CN111514159A - Application of miR-20a in promoting nerve regeneration and repairing nerve injury - Google Patents
Application of miR-20a in promoting nerve regeneration and repairing nerve injury Download PDFInfo
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Abstract
The invention discloses application of miR-20a in preparation of a medicine for promoting nerve regeneration and repairing nerve injury. The invention also discloses application of the miR-20a in promoting regeneration of DRG neuron axons and repair of peripheral nerve injury, which is characterized by comprising the following steps of: s1, detecting the expression change of miR-20a in DRG tissue after rat sciatic nerve injury; s2, in vitro miR-20a overexpression promotes growth of DRG neuron axons; s3, and the in vivo overexpression of miR-20a promotes the growth of DRG neuron axons. In the embodiment of the invention, miR-20a can participate in peripheral nerve injury repair by regulating the growth of DRG neuron axons, is beneficial to better understanding of the important role of miRNA in the nerve injury repair process, and provides a new target point for treatment after nerve injury.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to application of miR-20a in preparation of a medicine for promoting nerve regeneration and repairing nerve injury, in particular to application of miR-20a in preparation of a medicine for promoting nerve regeneration and repairing nerve injury, and application of miR-20a in promotion of DRG neuron axon regeneration and peripheral nerve injury repair.
Background
Peripheral nerve injury repair is a clinical problem, and causes great burden to society and families. Peripheral nerves can spontaneously regenerate after damage, but their function is difficult to recover due to limited regeneration rate, relative to central nerves. The cell and molecular mechanism of peripheral nerve injury regeneration is fully and deeply explored, the peripheral nerve function repair is promoted, and a theoretical basis is provided for clinical treatment.
microRNA (miRNA) is endogenous small non-coding RNA, is 21-23 nucleotides in length, and has the main function of inhibiting the translation of a target gene or directly degrading the target gene by completely or incompletely combining with a non-translation region at the 3' end of the target gene. The miRNA can inhibit the apoptosis of neurons and promote the regeneration of neuron axons in the peripheral nervous system, and can also participate in the regulation of the proliferation and migration of glial cells. In the early stage of the invention, a series of miRNAs (including miR-20 a) with differential expression are obtained by carrying out chip analysis on microRNAs expressed by DRG neurons cultured in vitro at different time points. However, there are few reports about the role of miR-20a in peripheral nerve injury, and the invention aims to further explore the regulation and control effect of differentially expressed miR-20a on DRG neurons in the peripheral nerve injury repair process.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides application of miR-20a in preparation of a medicine for promoting nerve regeneration and repairing nerve injury, application of miR-20a in promotion of DRG neuron axon regeneration and peripheral nerve injury repair, and researches the regulation and control effect of miR-20a on DRG neurons in the peripheral nerve injury repair process.
In order to achieve the purpose, the embodiment of the invention provides application of miR-20a in preparation of a medicine for promoting nerve regeneration and repairing nerve injury.
Further, the nerve injury is a sciatic nerve injury of the peripheral nervous system.
Furthermore, the drug takes miR-20a as a molecular intervention target, over-expresses miR-20a, and promotes the growth of DRG neuron axons.
The embodiment of the invention also provides application of miR-20a in promoting regeneration of DRG neuron axons and repair of peripheral nerve injury, which is characterized by comprising the following steps: s1, preparing a rat sciatic nerve injury model; detecting the expression change of miR-20a in DRG tissue after rat sciatic nerve injury; s2, extracting DRG neurons from adult male rats, and carrying out in-vitro miR-20a overexpression to promote the growth of DRG neuron axons; s3, selecting adult healthy male rats, and over-expressing miR-20a in vivo to promote the growth of DRG neuron axons.
