CN111330009B - Application of m6A modified related gene ALKBH5 in promotion of nerve axon injury repair - Google Patents

Abstract

The invention discloses application of a m6A modified related gene ALKBH5 in preparation of a medicine for treating diseases related to axonal injury. The invention also discloses an application of the m6A modified related gene ALKBH5 in promoting the repair of axonal injury, which is characterized by comprising the following steps: s1, detecting the expression and positioning of ALKBH 5; s2, interfering with the effect of ALKBH5 expression on neuronal axon growth; s3, interfering with the effect of ALKBH5 expression on sciatic nerve axon growth; s4, and ALKBH5 target gene LPIN 2. The invention verifies that the in vitro and in vivo interference of the ALKBH5 gene can obviously promote the growth of neuron axons and promote the recovery of sciatic nerve functions; and through transcriptome analysis and target gene verification after ALKBH5 interference, a target gene LPIN2 regulated by ALKBH5 is searched, and the effect of regulating and controlling the growth of neuron axons is verified.

Description

Application of m6A modified related gene ALKBH5 in promotion of nerve axon injury repair

Technical Field

The invention belongs to the technical field of biomedical research, and particularly relates to application of a m6A modified related gene ALKBH5 in promoting repair of axonal injury.

Background

With the development of human society, peripheral nerve injuries caused by war, car accidents, sports injuries and the like have high morbidity, great injuries are brought to patients, and serious social and family burdens are also caused. At present, the application of microsurgery, local peripheral nerve transplantation or tissue engineering materials has great limitation on the treatment of peripheral nerve injury, mainly shows that the treatment of long-distance peripheral nerve injury is difficult and partial loss of nerve function is caused, and the key reason is that the key molecular target point and the molecular mechanism for regulation in the nerve injury repair are not completely clarified.

Disclosure of Invention

The invention aims to solve the technical problem of providing application of a m6A modified related gene ALKBH5 in preparation of a medicine for treating diseases related to axonal injury of nerves, so as to solve the problems in the background technology.

In order to solve the technical problems, the embodiment of the invention provides an application of a m6A modification related gene ALKBH5 in preparation of a medicament for treating diseases related to axonal injury.

Further, the diseases related to the axonal damage of the nerve comprise peripheral and central nerve damages.

The embodiment of the invention also provides an application of the m6A modification related gene ALKBH5 in promoting the repair of axonal injury, which is characterized by comprising the following steps:

s1, detecting the expression and location of ALKBH 5: preparing a rat sciatic nerve injury model, and detecting the expression change of ALKBH5 in DRG tissues in the rat sciatic nerve injury model through Western blot; detecting ALKBH5 expression and positioning of a rat model with sciatic nerve injury in DRG at different time points after operation through immunofluorescence;

s2, interference with the effect of ALKBH5 expression on neuronal axonal growth: separating and culturing DRG neurons; interfering with the expression of ALKBH5 by infecting DRG neuronal virus by injection of AAV virus; in vitro neuron axon growth experiments detect the influence of interference ALKBH5 on the neuron axon growth;

s3, interference with the effect of ALKBH5 expression on sciatic nerve axonal growth: selecting an experimental animal SD rat, and injecting an interference virus intrathecally; performing intrathecal injection for 2 weeks, and then performing sciatic nerve clamping injury; perfusing and dehydrating the rat with the injury of the sciatic nerve; sciatic nerve axon staining experiments were performed to detect the effect of in vivo interference of ALKBH5 on sciatic nerve axon growth;

s4, ALKBH5 target gene LPIN2 screening and function detection: screening ALKBH5 target gene LPIN 2; separating and culturing DRG neurons; transfection of LPIN2 interfering RNA in DRG neurons; in vitro neuron axon growth experiment, ALKBH5 target gene LPIN2 is interfered, and the influence on the neuron axon growth is detected.

In step S1, the rat sciatic nerve clamp wound model is prepared as follows:

selecting experimental SD rats with 15 rats in each batch, adult male rats with the weight of 180-; the rats were divided into 3 groups of 5 rats each; before an operation, firstly, carrying out abdominal anesthesia on a rat, and then disinfecting the hair of the left hind limb; exposing skin with surgical scissors, bluntly separating muscle and basement membrane covering sciatic nerve with ophthalmic scissors, clamping the proximal end of sciatic nerve with a clamp wound forceps, holding the clamp wound position 3mm wide for 30s, withdrawing sciatic nerve under muscle after the clamp wound is finished, and finally suturing the wound; keeping warm for the postoperative rat, and injecting 1ml of normal saline to accelerate the metabolism of the anesthetic out of the body; and 3d after 3d of pinching, smearing iodophor on the pinching suture part, cutting the skin by using surgical scissors, bluntly separating the basement membrane and the muscle covering the sciatic nerve by using an ophthalmology scissors, finding the pinching position after exposing the sciatic nerve, and collecting sciatic nerve tissues and placing the sciatic nerve tissues in a formaldehyde solution by using the pinching position as a mark.

In the step S1, the DRG sample protein extraction, Western blot detection and immunofluorescence detection specifically include the following steps:

DRG sample protein extraction and Western blot experiment: after preparing a rat sciatic nerve clamp injury model, taking L4-L6 Dorsal Root Ganglion (DRG) tissues at the sciatic nerve clamp injury sides of 0h, 1d and 3d after operation, and preparing a tissue protein sample by using a tissue sample protein extraction kit; transferring the L4 and L5 DRG tissues at different time points from the cryopreservation tube to a 1.5 ml EP tube by using a suction head; adding 150ul of tissue lysate into the tube, beating the tissue into single cell suspension by using a homogenizer, placing on ice for cracking for 15min, and centrifuging for 10 min; taking out the centrifuge tube, sucking the supernatant into a new EP tube, detecting the protein concentration in the sample, determining and recording the concentration of the protein sample, sucking a part of the sample for protein denaturation, marking the rest sample and storing at-80 ℃; adding a proper amount of 6Xloading protein loading buffer solution according to the protein concentration of a detected sample, uniformly mixing, and performing denaturation at 95 ℃ for 10 min; preparing polyacrylamide gel, performing SDS-PAGE electrophoresis on a protein sample, performing 120V 100min, performing membrane transfer for 100V 40min, sealing with 5% skim milk at room temperature for 2h, incubating primary antibody ALKBH5 at 4 ℃, after GAPDH is kept overnight, incubating secondary antibody Rabbit-HRP at room temperature for 2h, and developing with ECL;

and (3) performing immunofluorescence detection: immunofluorescence detection of ALKBH5 in DRG: after preparing a rat sciatic nerve clamp injury model, taking L4-L6 Dorsal Root Ganglion (DRG) tissues at the sciatic nerve clamp injury side of 0h and 3d after operation, and preparing a frozen section after fixing by 4% PFA; the FISH detection is carried out by firstly washing a sliced sample by 0.3 percent TBST at room temperature for 2 times and 10 min/time, incubating primary antibody ALKBH5 at 4 ℃ after being sealed for 1h at room temperature, incubating fluorescent secondary antibodies Rabbit-488 and Mouse-594 for 2h at room temperature in the dark after Neun stays overnight, and observing the sliced sample by a fluorescence microscope.

