B shows that all nine genes promote cell migration, among which MKX and RUNX1 genes have the most significant effects.

[0078] Example 7

[0079] The mice used in this example are female C57BL / 6 mice, 8 to 10 weeks old, weighing 20 to 25 g, and are sourced from Liaoning Changsheng Biotechnology Co., Ltd.

[0080] (A) Experimental groups

[0081] Group A: simple SCI group (SCI group);

[0082] Group B: SCI+transfected with adeno-associated virus empty vector group (AAV group);

[0083] Group C: SCI+transfection of target gene overexpression adeno-associated virus vector group (AAV-MKX group).

[0084] (B) The membrane of mouse spinal cord injury was constructed as follows:

[0085] a. Anesthetize the mice by injecting tribromoethanol (dose 0.2ml / 10g);

[0086] b. Make an incision on the skin above the T10 vertebra of the mouse, disinfect the surgical area with 75% ethanol first, and then disinfect it again with betadine solution. Then, peel off the fascia and muscles to expose the lower vertebral body;

[0087] c. Perform T10 laminectomy to completely expose the spinal cord, then let a 10g weight fall freely from a height of 15mm to hit the exposed spinal cord, causing spinal cord contusion;

[0088] d. The treatment of mice in the sham operation group was exactly the same as that of the contusive spinal cord injury group, with the only difference being that no heavy objects were applied to hit the exposed spinal cord;

[0089] e. After suturing the spinal cord incision, the animal was allowed to recover naturally. Manual bladder management was performed twice a day until the mouse was able to recover its urination reflex naturally.

[0090] (C) This experiment uses the BBB scoring system to evaluate the recovery of motor function after spinal cord injury in mice. BBB scoring was performed before spinal cord injury and 7, 14, 21, and 28 days after injury. The scoring was performed by two people who were proficient in the scoring standard, using a double-blind method and independent observation and recording. The average score of the two researchers was finally taken and recorded as the score of one animal.

[0091] The BBB rating standards are as follows:

[0092] 0 points: no hind limb movement;

[0093] 1 point: Slight movement of one or two joints, usually the hip and knee joints;

[0094] 2 points: one joint moves greatly or one joint moves greatly and another joint moves slightly;

[0095] 3 points: wide range of motion of both joints;

[0096] 4 points: All three joints of the hind limbs can move slightly;

[0097] 5 points: Two joints can move slightly, and the third joint can move widely;

[0098] 6 points: Two joints can move widely, and the third joint can move slightly;

[0099] 7 points: All three joints of the hind limbs can move to a large extent;

[0100] 8 points: The paw can touch the ground without bearing weight;

[0101] 9 points: The palm surface of the claw bears weight or the back of the claw bears weight and moves, and there is no palm surface of the claw supporting movement;

[0102] 10 points: Occasionally the palm surface of the paw moves under weight, but there is no coordinated movement of the front and rear limbs;

[0103] 11 points: The palm surface of the paw can be seen to bear weight and move more, but there is no coordinated movement of the front and rear limbs;

[0104] 12 points: The palm surface of the paw can be seen to bear weight and move more frequently, and coordinated movements of the front and rear limbs can be seen occasionally;

[0105] 13 points: The palm surface of the paw can be seen to bear weight and move more often, and coordinated movements of the front and rear limbs are common:

[0106] 14 points: There are continuous weight-bearing movements of the palm surface and coordinated movements of the front and back limbs, or common palm surface movements, continuous coordinated movements of the front and back limbs, and occasional dorsal movement of the paw;

[0107] 15 points: continuous palmar movement and continuous coordinated movements of the forelimbs and hindlimbs, with or without forelimb grasping during forward movement, and the active claw position is parallel to the body at the time of initial contact;

[0108] 16 points: Continuous palm movement and continuous coordinated movements of the forelimbs and hindlimbs can be seen in the gait. The claws are often seen grasping the ground during the forward movement of the forelimbs. The active claw position is parallel to the body at the initial contact, and the weight is transferred and then rotated;

[0109] 17 points: Continuous palmar movement and coordinated forelimb and hindlimb movements can be seen in the gait. The claws are often seen grasping the ground during the forward movement of the forelimbs. The active claw position is parallel to the body at the initial contact and after the weight transfer.