Further, detecting the expression change of miR-20a in DRG tissue after rat sciatic nerve injury in S1 specifically comprises the following steps:
s1-1, DRG tissue RNA extraction after rat sciatic nerve injury and qRT-PCR
S1-1-1, taking L4-6 DRG tissues of SD rats at different time points after 0 h, 3 h, 9 h, 1 d, 4 d and 7 d sciatic nerve bruises, putting the tissues into liquid nitrogen, and waiting for extracting RNA; extracting tissue RNA according to TRIZOL T REAgent (Invitrogen) specifications;
s1-1-2, carrying out reverse transcription by using a TaqMan reverse transcription kit and a miR-20a specific reverse transcription primer;
s1-1-3, carrying out qRT-PCR by using SYBR PrimeScript RT-PCR Kit (Takara) after reverse transcription, and carrying out the operation according to the Kit instruction (taking GAPDH as an internal reference);
wherein, the reaction program of the PCR instrument is as follows: stage 1: 95 ℃ for 2 min, Stage 2 (Cycle: 40): 95 ℃ for 15 s and 60 ℃ for 1 min; stage 3: 95 ℃ for 15 s, 60 ℃ for 1 min and 95 ℃ for 15 s;
miR-20a primer sequence forward: 5'-UAAAGUGCUUAUAGUGCAGGUAG-3' and miR-20a primer sequence reverse: 5'-CUACCUGCACUAUAAGCACUUUA-3';
s1-2, frozen section and in situ hybridization
S1-2-1, taking out the L4-6 DRG tissue from the rat which is filled with 0 d and 4 d sciatic nerve clamp injury, transferring the tissue into cane sugar to perform gradient dehydration, putting the tissue into a plastic mould when the tissue is completely sunk to the bottom of liquid, adding OCT, putting the plastic mould into liquid nitrogen to perform quick freezing;
s1-2-2, slicing by using a Leica CM3050S freezing microtome, drying the slices, and storing at-80 ℃;
s1-2-3, preparing a specific probe aiming at miR-20a, wherein the probe sequence is as follows: 5'-CUACCUGCACUAUAAGCACUUUA-3' DRG tissue sections at both time points 0 d and 4 d after sciatic nerve injury in adult rats were hybridized in situ using 50 nM of target probe and negative control according to the protocol of the mircurY LNAmicroRNA ISH Optimization Kit.
Further, in S2, the in vitro overexpression of miR-20a promotes the growth of DRG neuron axons, which specifically includes the following processes:
s2-1, extraction of primary DRG neuron cells
S2-1-1, wherein the DRG neuron is from adult male rat, the dorsal root ganglion is taken out and put into the dissecting fluid HA, and a proper amount of collagenase of 3.3mg/ml is digested at 37 ℃ for 90 min;
s2-1-2, discarding collagenase, adding appropriate amount of 0.25% pancreatin for digestion, at 37 deg.C for 20 min;
s2-1-3, terminating the pancreatin action by PBS containing 10% fetal calf serum, centrifuging and then removing the supernatant;
s2-1-4, suspending cells by using 15% bovine serum albumin, centrifuging, and removing supernatant;
s2-1-5, suspending cells by using neuron culture medium, sieving the cells by using a 200-mesh sieve, and then planting the cells into a plate hole coated by polylysine;
s2-2, transient electrocution of neuron cell
S2-2-1, using Rat Neuron NucleofectorTMThe DRG neuron cell which is well suspended and digested by the Solution is filled with 1 mug of green fluorescent plasmid, miR-20a mimics and negative cellsComparing, mixing, inserting the electric transfer groove into a Lonza AMAXA Lonza Nucleofector II nuclear transfectator, selecting G-013 Neurons Rat DRG program, and starting instantaneous electric transfer;
s2-2-2, adding low-calcium HA for balancing after completion, and inoculating the mixture into the plate holes;
s2-2-3, culturing for 4 h, and then changing into a normal culture medium;
s2-3, cellular immunofluorescent staining and axon growth length measurement
S2-3-1, after DRG neuron cells are subjected to electrotransformation for 72 h, discarding the cell culture medium, rinsing with PBS, adding 4% paraformaldehyde, and fixing for 30 min; after discarding paraformaldehyde, washing with PBS for three times, each for 5 min;
s2-3-2, adding immunohistochemical blocking liquid, and blocking for 1 h at room temperature;
s2-3-3, diluting the primary anti-Tuj1, adding the primary anti-anti;
s2-3-4, discarding the primary antibody, washing 3 times with PBS for 5min each time;
s2-3-5, diluting a fluorescent secondary antibody Cy3 sheet anti-rabbitIgG by using an immunohistochemical secondary antibody diluent, adding the secondary antibody, and keeping away from light for 2 hours at room temperature;
s2-3-6, discarding the secondary antibody, washing 3 times with PBS for 5min each time;
s2-3-7, diluting Hoechest with PBS, adding Hoechest, and keeping the temperature at room temperature for 10 min;
s2-3-8, discarding Hoechest, washing 3 times with PBS for 5min each time;
s2-3-9, adding a proper amount of fluorescent mounting solution, observing the ZEISS under a positive fluorescence microscope, and taking a picture.