In the step S2 and the step S4, the process of isolating and culturing the DRG neuron specifically includes the following steps:

placing the dissecting liquid into a small dish, adding antibiotics (penicillin and streptomycin), and placing on ice for precooling; anesthetizing 3 rats in abdominal cavity, cutting skin from tail part along vertebra to head part with surgical scissors, removing whole spinal column, opening vertebral plate from neck part, pulling out all DRG tissue with micro forceps, and placing into dissection liquid; discarding the dissecting solution, and rinsing the tissue for 2 times by using PBS; discarding PBS, adding 2ml collagenase, transferring the tissue and digestive juice to a 5ml centrifuge tube; sufficiently shearing the tissue by using a micro-scissors, putting the tissue into a cell culture box, and digesting for 90 min; centrifuging to remove collagenase, adding 1ml pancreatin digestive juice, blowing for 1min with a gun until the tissue is dispersed uniformly, putting into a cell culture box for digestion for 10min, taking out every 5min, blowing uniformly, and then putting back into the culture box; adding 3ml of digestion stop solution into the centrifuge tube to stop digestion when the tissue is digested to no obvious tissue block; blowing with a gun for 1min, sieving with a mesh, filtering to remove excessive tissue, collecting cell suspension, centrifuging in a new 5ml centrifuge tube, and removing supernatant; adding 4ml of pre-preheated BSA solution into a centrifuge tube, resuspending the cells, centrifuging at 9000rpm for 5min, and removing floating mixed cells by suction; the process is repeated again; adding preheated neuron culture medium, blowing and beating cells uniformly, inoculating into 6-well plate, cross mixing cells uniformly, and adding 5% CO₂Incubator at 37 ℃.

Wherein, in step S2, the DRG neuron virus infection comprises the following processes: after the observation of good cell adherence, AVV virus is added in 24h of cell inoculation, and the virus infection amount is controlled at 2 x 10⁴ v.g./cell; the virus is added into the culture medium for 24 hours, the infection culture medium is discarded, and is replaced by a fresh complete culture medium; cells were harvested 96h after viral infection when strong GFP fluorescence was observed for cell expression.

Wherein, in the step S2 and the step S4, the in vitro neuron axon growth experiment specifically comprises the following steps:

before dyeing, the cells are subjected to virus pre-infection for 96 hours, and the cells can be dyed after the fluorescence brightness of virus expression GFP is observed to be brighter; discarding the cell culture medium, and adding preheated 1 XPBS to rinse the cells;

discarding 1 × PBS, adding pre-cooled 4% paraformaldehyde, and fixing on ice for 20 min; discarding formaldehyde, rinsing with 1 × PBS, washing at room temperature for 3 times and 5 min/time;

after the washing is finished, removing PBS, dropwise adding confining liquid, keeping standing at room temperature for 40min, wherein each hole is 200 mu l;

discarding the confining liquid, slightly dropwise adding Anti-beta-Tublin III antibody diluted by primary Anti-diluent, keeping standing at 4 ℃ for incubation overnight, wherein each well is 200 mu l;

discarding the primary antibody, rinsing with 1 × PBS, washing at room temperature for 3 times, 5 min/time;

discarding PBS, gently dripping Alexa fluor 647 goat anti-rabbitt diluted by a secondary antibody diluent, keeping the solution at room temperature for incubation for 2 hours, wherein each well is 200 mu l;

discarding the secondary antibody, rinsing with 1 × PBS, washing at room temperature for 3 times, 5 min/time;

after the washing, the round glass slide is taken out of the hole, the side with the cells faces downwards is covered on the glass slide on which the mounting liquid is dripped, and the glass slide is placed in a wet box and stored at 4 ℃.

Wherein, in the step S3, the interfering with intrathecal injection of the virus, tissue perfusion and dehydration, and sciatic nerve immunochemical staining specifically comprises the following steps:

interfering with intrathecal injection of the virus: selecting experimental SD rats, 15 adult male rats in each batch; dividing the experimental mice into 3 groups at random, wherein each group comprises 5 mice; before an operation, firstly carrying out abdominal anesthesia on a rat, then disinfecting the vicinity of ilium on the back of the rat, then exposing the skin by using an operating scissors, cutting muscle tissues along the ilium by using an ophthalmic scissors, replacing the bone scissors to cut off the protuberant spinous process, gently wiping off blood and minced meat by using a small cotton ball stained with physiological saline, clearly exposing intervertebral foramen of L4 and L5 DRG, and injecting 2/8 type AAV virus packaged with shRNA into the intervertebral foramen at a constant speed by using a microsyringe; after the injection is finished, the rat is kept flat for 2min and then the wound is sutured; sciatic nerve clamp injury was performed on virus-injected rats 2 weeks after intrathecal injection;

tissue perfusion and dehydration: placing the prepared 4% formaldehyde solution on ice for precooling for later use; firstly, performing abdominal anesthesia on a rat, fixing the rat, cutting off abdominal skin in the heart direction by using an operation scissors, cutting off the abdominal skin along the edge after a diaphragm is exposed, exposing the heart, peeling off adipose tissues covered on the heart to expose the artery, inserting a needle tube into the artery from the heart apex position, starting normal saline perfusion, cutting off the right auricle by using an ophthalmic scissors, opening formaldehyde perfusion after all blood in the rat body is replaced by the normal saline, and performing material drawing operation after observing that the four limbs of the rat are stiff; soaking the tissue taken out in 4% formaldehyde solution, removing the formaldehyde solution after 8h, washing residual formaldehyde with 1 XPBS, and finally replacing with 30% sucrose solution to carry out tissue dehydration; observing that the tissue sinks in a sucrose solution, namely finishing dehydration, taking out the tissue, placing the tissue under a dissecting mirror, trimming redundant muscle tissue by using micro-forceps, then straightening and placing the tissue on a freezing table attached with the sucrose solution, adding the sucrose solution for quick freezing, and storing the tissue after the freezing table is manufactured at-20 ℃ to be sliced; preparing a slide glass coated by PLL (phase locked loop) before slicing, and starting slicing after the thickness of the slice is set according to experiment needs; after slicing, drying in a 50 ℃ drying oven for 2h, sealing after drying, and storing at-80 ℃;