[0110] 18 points: Continuous palm movement and coordinated movements of the front and rear limbs can be seen in the gait. The front limbs can continuously grasp the ground with their claws. The active claws are parallel to the body at the initial contact and after the weight transfer. The tail is sometimes or always drooping.

[0111] 19 points: Continuous palm movement and coordinated movements of the forelimbs and hindlimbs can be seen in the gait. The forelimbs can continuously grasp the ground with their claws during forward movement. The active claws are parallel to the body at the initial contact and after the weight transfer. The tail is sometimes or always drooping.

[0112] 20 points: continuous palmar movement, continuous coordinated gait, continuous toe gripping, active claw position parallel to the body at initial contact and after weight transfer, unstable trunk, and continuous tail lift;

[0113] 21 points: Continuous palm movement, continuous coordinated gait, toes continuously gripping the ground, the active claw position is always parallel to the body during the activity, the trunk is continuously stable, and the tail is continuously lifted.

[0114] Fig. 9Figure 2 is the BBB score of mice in each group. Before modeling, the BBB score of all rats was about 21 points. After recovery from anesthesia, the BBB scores of the SCI group, AAV group, and AAV-MKX group decreased significantly. The BBB scores of the AAV-MKX group at 14d, 21d, and 28d after injury were significantly increased compared with those of the SCI group and AAV group, and the differences were statistically significant (P<0.05).

[0115] Example 8: Western Blot detection of MKX protein expression in spinal cord injury tissue of mice

[0116] The steps of this embodiment are as follows:

[0117] a. Take the spinal cord tissues of mice in group B and group C in Example 7, grind them into powder under liquid nitrogen freezing, transfer them into centrifuge tubes after the liquid nitrogen evaporates, add RIPA lysis buffer and PMSF to dissolve them, fully lyse them, heat them in a 100°C water bath for 10 min, centrifuge them at 12000 r / min for 10 min, and transfer the supernatant into test tubes for later use;

[0118] b. Take an equal amount of the protein samples obtained in the above steps, separate the proteins by SDS-PAGE electrophoresis, transfer to PVDF membrane at 30V for 40min, take the membrane and add blocking solution to block at room temperature, add primary antibody and secondary antibody to bind, wash 3 times with PBST, 10min each time, add ECLPlus reagent after draining, and develop in an automatic developer.

[0119] Figure 8 This is the result of Western Blot detection of the expression of MKX protein in the spinal cord injury tissue of each group of mice. As can be seen from the figure, it was verified that the MKX gene has been expressed in the mouse spinal cord.

[0120] Example 9: Effect of transcription factor MKX on neuronal survival

[0121] Take the spinal cord tissue paraffin sections of groups A, B, and C in Example 7 that were stored at low temperature, place them at room temperature for 30 minutes, dewax them in a conventional gradient of xylene and ethanol, and then put them in a pH 9.0 citrate buffer for antigen repair at 80°C for 5 minutes. After cooling at room temperature, the sections were soaked in PBS for 5 minutes, rat anti-NeuN primary antibody (1:100) was added, and the sections were kept in a refrigerator at 4°C overnight. After washing with PBS, AlexaFluor488-labeled goat anti-mouse IgG secondary antibody was added and incubated at room temperature for 1 hour. Hoechst staining for 30 minutes, PBS washing, anti-fluorescence attenuation sealing liquid sealing the sections, and the sections were observed and images were collected under a fluorescence microscope to observe the number of fluorescent signals. Three fields of view were randomly selected around the spinal cord injury for each section, and the number of positive cells was counted.

[0122] Fig.10Immunofluorescence was used to detect the survival of spinal cord neurons in each group of mice. At 28 days after SCI, the number of NeuN-positive neurons in the AAV-MKX group increased significantly compared with the SCI group and the AAV group, and the difference was statistically significant (P<0.05).

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of lamprey transcription factor MKX in the preparation of drugs to promote spinal cord injury repair.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the transcription factor MKX is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The drug for promoting spinal cord injury repair comprises a viral vector and a transcription factor MKX loaded on the viral vector.

4. The use according to claim 1, characterized in that: The viral vector is adeno-associated virus AAV.

5. The use according to claim 1, characterized in that: The concentration of the adeno-associated virus in the drug for promoting spinal cord injury repair is 1x10 13 PFU / ml.

6. The use according to claim 1, characterized in that: The drug is administered by injection at a fixed point.

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