Further, in the S3, the in vivo overexpression of miR-20a promotes the growth of DRG neuron axons, which specifically includes the following processes:
s3-1, selecting adult healthy male rats, anesthetizing with a compound anesthetic, shaving the backs of the rats, disinfecting with iodophor, cutting the skin, cutting the muscles at the ilium to expose the spine at the L5, injecting 30 mu L of control or Cy 3-labeled chemical mimic agomir of miR-20a through BD needle tubes, and suturing the muscles and the skin;
s3-2, feeding the rats in a proper environment, feeding and drinking water freely, and illuminating for 12 hours each day; 48 h later, a second injection of 30. mu.l of control or the Cy 3-labeled chemical mimic of miR-20a, agomir;
s3-3, performing sciatic nerve clamping injury for 72 h, shaving the mouse hair around the left leg with a razor, disinfecting with iodophors, cutting skin, further cutting muscle, and exposing the left sciatic nerve;
s3-4, selecting the upper end of the bifurcation as a clamping position, wherein the clamping width is 2 mm, and the clamping width is 30S, and the whole teeth are formed; after the operation is finished, the muscle and the skin are sutured;
s3-5, fixing paraformaldehyde 96 h after injury, taking sciatic nerve, freezing section and immunohistochemistry, carrying out primary anti-Tuj1 anti-body and SCG10, carrying out secondary antibody Cy3 sheet anti-body IgG and FITC goat anti-body IgG, observing under a microscope, and taking pictures.
The technical scheme of the invention has the following beneficial effects:
(1) in the early stage of the invention, a series of miRNAs with differential expression are obtained by carrying out chip analysis on microRNAs expressed by DRG neurons cultured in vitro at different time points. Wherein miR-20a increases in expression in DRG at different time points. In the embodiment of the invention, a rat model is prepared, and qRT-PCR and in-situ hybridization are utilized to find that miR-20a expression in a DRG tissue is remarkably increased after sciatic nerve injury of a rat. In vitro DRG transfects miR-20a mimic, and the overexpression of miR-20a can obviously promote the growth of primary culture DRG neuron axons. Rats are injected with cy 5-labeled miR-20a agomir intrathecally to promote the growth of axons after sciatic nerve injury. In the embodiment of the invention, miR-20a can participate in peripheral nerve injury repair by regulating the growth of DRG neuron axons, is beneficial to better understanding of the important role of miRNA in the nerve injury repair process, and provides a new target point for treatment after nerve injury.
(2) The miR-20a is used for preparing the medicine for promoting nerve regeneration and repairing nerve injury, the miR-20a is used as a molecular intervention target point, the miR-20a is over-expressed, the growth of DRG neuron axon is promoted, and a medicine target point and a treatment direction are provided for promoting nerve regeneration and repairing nerve injury.
Drawings
FIG. 1 is a graph showing the results of qRT-PCR and in situ hybridization of miR-20a in DRG tissue after sciatic nerve injury in rat in example 1 of the present invention. Wherein, FIG. 1A is a graph of miR-20a expression at different time points detected by qRT-PCR after rat sciatic nerve injury, and the internal reference is U6; FIG. 1B is a graph showing in situ hybridization expression of miR-20a in DRG tissues 0 d and 4 d after sciatic nerve injury in rats.