sciatic nerve immunochemical staining: taking out the tissue section from-80 ℃ 1h before dyeing, and placing the tissue section in room temperature for rewarming for 1 h; selecting tissues which are not wrinkled, complete in shape and free of bubbles, putting the tissues into a washing tank containing 1 multiplied by PBS, and placing the tissues on a shaking table for low-speed washing for 15 min; taking out the section from the washing tank, wiping off water stain outside the tissue, separating the tissue by using a circle of a composition pen, adding a proper amount of immunostaining sealing liquid into the circle, and standing for 1h at room temperature in a wet box; before adding the confining liquid, the tissue is kept moist as much as possible, otherwise the dyeing effect is influenced; discarding confining liquid, wiping off water stain on tissue edges, dropwise adding SCG 10-diluted by primary anti-dilution liquid into a grouping ring, and standing overnight at 4 ℃ in a wet box; the next day, the wet box is placed at room temperature and kept stand for 1 hour; discarding the primary antibody, placing the slices in a washing tank containing 1 × PBS, and washing on a shaking table at low speed for 3 times and 15 min/time; taking out the section from the washing tank, wiping water stain outside the tissue, dripping Alexa fluor 647 coat anti-rabbitt diluted by a second antibody diluent into the grouping ring under the condition of keeping out of the light, and placing the grouping ring in a light-proof wet box for incubation for 2 hours at room temperature; discarding the secondary antibody, placing the slice in a washing tank containing 1 × PBS, and washing on a shaking table at low speed for 3 times and 15 min/time; taking out the slices from the washing tank under the condition of keeping out of the light, wiping water stains outside the tissues, dripping fluorescent mounting liquid into the grouping ring, mounting the sections by using a cover glass, and storing the sections in a wet box.

In step S4, the screening of the target gene and the transfection of the LPIN2 interfering RNA in the DRG neuron specifically include the following steps:

screening of target genes: extracting total RNA from DRG neurons interfering with ALKBH5 expression and a control group, and sequencing transcriptome; carrying out GO pathway analysis on the differential expression genes, and screening genes related to axon growth to obtain 10 genes; qRT-PCR verification is carried out on DRG neurons interfering ALKBH5, and 5 genes are found to be obviously reduced in expression: ALDH3B1, GALNS, LPIN2, COX8A, and ST6GA 1;

transfection of LPIN2 interfering RNA in DRG neurons: after the DRG neuron is separated and inoculated to a cell culture plate, the transfection of interfering RNA can be carried out; adding 1ul of transfection reagent into 25ul of transfection medium in a centrifuge tube A, gently mixing, preparing another centrifuge tube B, adding 2.5ul of interference RNA into 25ul of transfection medium, gently mixing, slowly adding the liquid in the centrifuge tube A into the centrifuge tube B, gently mixing, standing for 15min, uniformly adding into a cell culture plate pre-inoculated with DRG neurons, uniformly mixing cells, and putting into a 5% CO2 incubator at 37 ℃; cells were harvested 48h after transfection.

The technical scheme of the invention has the following beneficial effects:

(1) the invention detects the expression distribution of ALKBH5 in DRG samples at different time points after the injury of the sciatic nerve of SD rats, and further analyzes the function of the ALKBH5 in the growth of neuron axons; the embodiment of the invention verifies that the in vitro and in vivo interference of the ALKBH5 gene can obviously promote the growth of neuron axons and promote the recovery of sciatic nerve functions; and further through transcriptome analysis and target gene verification after ALKBH5 interference, a target gene LPIN2 regulated by ALKBH5 is searched, and the effect of regulating and controlling the growth of neuron axons is further verified. The invention suggests that ALKBH5 may influence the function of LPIN2 by adjusting the m6A modification level of LPIN2, and finally regulates the growth of neuron axons, and the ALKBH5 has the potential to become an important molecular target for nerve injury repair.

(2) The invention establishes experimental verification based on a rat sciatic nerve injury animal model, and further studies the effect of the rat sciatic nerve injury animal model on treating peripheral nerve injury in large animals and non-human primates in the later period.

Drawings

FIG. 1 is a graph showing the expression and localization of ALKBH5 detected in example 1 of the present invention; wherein, FIG. 1A is a Western blot detection expression change diagram of ALKBH5 in DRG tissues in a sciatic nerve injury model, and FIG. 1B is a statistical analysis diagram of gray values in FIG. 1A; FIG. 1C is a graph showing the expression of ALKBH5 in DRGs at 0d and 3d after sciatic nerve injury detected by immunofluorescence;

FIG. 2 is a graph of the effect of interfering ALKBH5 on neuronal axon growth in example 2 of the present invention; wherein, FIG. 2A is a graph of neurite outgrowth of neurons after interference with ALKBH 5; FIG. 2B is a statistical plot of the total and longest axons of neurons;

FIG. 3 is a graph showing the effect of interference ALKBH5 on sciatic nerve injury repair in example 3 of the present invention; wherein, FIG. 3A is a graph showing the effect of interfering ALKBH5 on sciatic nerve axon growth in vivo in sciatic nerve axon staining experiments; FIG. 3B is a statistical plot of fluorescence intensity of sciatic nerve regenerated axons; FIG. 3C is a statistical plot of the longest length of regenerating axons of sciatic nerves;

FIG. 4 is a functional diagram of the target gene LPIN2 of ALKBH5 according to the present invention; wherein, FIG. 4A is a functional analysis diagram of a target gene after expression of interference ALKBH5 in DRG; FIG. 4B is a qRT-PCR validation of the expression of the ALKBH5 target gene in neurons; FIG. 4C is a graph of the effect of interfering with the detection of the ALKBH5 target gene LPIN2 on neuronal axonal growth; FIG. 4D is a statistical plot of the total and longest axons of neurons.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

Example 1 detection of the expression and localization of ALKBH5

(1) DRG sample protein extraction and Western blot

After preparing rat sciatic nerve clamp wound model, taking L4-L6 Dorsal Root Ganglion (DRG) tissues at the sciatic nerve clamp wound side of 0h, 1d and 3d after operation, and preparing tissue protein sample by using a tissue sample protein extraction kit: the L4, L5 DRG tissues at different time points were transferred from the cryopreserved tubes to 1.5 ml RNase-free EP tubes using RNase-free tips. 150ul of tissue lysate is added to the tube, protease inhibitor is added, the tissue is beaten into single cell suspension by a homogenizer, and then the single cell suspension is placed on ice for lysis for 15min, and then centrifugation is carried out at 12000 rpm at 4 ℃ for 10min (the centrifugation temperature is 4 in the preparation operation of protein samples). Carefully taking out the centrifuge tube, sucking the supernatant into a new EP tube, detecting the protein concentration in the sample by a protein concentration detection kit, measuring and recording the concentration of the protein sample, sucking a part of the sample for protein denaturation, marking the rest of the sample and storing at-80 ℃.

According to the protein concentration of the tested sample, adding a proper amount of 6Xloading protein loading buffer solution, uniformly mixing, and then denaturing at 95 ℃ for 10 min. Preparing polyacrylamide gel, wherein the concentration of the separation gel is 10 percent, and the concentration of the concentration gel is 5 percent. Protein samples were subjected to SDS-PAGE at 120V for 100min, transferred to membrane for 100V 40min, blocked with 5% skim milk at room temperature for 2h, incubated with primary anti-ALKBH 5(1:1000) and GAPDH (1:10000) overnight at 4 ℃ and then incubated with secondary anti-Rabbit-HRP (1:20000) at room temperature for 2h, and developed with ECL. As a result, as shown in fig. 1A and fig. 1B, the expression change of ALKBH5 (GAPDH is an internal reference) in DRG tissues in the sciatic nerve injury model was detected by Western blot, and the expression of ALKBH5 was gradually decreased after nerve injury.

(2) ALKBH5 in DRG

After preparing rat sciatic nerve clamp wound model, taking L4-L6 Dorsal Root Ganglion (DRG) tissues at the side of sciatic nerve clamp wound for 0h and 3d after operation, fixing by 4% PFA, and making frozen section. The FISH detection is carried out by firstly washing a section sample with 0.3 percent TBST at room temperature for 2 times and 10 min/time, sealing the section sample at room temperature for 1h, then incubating primary antibody ALKBH5(1:400) at 4 ℃, after Neun (1:500) stays overnight, incubating fluorescent secondary antibodies Rabbit-488(1:500) and Mouse-594(1:500) at room temperature in the dark for 2h, and observing and shooting the section sample by a fluorescence microscope. The photograph result is shown in fig. 1C, and the expression of ALKBH5 in DRGs at 0d and 3d after sciatic nerve injury is detected by immunofluorescence, and Neun is used as the reference of the nuclear gene. Neun is predominantly expressed in the nucleus, whereas ALKBH5 is predominantly expressed in the cytoplasm, indicating that ALKBH5 is predominantly expressed in the cytoplasm.

Example 2 interference of the Effect of ALKBH5 expression on neuronal axonal growth

(3) And separating and culturing DRG neuron

The dissecting solution was prepared and placed in a small dish, and after the antibiotics (penicillin and streptomycin) were added, it was placed on ice for precooling. 3 rats were anesthetized with abdominal cavity, the skin was cut open from the tail along the spine towards the head with surgical scissors, the entire spine was carefully removed, the vertebral plate was opened from the neck, and all DRG tissues were pulled out with micro-forceps and placed in the dissecting fluid. All DRG tissues were removed and the dissecting solution was discarded, the tissues were rinsed 2 times with cell-grade PBS, and excess tissue and blood traces were washed away. PBS was discarded, 2ml collagenase (3mg/ml) was added and the tissue and digest were transferred to a 5ml centrifuge tube. The tissue is cut into pieces by micro-scissors, and then placed in a cell culture box for digestion for 90 min. Centrifuging to remove collagenase, adding 1ml pancreatin digestive juice, blowing for about 1min with a gun until the tissue is dispersed uniformly, then placing into a cell culture box for digestion for 10min, taking out every 5min, blowing uniformly, and then placing back into the culture box. When the tissue is digested to no obvious tissue block, 3ml of digestion stop solution is added into the centrifuge tube to stop the digestion. Blowing with a gun for about 1min, sieving with a screen (200 mesh), filtering to remove excess tissue, collecting cell suspension, transferring into a new 5ml centrifuge tube at 1100rpm for 5min, and discarding supernatant. 4ml of pre-warmed BSA solution was added to the centrifuge tube, the cells were resuspended, centrifuged at 9000rpm for 5min, and the floating contaminating cells were carefully aspirated away. This step is repeated again. Adding preheated neuron culture medium, blowing and beating cells uniformly, inoculating into a 6-well plate, mixing the cells uniformly in a cross shape, and putting into an incubator at 37 ℃ with 5% CO 2.

(4) DRG neuronal viral infection

After the cell adherence condition is observed to be good, the virus can be added, and the virus infection amount is controlled to be 2 x 104 v.g./cell. Note that during the virus infection operation, the tips that are contaminated with virus are soaked in 84 disinfectant. The original medium was discarded first, the cells were rinsed with pre-warmed PBS, half the volume of virus infection medium was added, and the corresponding titer of virus was added. Note that the virus needs to be added within 24h of cell inoculation, otherwise the virus infection effect is influenced. The virus was added over 24h and the infection medium was discarded and replaced with fresh complete medium. After the virus is infected for 96 hours, the cells can be harvested when the cells express strong GFP fluorescence. After 96h of viral infection if necessary for subsequent staining, the resuspended cells were digested with 0.025% pancreatin and reseeded into 24-well plates. Cells were collected within 12-18 h after inoculation.

(5) In vitro neuronal axon growth assay

Before dyeing, the cells are subjected to virus pre-infection for 96h, and the cells can be dyed by observing the fact that the fluorescence brightness of virus-expressed GFP is brighter. The cell culture medium was discarded and the cells were rinsed with 1 × PBS after preheating. Discard 1 XPBS, add pre-cooled 4% paraformaldehyde and fix on ice for 20 min. After discarding formaldehyde, the column was rinsed with 1 XPBS for 3 times at room temperature for 5 min. After the washing, the PBS was discarded, and the blocking solution was gently added dropwise to 200. mu.l/well, and allowed to stand at room temperature for 40 min. The blocking solution was discarded, and Anti-. beta. -Tublin III antibody (1:400) diluted with primary antibody was gently added dropwise thereto at 200. mu.l per well, and incubated at 4 ℃ overnight. The primary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. PBS was discarded, and Alexa fluor 647 goat anti-rabbitt (1:1000) diluted with a secondary antibody diluent was gently added dropwise thereto at 200. mu.l per well, followed by incubation at room temperature for 2 hours. The process is protected from light. The secondary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. The process is protected from light. After the washing, the round glass slide is taken out of the hole, the side with the cells faces downwards is covered on the glass slide on which the mounting liquid is dripped, and the glass slide is placed in a wet box and stored at 4 ℃. Care was taken not to generate bubbles in the process. The process is protected from light. The neuron axon staining experiment detects the influence of interference ALKBH5 on the neuron axon growth, the experimental detection results are shown in fig. 2A and fig. 2B, and the axon growth condition of the neuron after interference of ALKBH5 in fig. 2A and the total length and longest length of the neuron axon in fig. 2B are both shown to remarkably promote the neuron axon growth after interference of ALKBH 5.