FIG. 2 is a graph of the in vitro overexpression of miR-20a in example 2 of the present invention can significantly promote the growth of DRG neuronal axons. Wherein, FIG. 2A is a staining pattern of cell immunohistochemistry after 72 h of DRG neurons were cultured in vitro with cotransfer of green fluorescent plasmid (pmax GFP) and control (negative control) or miR-20amimics, Bar =50 μm; FIG. 2B is a graph of in vitro DRG neurons transfected with control and miR-20a mimics, after which axonal length distribution of all neurons was measured; FIG. 2C is the average of the longest axons of all neurons after in vitro DRG neurons were transfected with control and miR-20a mimics. P <0.05, P < 0.001.
FIG. 3 is a diagram showing that miR-20a can promote regeneration of damaged nerves in vivo in example 3 of the present invention. FIG. 3A is a graph of immunohistochemical results of axonal growth after intrathecal injection of over-expressed miR-20a, sciatic nerve pinches, Bar =1000 μm, right graph is a partial magnification of the left white dashed box; FIG. 3B, statistical plot of the sciatic nerve regeneration length in vivo. FIG. 3C is a statistical plot of fluorescence intensity of regenerating axons at different distances from the injury site. P < 0.01.
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 examination of miR-20a expression Change in DRG tissue after injury of rat sciatic nerve
Firstly, preparing rat sciatic nerve injury model
Adult healthy male C57BL/6J mice, weighing 20 g, were randomly assigned to 6 groups of 6 mice each. After anesthesia with the compound anesthetic, the skin of the left lower limb was incised, the sciatic nerve on the left side parallel to the mid-femur was exposed, incised with toothless forceps for 30s, and then sutured. After surgery, the animals were kept under appropriate conditions to relieve pain, 12 h daily, free to drink and eat. The tissues of the sciatic nerve of the rats from L4 to L6 DRG on the sides of the injury are taken at 0 h, 3 h, 9 h, 1 d, 4 d and 7 d after the injury.
Second, DRG tissue RNA extraction and qRT-PCR after rat sciatic nerve injury
Taking L4-6 DRG tissues at different time points after 0 h, 3 h, 9 h, 1 d, 4 d and 7 d sciatic nerve clamping injury of SD rats, putting the tissues into liquid nitrogen, and waiting for RNA extraction.
Tissue RNA was extracted according to TRIZOL Reagent (Invitrogen) instructions. Reverse transcription is carried out by using a TaqMan reverse transcription kit and a miR-20a specific reverse transcription primer.
After reverse transcription, qRT-PCR was performed using SYBR PrimeScript RT-PCR Kit (Takara) according to the Kit instructions (GAPDH as reference).
Reaction program of PCR instrument: stage 1: 95 ℃ for 2 min, Stage 2 (Cycle: 40): 95 ℃ for 15 s and 60 ℃ for 1 min; stage 3: 95 ℃ for 15 s, 60 ℃ for 1 min and 95 ℃ for 15 s.
miR-20a primer sequence forward: 5'-UAAAGUGCUUAUAGUGCAGGUAG-3' and miR-20a primer sequence reverse: 5'-CUACCUGCACUAUAAGCACUUUA-3'.
The qRT-PCR results are shown in fig. 1A, which shows sustained upregulation of miR-20a expression in DRG tissues after 3 h, 9 h, 1 d, 4 d, and 7 d injury compared to 0 d.
Three, frozen section and in situ hybridization
Taking out the L4-6 DRG tissue from the rat with the injury of sciatic nerve of 0 d and 4 d, transferring into sucrose for gradient dehydration, placing into a plastic mold when the tissue is completely sunk to the bottom of the liquid, adding OCT, and placing into liquid nitrogen for quick freezing.
The slices were taken on a Leica CM3050S cryomicrotome, dried and stored at-80 ℃.