Example 3 interference of ALKBH5Effect of expression on sciatic nerve axon growth

(6) Interfering with intrathecal injection of viruses

Experimental animals SD rats were provided from the experimental animal center, 15 adult male rats per batch, about 200 g. The experimental mice were randomly divided into 3 groups of 5 mice each. Before operation, a rat is subjected to abdominal anesthesia, then the part, close to the ilium, of the back of the rat is disinfected by hair, skin is exposed by using surgical scissors, muscle tissues are cut along the ilium by using ophthalmic scissors, protruded spinous processes are cut by using bone scissors, blood and minced meat are gently wiped off by using a small cotton ball stained with physiological saline, intervertebral foramen of L4 and L5 DRG can be clearly exposed, and the 2/8 AAV packaged with shRNA is injected into the intervertebral foramen at a constant speed by using a microsyringe device. To avoid the virus from exiting the intervertebral foramen for human reasons, the mice were placed flat for 2min after injection and the wounds were sutured closed. In addition, a dye can be added into the virus to judge whether the virus enters DRG tissues or not, and the injection amount of the virus is controlled to be 4 x 1010 v.g./virus. To allow the virus to better infect the DRG tissue, sciatic nerve clamp injury was performed 2 weeks after intrathecal injection. And 3d after 3d of traumatic injury, cutting skin along the suture of the traumatic injury to expose sciatic nerve, cutting the sciatic nerve along the sciatic nerve to the spine, cutting ilium by using surgical scissors to expose intervertebral foramen, carefully pulling out the DRG tissue to cut the front and back heel tissues, and placing the DRG tissue in liquid nitrogen or formaldehyde.

(7) Sciatic nerve clamp injury

Experimental animals SD rats were provided from the experimental animal center, 15 adult male rats per batch, about 200 g. The mice were divided into 3 groups of 5 mice each. Before operation, the rat is firstly subjected to abdominal anesthesia, and then the left hind limb peritrichous is disinfected. Exposing skin with surgical scissors, separating muscle and basement membrane covering sciatic nerve with ophthalmic scissors, clamping sciatic nerve proximal end with clamping wound forceps 3mm wide for 30s, withdrawing sciatic nerve under muscle after clamping wound, and suturing wound. The mice after the operation pay attention to the warm keeping, and 1ml of normal saline is injected into the mice to accelerate the metabolism of the anesthetic out of the body. And 3d after 3d of pinching, smearing iodophor on the pinching suture part, cutting the skin by using surgical scissors, bluntly separating the basement membrane and the muscle covering the sciatic nerve by using an ophthalmology scissors, finding the pinching position after exposing the sciatic nerve, and collecting sciatic nerve tissues and placing the sciatic nerve tissues in a formaldehyde solution by using the pinching position as a mark.

(8) Tissue perfusion and dehydration

The now prepared 4% formaldehyde solution is placed on ice for precooling for standby. Firstly, performing abdominal anesthesia on a rat, fixing the rat, cutting off abdominal skin in the direction of the heart by using surgical scissors, cutting off the abdominal skin along the edge after the diaphragm is exposed, exposing the heart, peeling off adipose tissues covered on the heart to expose the artery, inserting a needle tube into the artery from the apex of the heart, starting normal saline perfusion, cutting off the right auricle by using ophthalmic scissors, opening formaldehyde perfusion after all blood in the rat is replaced by the normal saline, and observing that the limbs of the rat are all stiff, wherein the operation of taking materials can be performed. Note that the pre-exposure procedure cannot cut into the viscera or otherwise affect perfusion. Soaking the tissue in 4% formaldehyde solution for 8 hr, removing formaldehyde solution, washing with 1 × PBS to remove residual formaldehyde, and dewatering with 30% sucrose solution. Observing that the tissue sinks in the sucrose solution, namely finishing dehydration, taking out the tissue, placing the tissue under a dissecting mirror, trimming redundant muscle tissue by using micro forceps, carefully straightening, placing the tissue on a freezing table attached with the sucrose solution, adding the sucrose solution for quick freezing, and storing the tissue after the freezing table is manufactured at-20 ℃ for processing slices. Before slicing, a slide coated with PLL is prepared, and slicing can be started after the thickness of the slice is set according to experiment needs. After the slicing is finished, the slices are placed in a 50 ℃ oven to be dried for 2h, and the slices are sealed after being dried and can be stored at minus 80 ℃.

(9) Sciatic nerve immunochemical staining

Taking out the tissue section from-80 ℃ 1h before staining, and placing the tissue section in room temperature for rewarming for 1 h. Selecting tissues without wrinkles, complete shape and bubbles, putting the tissues into a washing tank containing 1 XPBS, and placing the tissues on a shaking table to wash for 15min at a low speed. Taking out the section from the washing tank, wiping off water stain outside the tissue, separating the tissue by using a circle of a tissue forming pen, adding a proper amount of immunostaining sealing liquid into the circle, and standing for 1h at room temperature in a wet box. Before adding the sealing liquid, the tissue is kept moist as much as possible, otherwise, the dyeing effect is influenced. The blocking solution was discarded, the tissue edges were wiped off of water, SCG10 (1:400) diluted with primary anti-diluent was added dropwise to the ring, and the mixture was left to stand overnight in a wet box at 4 ℃. The next day, the wet box was left to stand at room temperature for 1 h. Primary antibody was discarded and the sections were placed in a wash tank containing 1 XPBS and washed 3 times 15 min/time on a shaker at low speed. The section is taken out from the washing tank, water stain outside the tissue is wiped dry, Alexa fluor 647 coat anti-rabbitt (1:1000) diluted by secondary antibody diluent is dripped into the combined ring under the condition of keeping out of the light, and the combined ring is placed in a light-proof wet box for incubation for 2 hours at room temperature. The secondary antibody was discarded and the sections were placed in a wash tank containing 1 × PBS and washed 3 times 15 min/time on a shaker at low speed. Taking out the slices from the washing tank under the condition of keeping out of the light, wiping water stain outside the tissues, dripping fluorescent sealing liquid into the grouping ring, sealing the slices by using a special cover glass, and storing the slices in a wet box without generating bubbles in the process. The staining experiment is carried out on the sciatic nerve axons, the influence of interference ALKBH5 on the sciatic nerve axon growth in a specimen is detected, the staining experiment result is shown in figures 3A-3C, and the ALKBH5 significantly promotes the sciatic nerve axon growth after interference.

Example 4 screening and function of ALKBH5 target Gene LPIN2

(10) Screening of target Gene

And extracting total RNA from the DRG neuron interfering the ALKBH5 expression and a control group, and performing transcriptome sequencing. GO pathway analysis is carried out on the differentially expressed genes, the analysis result of the GO pathway analysis is shown in figure 4A, and the differentially expressed genes are mainly related to the metabolism of neurons. Further, 10 genes were selected from genes related to axon growth by consulting the literature. We subsequently performed qRT-PCR validation on DRG neurons interfering with ALKBH5, and found that there were 5 genes with significantly reduced expression, as shown in fig. 4B, including the following 5 genes: ALDH3B1, GALNS, LPIN2, COX8A, and ST6GA 1.

(11) And separating and culturing DRG neuron

The dissecting solution was prepared and placed in a small dish, and after the antibiotics (penicillin and streptomycin) were added, it was placed on ice for precooling. 3 rats were anesthetized with abdominal cavity, the skin was cut open from the tail along the spine towards the head with surgical scissors, the entire spine was carefully removed, the vertebral plate was opened from the neck, and all DRG tissues were pulled out with micro-forceps and placed in the dissecting fluid. All DRG tissues were removed and the dissecting solution was discarded, the tissues were rinsed 2 times with cell-grade PBS, and excess tissue and blood traces were washed away. PBS was discarded, 2ml collagenase (3mg/ml) was added and the tissue and digest were transferred to a 5ml centrifuge tube. The tissue is cut into pieces by micro-scissors, and then placed in a cell culture box for digestion for 90 min. Centrifuging to remove collagenase, adding 1ml pancreatin digestive juice, blowing for about 1min with a gun until the tissue is dispersed uniformly, then placing into a cell culture box for digestion for 10min, taking out every 5min, blowing uniformly, and then placing back into the culture box. When the tissue is digested to no obvious tissue block, 3ml of digestion stop solution is added into the centrifuge tube to stop the digestion. Blowing with a gun for about 1min, sieving with a screen (200 mesh), filtering to remove excess tissue, collecting cell suspension, transferring into a new 5ml centrifuge tube at 1100rpm for 5min, and discarding supernatant. 4ml of pre-warmed BSA solution was added to the centrifuge tube, the cells were resuspended, centrifuged at 9000rpm for 5min, and the floating contaminating cells were carefully aspirated away. This step is repeated again. Adding preheated neuron culture medium, blowing and beating cells uniformly, inoculating into a 6-well plate, mixing the cells uniformly in a cross shape, and putting into an incubator at 37 ℃ with 5% CO 2.

(12) LPIN2 interference RNA transfection in DRG neurons

After the DRG neuron is separated and inoculated to a cell culture plate, the transfection of the interfering RNA can be carried out. Adding 1ul of transfection reagent into 25ul of transfection medium in a centrifuge tube A, mixing the reagent gently, preparing another centrifuge tube B, adding 2.5ul of interfering RNA into 25ul of transfection medium, mixing the reagent gently, slowly adding the liquid in the centrifuge tube A into the centrifuge tube B, mixing the reagent gently, standing for 15min, uniformly adding the reagent into a cell culture plate pre-inoculated with DRG neurons, mixing the cells uniformly, and putting the mixture into a 5% CO2 incubator at 37 ℃. Cells were harvested 48h after transfection.

(13) In vitro neuronal axon growth assay

The cell culture medium was discarded and the cells were rinsed with 1 × PBS after preheating. Discard 1 XPBS, add pre-cooled 4% paraformaldehyde and fix on ice for 20 min. After discarding formaldehyde, the column was rinsed with 1 XPBS for 3 times at room temperature for 5 min. After the washing, the PBS was discarded, and the blocking solution was gently added dropwise to 200. mu.l/well, and allowed to stand at room temperature for 40 min. The blocking solution was discarded, and Anti-. beta. -Tublin III antibody (1:400) diluted with primary antibody was gently added dropwise thereto at 200. mu.l per well, and incubated at 4 ℃ overnight. The primary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. PBS was discarded, and Alexa fluor 647 goat anti-rabbitt (1:1000) diluted with a secondary antibody diluent was gently added dropwise thereto at 200. mu.l per well, followed by incubation at room temperature for 2 hours. The process is protected from light. The secondary antibody was discarded, rinsed with 1 XPBS, and washed 3 times 5 min/time at room temperature. The process is protected from light. After the washing, the round glass slide is taken out of the hole, the side with the cells faces downwards is covered on the glass slide on which the mounting liquid is dripped, and the glass slide is placed in a wet box and stored at 4 ℃. Care was taken not to generate bubbles in the process. The process is protected from light. Interfering with the ALKBH5 target gene LPIN2, detecting the influence on the neuron axon growth; the results are shown in fig. 4C and 4D, where fig. 4C is the axonal growth of neurons after interference with LPIN2, and fig. 4D is a statistical graph of the total and longest axons of neurons, both showing that LPIN2 significantly promoted the growth of neuronal axons after interference.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (9)

1. An application of a virus interfering with a m6A modification-related gene ALKBH5 in preparation of a medicine for treating sciatic nerve injury-related diseases is characterized in that the virus is a 2/8 type AAV virus packaged with shRNA.

2. The application of the m6A modification related gene ALKBH5 in promoting the repair of axonal injury of nerves is experimentally verified for non-diagnosis or treatment purposes, and the method is characterized by comprising the following steps:

s1, detecting the expression and location of ALKBH 5: preparing a rat sciatic nerve injury model, and detecting the expression change of ALKBH5 in DRG tissues in the rat sciatic nerve injury model through Western blot; detecting ALKBH5 expression and positioning of a rat model with sciatic nerve injury in DRG at different time points after operation through immunofluorescence;

s2, interference with the effect of ALKBH5 expression on neuronal axonal growth: separating and culturing DRG neurons; interfering with the expression of ALKBH5 by infecting DRG neuronal virus by injection of AAV virus; in vitro neuron axon growth experiments detect the influence of interference ALKBH5 on the neuron axon growth;

s3, interference with the effect of ALKBH5 expression on sciatic nerve axonal growth: selecting an experimental animal SD rat, and injecting an interference virus intrathecally; performing intrathecal injection for 2 weeks, and then performing sciatic nerve clamping injury; perfusing and dehydrating the rat with the injury of the sciatic nerve; sciatic nerve axon staining experiments were performed to detect the effect of in vivo interference of ALKBH5 on sciatic nerve axon growth;

s4, ALKBH5 target gene LPIN2 screening and function detection: screening ALKBH5 target gene LPIN 2; separating and culturing DRG neurons; transfection of LPIN2 interfering RNA in DRG neurons; in vitro neuron axon growth experiment, ALKBH5 target gene LPIN2 is interfered, and the influence on the neuron axon growth is detected.