Specific probes for miR-20a, Probe sequence 5'-CUACCUGCACUAUAAGCACUUUA-3', in situ hybridization of DRG tissue sections at two time points, 0 d and 4 d after sciatic nerve injury in adult rats, were performed according to the protocol of the mircurY LNAmicroRNA ISH Optimization Kit (Exiqon Co.) using 50 nM of the probe of interest and negative control.
Microscopic observation and photographing show that the expression level of miR-20a in DRG tissue is significantly increased 4 d after sciatic nerve injury of rats compared with 0 d, and negative control (conorl) is unchanged as shown in FIG. 1B.
Example 2 in vitro overexpression of miR-20a promotes DRG neuronal axon growth
Extraction of primary DRG neuronal cells
DRG neurons were obtained from adult male rats, dorsal root ganglia were removed and placed in dissecting fluid HA, digested with appropriate amount of collagenase 3.3mg/ml, at 37 deg.C for 90 min. Discarding collagenase, adding appropriate amount of 0.25% pancreatin for digestion at 37 deg.C for about 20 min.
Pancreatin was stopped with PBS containing 10% fetal bovine serum, and the supernatant was discarded after centrifugation. To remove the contaminating glial cells, the cells were suspended with 15% bovine serum albumin and centrifuged, and the supernatant was discarded. Cells were resuspended in neuronal medium, screened through a 200 mesh screen, and plated into wells coated with polylysine.
Second, transient electrocution of neuronal cells
The digested DRG neuronal cells were suspended in Rat Neuron nuclear emission Solution (Lonza), 1. mu.g of a green fluorescent plasmid (pmax GFP) was added to the cell suspension, as well as miR-20a mimics and a negative control (20 nM concentration, Campylobacter, Guangzhou), and after mixing, the cells were inserted into a Lonza AMAXA Lonza Nuclear emission II nuclear transfectator (Lonza), and the G-013 Nerons Rat DRG program was selected to initiate transient electrotransformation. After completion, low calcium HA was added to equilibrate and plated into the wells. After 4 h of culture, the medium was changed to normal medium.
Third, cellular immunofluorescent staining and axon growth length measurement
After 72 h of DRG neuron cell electrotransformation, the cell culture medium was discarded, rinsed once with PBS, and fixed for 30 min with 4% paraformaldehyde. After discarding paraformaldehyde, the PBS was washed three times for 5min each. Adding immunohistochemical blocking solution, and blocking at room temperature for 1 hr. Primary anti-Tuj1 antibody (1: 200, Sigma) was diluted with immunohistochemical primary antibody diluent and added overnight at 4 ℃. Primary antibody was discarded and washed 3 times with PBS for 5min each. The fluorescent secondary antibody Cy3 sheepanti-rabbitIgG (1: 400, Sigma) was diluted with an immunohistochemical secondary antibody diluent, and after addition of the secondary antibody, it was protected from light for 2 hours at room temperature. The secondary antibody was discarded and washed 3 times with PBS for 5min each time. Hoechest was diluted with PBS and, after addition, 10 min at room temperature. Hoechest was discarded and washed 3 times with PBS for 5min each time. Adding a proper amount of fluorescent mounting solution, observing by a ZEISS (zero-electron fluorescence microscope) under a positive fluorescent microscope, and taking a picture. And observing the growth condition of the protrusions, photographing and counting the longest protrusion length of each group and the distribution of the protrusion length of each group.