3. The experimental verification of the use of the m6A modification-related gene ALKBH5 in promoting axon damage repair according to claim 2, wherein in step S1, the rat sciatic nerve injury model is prepared as follows:

selecting experimental SD rats with 15 rats in each batch, adult male rats with the weight of 180-; the rats were divided into 3 groups of 5 rats each; before an operation, firstly, carrying out abdominal anesthesia on a rat, and then disinfecting the hair of the left hind limb; exposing skin with surgical scissors, bluntly separating muscle and basement membrane covering sciatic nerve with ophthalmic scissors, clamping the proximal end of sciatic nerve with a clamp wound forceps, holding the clamp wound position 3mm wide for 30s, withdrawing sciatic nerve under muscle after the clamp wound is finished, and finally suturing the wound; keeping warm for the postoperative rat, and injecting 1ml of normal saline to accelerate the metabolism of the anesthetic out of the body; and 3d after 3d of pinching, smearing iodophor on the pinching suture part, cutting the skin by using surgical scissors, bluntly separating the basement membrane and the muscle covering the sciatic nerve by using an ophthalmology scissors, finding the pinching position after exposing the sciatic nerve, and collecting sciatic nerve tissues and placing the sciatic nerve tissues in a formaldehyde solution by using the pinching position as a mark.

4. The application of the m6A modification-related gene ALKBH5 in promotion of axon injury repair of a nerve according to claim 2, wherein the DRG sample protein extraction, Western blot detection and immunofluorescence detection in the step S1 further comprise the following steps:

DRG sample protein extraction and Western blot detection: after preparing a rat sciatic nerve clamp injury model, taking L4-L6 Dorsal Root Ganglion (DRG) tissues at the sciatic nerve clamp injury sides of 0h, 1d and 3d after operation, and preparing a tissue protein sample by using a tissue sample protein extraction kit; transferring the L4 and L5 DRG tissues at different time points from the cryopreservation tube to a 1.5 ml EP tube by using a suction head; adding 150ul of tissue lysate into the tube, beating the tissue into single cell suspension by using a homogenizer, placing on ice for cracking for 15min, and centrifuging for 10 min; taking out the centrifuge tube, sucking the supernatant into a new EP tube, detecting the protein concentration in the sample, determining and recording the concentration of the protein sample, sucking a part of the sample for protein denaturation, marking the rest sample and storing at-80 ℃; adding a proper amount of 6Xloading protein loading buffer solution according to the protein concentration of a detected sample, uniformly mixing, and performing denaturation at 95 ℃ for 10 min; preparing polyacrylamide gel, performing SDS-PAGE electrophoresis on a protein sample, performing 120V 100min, performing membrane transfer for 100V 40min, sealing with 5% skim milk at room temperature for 2h, incubating primary antibody ALKBH5 at 4 ℃, after GAPDH is kept overnight, incubating secondary antibody Rabbit-HRP at room temperature for 2h, and developing with ECL;

and (3) performing immunofluorescence detection: immunofluorescence detection of ALKBH5 in DRG: after preparing a rat sciatic nerve clamp injury model, taking L4-L6 Dorsal Root Ganglion (DRG) tissues at the sciatic nerve clamp injury side of 0h and 3d after operation, and preparing a frozen section after fixing by 4% PFA; the FISH detection is carried out by firstly washing a sliced sample by 0.3 percent TBST at room temperature for 2 times and 10 min/time, incubating primary antibody ALKBH5 at 4 ℃ after being sealed for 1h at room temperature, incubating fluorescent secondary antibodies Rabbit-488 and Mouse-594 for 2h at room temperature in the dark after Neun stays overnight, and observing the sliced sample by a fluorescence microscope.

5. The experimental verification of the m6A modification-related gene ALKBH5 for promoting the repair of axonal injury of a nerve according to claim 2, wherein the step S2 and the step S4 are used for isolating and culturing DRG neurons, and the method specifically comprises the following steps:

placing the dissection liquid into a small dish, adding a double antibody, and then placing on ice for precooling; anesthetizing 3 rats in abdominal cavity, cutting skin from tail part along vertebra to head part with surgical scissors, removing whole spinal column, opening vertebral plate from neck part, pulling out all DRG tissue with micro forceps, and placing into dissection liquid; discarding the dissecting solution, and rinsing the tissue for 2 times by using PBS; discarding PBS, adding 2ml collagenase, transferring the tissue and digestive juice to a 5ml centrifuge tube; sufficiently shearing the tissue by using a micro-scissors, putting the tissue into a cell culture box, and digesting for 90 min; centrifuging to remove collagenase, adding 1ml pancreatin digestive juice, blowing for 1min with a gun until the tissue is dispersed uniformly, putting into a cell culture box for digestion for 10min, taking out every 5min, blowing uniformly, and then putting back into the culture box; adding 3ml of digestion stop solution into the centrifuge tube to stop digestion when the tissue is digested to no obvious tissue block; blowing with a gun for 1min, sieving with a mesh, filtering to remove excessive tissue, collecting cell suspension, centrifuging in a new 5ml centrifuge tube, and removing supernatant; adding 4ml of pre-preheated BSA solution into a centrifuge tube, resuspending the cells, centrifuging at 9000rpm for 5min, and removing floating mixed cells by suction; the process is repeated again; adding preheated neuron culture medium, blowing and beating cells uniformly, inoculating into 6-well plate, cross mixing cells uniformly, and adding 5% CO₂Incubator at 37 ℃.

6. The experimental validation of the m6A modification-related gene ALKBH5 for promoting the repair of axonal injury in the non-diagnostic or therapeutic purpose of claim 2, wherein the step S2 is a DRG neuron virus infection, which comprises the following steps: after the observation of good cell adherence, AVV virus is added in 24h of cell inoculation, and the virus infection amount is controlled at 2 x 10⁴ v.g./cell; the virus is added into the culture medium for 24 hours, the infection culture medium is discarded, and is replaced by a fresh complete culture medium; cells were harvested 96h after viral infection when strong GFP fluorescence was observed for cell expression.