The result is shown in figure 2, and the miR-20a (miR-20a mimics) is over-expressed to remarkably promote the growth of DRG neuron axons. FIG. 2A is a staining diagram of cell immunohistochemistry after DRG neurons were cultured in vitro for 72 h in cotransfer of green fluorescent plasmid (pmax GFP) and control (negative control) or miR-20amimics, and it can be seen that the DRG neurons in vitro overexpress miR-20a and increase the axon growth length. Tuj1 in FIG. 2A labels neuronal axons, GFP labels neuronal cells transfected with miR-20amimics or its control. Neuronal cells co-targeted with Tuj1 and GFP were selected for subsequent neuronal axonal growth statistics. FIG. 2B is a graph showing the axon length distribution of all neurons measured after in vitro DRG neurons were transfected with control and miR-20a mimics; the longest axon lengths of all neurons were counted and grouped by 0-100 μm, 100-200 μm, 200-300 μm, 300-400 μm, 400-500 μm, 500-600 μm, 600-700 μm and 700 μm or more. After miR-20a is transfected, the length of axon growth is biased to be distributed in a long interval, and the fact that in vitro DRG neurons over-express miR-20a can promote axon growth is shown. FIG. 2C is a statistical plot of the mean of the longest axons of all neurons after in vitro DRG neurons were transfected with control and miR-20a mimics. P <0.05, P < 0.001; as can be seen, the average value of the longest axon length of the neurons is obviously increased after the DRG neurons overexpress miR-20a in vitro, which indicates that miR-20a can promote axon growth.
Example 3 in vivo overexpression of miR-20a promotes DRG neuronal axon growth
Adult healthy male rats were selected, anesthetized with a compound anesthetic, and then the backs of the rats were shaved, sterilized with iodophor, the skin was cut, the muscles were cut at the iliac bones to expose the spinal column at L5, 30 μ L of control or a chemomimetic agomir of Cy 3-labeled miR-20a was injected with a BD needle tube, and then the muscles and the skin were sutured. Rats were kept in the appropriate environment with free access to food and water and light exposure for 12 h per day. A second injection was given 48 h later, as was the first. Sciatic nerve injury was performed over 72 hours (sciatic nerve injury model was prepared in the same manner as in example 1), left lateral peri-lateral leg rat hair was shaved with a razor, skin was cut after iodophor sterilization, and muscle was further cut to expose the left sciatic nerve. The upper end of the bifurcation is generally selected as a clamping position, the width of the clamping wound is 2 mm, the width of the clamping wound is 30s, and the whole tooth is formed. After the operation is completed, the muscle and the skin are sutured. Fixation with paraformaldehyde was performed at 96 h post-injury, sciatic nerve was taken, frozen sections and immunohistochemistry, primary anti-Tuj1 antibody (1: 200, Sigma) and SCG10 (1: 100, Proteintetech), secondary antibodies Cy3 sheet anti-rabbitIgG (1: 400, Sigma) and FITOAT anti-mouse IgG (1: 200, Sigma), and observed under a microscope and photographed.
The results are shown in figure 3, and it can be seen that miR-20a can promote the regeneration of damaged nerves in vivo. Specifically, as can be seen from FIG. 3A, the length of SCG-labeled regenerated axons of the experimental group injected with miR-20a agomir was significantly longer than that of the control group injected with agomir control, in which SCG10 labeled regenerated axons and Tuj1 labeled all axons. As can be seen from the statistical graph of the regeneration length of sciatic nerve in rats in FIG. 3B, the sciatic nerve length in rats injected intrathecally with miR-20a is significantly longer than that in rats of negative control (contorl), confirming that miR-20a can promote the regeneration of injured nerve in vivo. As can be seen from the fluorescence intensity statistical chart in FIG. 3C, after miR-20a is overexpressed, the fluorescence intensity of axons at the same distance is also superior to that of a control group, which indicates that the number of regenerated axons is more than that of the control group, and the axon regeneration of damaged neurons can be promoted by overexpression of miR-20a in vivo.
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.
Sequence listing
<110> university of southeast Tong
Application of <120> miR-20a in promoting nerve regeneration and repairing nerve injury
<141>2020-06-02
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<170>SIPOSequenceListing 1.0
<210>1
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<213> Positive-strand sequence of miR-20a (miR-20a-F)
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<210>2
<211>23
<212>RNA
<213> reverse-chain sequence of miR-20a (miR-20a-R)
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cuaccugcac uauaagcacu uua 23
Claims (7)
1. Application of miR-20a in preparation of medicine for promoting nerve regeneration and repairing nerve injury is provided.