7. The experimental verification of the use of the m6A modification-related gene ALKBH5 for promoting repair of axonal injury in a non-diagnostic or therapeutic purpose according to claim 2, wherein the step S2 and the step S4 are in vitro neuron axonal growth experiments, and specifically comprise the following steps:

before dyeing, the cells are subjected to virus pre-infection for 96 hours, and the cells can be dyed after the fluorescence brightness of virus expression GFP is observed to be brighter; discarding the cell culture medium, and adding preheated 1 XPBS to rinse the cells;

discarding 1 × PBS, adding pre-cooled 4% paraformaldehyde, and fixing on ice for 20 min; discarding formaldehyde, rinsing with 1 × PBS, washing at room temperature for 3 times and 5 min/time;

after the washing is finished, removing PBS, dropwise adding confining liquid, keeping standing at room temperature for 40min, wherein each hole is 200 mu l;

discarding the confining liquid, slightly dropwise adding Anti-beta-Tublin III antibody diluted by primary Anti-diluent, keeping standing at 4 ℃ for incubation overnight, wherein each well is 200 mu l;

discarding the primary antibody, rinsing with 1 × PBS, washing at room temperature for 3 times, 5 min/time;

discarding PBS, gently dripping Alexa fluor 647 goat anti-rabbitt diluted by a secondary antibody diluent, keeping the solution at room temperature for incubation for 2 hours, wherein each well is 200 mu l;

discarding the secondary antibody, rinsing with 1 × PBS, washing at room temperature for 3 times, 5 min/time;

after the washing, the round glass slide is taken out of the hole, the side with the cells faces downwards is covered on the glass slide on which the mounting liquid is dripped, and the glass slide is placed in a wet box and stored at 4 ℃.

8. The experimental verification of the use of the m6A modification-related gene ALKBH5 for promoting nerve axon injury repair according to claim 2, wherein the step S3 is to interfere with virus intrathecal injection, tissue perfusion and dehydration and sciatic nerve immunochemical staining, and comprises the following steps:

interfering with intrathecal injection of the virus: selecting experimental SD rats, 15 adult male rats in each batch; dividing the experimental mice into 3 groups at random, wherein each group comprises 5 mice; before an operation, firstly carrying out abdominal anesthesia on a rat, then disinfecting the vicinity of ilium on the back of the rat, then exposing the skin by using an operating scissors, cutting muscle tissues along the ilium by using an ophthalmic scissors, replacing the bone scissors to cut off the protuberant spinous process, gently wiping off blood and minced meat by using a small cotton ball stained with physiological saline, clearly exposing intervertebral foramen of L4 and L5 DRG, and injecting 2/8 type AAV virus packaged with shRNA into the intervertebral foramen at a constant speed by using a microsyringe; after the injection is finished, the rat is kept flat for 2min and then the wound is sutured; sciatic nerve clamp injury was performed on virus-injected rats 2 weeks after intrathecal injection;

tissue perfusion and dehydration: placing the prepared 4% formaldehyde solution on ice for precooling for later use; firstly, performing abdominal anesthesia on a rat, fixing the rat, cutting off abdominal skin in the heart direction by using an operation scissors, cutting off the abdominal skin along the edge after a diaphragm is exposed, exposing the heart, peeling off adipose tissues covered on the heart to expose the artery, inserting a needle tube into the artery from the heart apex position, starting normal saline perfusion, cutting off the right auricle by using an ophthalmic scissors, opening formaldehyde perfusion after all blood in the rat body is replaced by the normal saline, and performing material drawing operation after observing that the four limbs of the rat are stiff; soaking the tissue taken out in 4% formaldehyde solution, removing the formaldehyde solution after 8h, washing residual formaldehyde with 1 XPBS, and finally replacing with 30% sucrose solution to carry out tissue dehydration; observing that the tissue sinks in a sucrose solution, namely finishing dehydration, taking out the tissue, placing the tissue under a dissecting mirror, trimming redundant muscle tissue by using micro-forceps, then straightening and placing the tissue on a freezing table attached with the sucrose solution, adding the sucrose solution for quick freezing, and storing the tissue after the freezing table is manufactured at-20 ℃ to be sliced; preparing a slide glass coated by PLL (phase locked loop) before slicing, and starting slicing after the thickness of the slice is set according to experiment needs; after slicing, drying in a 50 ℃ drying oven for 2h, sealing after drying, and storing at-80 ℃;

sciatic nerve immunochemical staining: taking out the tissue section from-80 ℃ 1h before dyeing, and placing the tissue section in room temperature for rewarming for 1 h; selecting tissues which are not wrinkled, complete in shape and free of bubbles, putting the tissues into a washing tank containing 1 multiplied by PBS, and placing the tissues on a shaking table for low-speed washing for 15 min; taking out the section from the washing tank, wiping off water stain outside the tissue, separating the tissue by using a circle of a composition pen, adding a proper amount of immunostaining sealing liquid into the circle, and standing for 1h at room temperature in a wet box; before adding the confining liquid, the tissue is kept moist as much as possible, otherwise the dyeing effect is influenced; discarding confining liquid, wiping off water stain on tissue edges, dropwise adding SCG 10-diluted by primary anti-dilution liquid into a grouping ring, and standing overnight at 4 ℃ in a wet box; the next day, the wet box is placed at room temperature and kept stand for 1 hour; discarding the primary antibody, placing the slices in a washing tank containing 1 × PBS, and washing on a shaking table at low speed for 3 times and 15 min/time; taking out the section from the washing tank, wiping water stain outside the tissue, dripping Alexa fluor 647 coat anti-rabbitt diluted by a second antibody diluent into the grouping ring under the condition of keeping out of the light, and placing the grouping ring in a light-proof wet box for incubation for 2 hours at room temperature; discarding the secondary antibody, placing the slice in a washing tank containing 1 × PBS, and washing on a shaking table at low speed for 3 times and 15 min/time; taking out the slices from the washing tank under the condition of keeping out of the light, wiping water stains outside the tissues, dripping fluorescent mounting liquid into the grouping ring, mounting the sections by using a cover glass, and storing the sections in a wet box.

9. The experimental verification of the use of the m6A modification-related gene ALKBH5 for promoting repair of axonal injury in non-diagnostic or therapeutic purposes of claim 2, wherein the step S4 comprises the following steps of screening target genes and transfecting LPIN2 interference RNA in DRG neurons:

screening of target genes: extracting total RNA from DRG neurons interfering with ALKBH5 expression and a control group, and sequencing transcriptome; carrying out GO pathway analysis on the differential expression genes, and screening genes related to axon growth to obtain 10 genes; qRT-PCR verification is carried out on DRG neurons interfering ALKBH5, and 5 genes are found to be obviously reduced in expression: ALDH3B1, GALNS, LPIN2, COX8A, and ST6GA 1;

transfection of LPIN2 interfering RNA in DRG neurons: after the DRG neuron is separated and inoculated to a cell culture plate, the transfection of interfering RNA can be carried out; adding 1ul of transfection reagent into 25ul of transfection medium in a centrifuge tube A, gently mixing, preparing another centrifuge tube B, adding 2.5ul of interference RNA into 25ul of transfection medium, gently mixing, slowly adding the liquid in the centrifuge tube A into the centrifuge tube B, gently mixing, standing for 15min, uniformly adding into a cell culture plate pre-inoculated with DRG neurons, uniformly mixing cells, and putting into a 5% CO2 incubator at 37 ℃; cells were harvested 48h after transfection.

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