2. The use of miR-20a in the preparation of a medicament for promoting nerve regeneration and repairing nerve injury according to claim 1, wherein the nerve injury is sciatic nerve injury of a peripheral nervous system.
3. The use of miR-20a in the preparation of a medicament for promoting nerve regeneration and repairing nerve injury according to claim 1, wherein the medicament takes miR-20a as a molecular intervention target, overexpresses miR-20a, and promotes DRG neuron axon growth.
The application of miR-20a in promoting axon regeneration of DRG neurons and repair of peripheral nerve injury is characterized by comprising the following processes: s1, preparing a rat sciatic nerve injury model; detecting the expression change of miR-20a in DRG tissue after rat sciatic nerve injury; s2, extracting DRG neurons from adult male rats, and carrying out in-vitro miR-20a overexpression to promote the growth of DRG neuron axons; s3, selecting adult healthy male rats, and over-expressing miR-20a in vivo to promote the growth of DRG neuron axons.
5. The application of the miR-20a in promoting the axon regeneration of DRG neurons and the repair of peripheral nerve injury according to claim 4, wherein the expression change of the miR-20a in the DRG tissues after rat sciatic nerve injury is detected in S1, and the method specifically comprises the following processes:
s1-1, DRG tissue RNA extraction after rat sciatic nerve injury and qRT-PCR
S1-1-1, taking L4-6 DRG tissues of SD rats at different time points after 0 h, 3 h, 9 h, 1 d, 4 d and 7 d sciatic nerve bruises, putting the tissues into liquid nitrogen, and waiting for extracting RNA; extracting tissue RNA according to TRIZOL Reagent specifications;
s1-1-2, carrying out reverse transcription by using a TaqMan reverse transcription kit and a miR-20a specific reverse transcription primer;
s1-1-3, carrying out qRT-PCR by using SYBR PrimeScript RT-PCR Kit (Takara) after reverse transcription, carrying out the operation according to the Kit instruction and taking GAPDH as an internal reference;
wherein, the reaction program of the PCR instrument is as follows: stage 1: 95 ℃ for 2 min, Stage 2 (Cycle: 40): 95 ℃ for 15 s and 60 ℃ for 1 min; stage 3: 95 ℃ for 15 s, 60 ℃ for 1 min and 95 ℃ for 15 s;
miR-20a primer sequence forward: 5'-UAAAGUGCUUAUAGUGCAGGUAG-3' and miR-20a primer sequence reverse: 5'-CUACCUGCACUAUAAGCACUUUA-3';
s1-2, frozen section and in situ hybridization
S1-2-1, taking out the L4-6 DRG tissue from the rat which is filled with 0 d and 4 d sciatic nerve clamp injury, transferring the tissue into cane sugar to perform gradient dehydration, putting the tissue into a plastic mould when the tissue is completely sunk to the bottom of liquid, adding OCT, putting the plastic mould into liquid nitrogen to perform quick freezing;
s1-2-2, slicing by using a Leica CM3050S freezing microtome, drying the slices, and storing at-80 ℃;
s1-2-3, preparing a specific probe aiming at miR-20a, wherein the probe sequence is as follows: 5'-CUACCUGCACUAUAAGCACUUUA-3' DRG tissue sections at both time points 0 d and 4 d after sciatic nerve injury in adult rats were hybridized in situ using 50 nM of target probe and negative control according to the protocol of the mircurY LNAmicroRNA ISH Optimization Kit.
6. The use of miR-20a in promoting regeneration of DRG neuron axons and repair of peripheral nerve injury according to claim 4, wherein in S2, in vitro overexpression of miR-20a promotes DRG neuron axon growth, and specifically comprises the following processes:
s2-1, extraction of primary DRG neuron cells
S2-1-1, wherein the DRG neuron is from adult male rat, the dorsal root ganglion is taken out and put into the dissecting fluid HA, and a proper amount of collagenase of 3.3mg/ml is digested at 37 ℃ for 90 min;
s2-1-2, discarding collagenase, adding appropriate amount of 0.25% pancreatin for digestion, at 37 deg.C for 20 min;
s2-1-3, terminating the pancreatin action by PBS containing 10% fetal calf serum, centrifuging and then removing the supernatant;
s2-1-4, suspending cells by using 15% bovine serum albumin, centrifuging, and removing supernatant;
s2-1-5, suspending cells by using neuron culture medium, sieving the cells by using a 200-mesh sieve, and then planting the cells into a plate hole coated by polylysine;
s2-2, transient electrocution of neuron cell
S2-2-1, suspending and digesting DRG Neuron cells by using Rat Neuron nuclear reactor solid Solution, adding 1 mu G of green fluorescent plasmid, miR-20a mimics and negative control into cell suspension, uniformly mixing, inserting an electrotransformation tank into a Lonza AMAXA Lonza nuclear reactor II cell nucleus transfectator, selecting a G-013 Neuron Rat DRG program, and starting instantaneous electrotransformation;
s2-2-2, adding low-calcium HA for balancing after completion, and inoculating the mixture into the plate holes;
s2-2-3, culturing for 4 h, and then changing into a normal culture medium;
s2-3, cellular immunofluorescent staining and axon growth length measurement
S2-3-1, after DRG neuron cells are subjected to electrotransformation for 72 h, discarding the cell culture medium, rinsing with PBS, adding 4% paraformaldehyde, and fixing for 30 min; after discarding paraformaldehyde, washing with PBS for three times, each for 5 min;
s2-3-2, adding immunohistochemical blocking liquid, and blocking for 1 h at room temperature;
s2-3-3, diluting the primary anti-Tuj1, adding the primary anti-anti;
s2-3-4, discarding the primary antibody, washing 3 times with PBS for 5min each time;
s2-3-5, diluting a fluorescent secondary antibody Cy3 sheet anti-rabbitIgG by using an immunohistochemical secondary antibody diluent, adding the secondary antibody, and keeping away from light for 2 hours at room temperature;
s2-3-6, discarding the secondary antibody, washing 3 times with PBS for 5min each time;
s2-3-7, diluting Hoechest with PBS, adding Hoechest, and keeping the temperature at room temperature for 10 min;
s2-3-8, discarding Hoechest, washing 3 times with PBS for 5min each time;
s2-3-9, adding a proper amount of fluorescent mounting solution, observing the ZEISS under a positive fluorescence microscope, and taking a picture.
7. The use of miR-20a in promoting regeneration of DRG neuron axons and repair of peripheral nerve injury according to claim 4, wherein in S3, in vivo overexpression of miR-20a promotes growth of DRG neuron axons, and specifically comprises the following processes:
s3-1, selecting adult healthy male rats, anesthetizing with a compound anesthetic, shaving the backs of the rats, disinfecting with iodophor, cutting the skin, cutting the muscles at the ilium to expose the spine at the L5, injecting 30 mu L of control or Cy 3-labeled chemical mimic agomir of miR-20a through BD needle tubes, and suturing the muscles and the skin;
s3-2, feeding the rats in a proper environment, feeding and drinking water freely, and illuminating for 12 hours each day; 48 h later, a second injection of 30. mu.l of control or the Cy 3-labeled chemical mimic of miR-20a, agomir;
s3-3, performing sciatic nerve clamping injury for 72 h, shaving the mouse hair around the left leg with a razor, disinfecting with iodophors, cutting skin, further cutting muscle, and exposing the left sciatic nerve;
s3-4, selecting the upper end of the bifurcation as a clamping position, wherein the clamping width is 2 mm, and the clamping width is 30S, and the whole teeth are formed; after the operation is finished, the muscle and the skin are sutured;
s3-5, fixing paraformaldehyde 96 h after injury, taking sciatic nerve, freezing section and immunohistochemistry, carrying out primary anti-Tuj1 anti-body and SCG10, carrying out secondary antibody Cy3 sheet anti-body IgG and FITC goat anti-body IgG, observing under a microscope, and taking pictures.
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