Application of Ankrd2 overexpression reagent in preparation of medicine for treating denervating amyotrophy

Abstract

The invention belongs to the technical field of biomedicine, and relates to application of an Ankrd2 overexpression reagent in preparation of a medicine for treating denervating amyotrophy. The preparation method comprises the following steps: constructing an over-expression lentivirus (LVs-Ankrd2) vector containing Ankrd2; the method comprises the following steps: injecting an overexpressed Ankrd2 lentivirus into tibialis anterior muscle to obtain skeletal muscle with overexpressed Ankrd2 under denervation; the skeletal muscle fiber basement membrane is marked by immunofluorescence staining to display the form and analyze the change of the sectional area of the muscle fiber, the effect of overexpression Ankrd2 on treating denervating amyotrophy is verified, the progress of skeletal amyotrophy can be relieved, and the Ankrd2 can be applied to preparation of drugs for treating denervating amyotrophy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to the application of an Ankrd2 overexpression reagent in the preparation of a drug for treating denervated muscle atrophy. Background Art

[0002] Denervated skeletal muscle atrophy is a muscle atrophy phenomenon caused by nerve injury. When skeletal muscle loses its nerve innervation and nerve signals cannot be effectively transmitted to the muscle, the physiological functions of muscle cells are disturbed, ultimately leading to a significant decline in muscle mass and function. Research shows that when denervation occurs, significant changes occur in the structure and function of muscle cells, including a reduction in cell volume, a decrease in muscle fiber diameter, and an exacerbation of muscle tissue fibrosis. In addition, the protein synthesis rate in muscle cells decreases, while the protein degradation rate increases, and this imbalance is the fundamental cause of overall muscle atrophy. Therefore, in-depth study of the mechanism of denervated skeletal muscle atrophy is of great significance for developing effective treatment strategies. Summary of the Invention

[0003] The main object of the present invention is to provide the application of an Ankrd2 overexpression reagent in the preparation of a drug for treating denervated muscle atrophy.

[0004] In the first aspect of the present invention, there is provided the application of an Ankrd2 overexpression reagent in the preparation of a drug for treating denervated muscle atrophy, and the Ankrd2 overexpression reagent is an overexpression vector containing the Ankrd2 gene.

[0005] Further, the denervated muscle atrophy is skeletal muscle atrophy caused by sciatic nerve transection.

[0006] Further, the Ankrd2 overexpression reagent is an overexpression plasmid containing the Ankrd2 gene.

[0007] Further, the Ankrd2 overexpression reagent is a lentiviral vector containing the Ankrd2 gene.

[0008] Compared with the prior art, the present application has verified through experiments that Ankrd2 can alleviate denervation-induced skeletal muscle atrophy. Overexpression of Ankrd2 has a protective effect on denervated skeletal muscle atrophy. The present invention provides a new direction for the research and development of drugs for regulating denervated skeletal muscle atrophy. Brief Description of the Drawings

[0009] The technical solutions of the present invention will be further specifically described below through examples and in conjunction with the drawings.

[0010] The following description of the embodiments of the present invention with reference to the drawings is intended to explain the general inventive concept of the present invention and should not be construed as a limitation of the present invention. In the drawings:

[0011] Figure 1 It is a schematic diagram of constructing an Ankrd2 gene vector in Example 1 of the present invention;

[0012] Figure 2 It is a result graph and statistical graph of detecting the expression of Ankrd2 by western blot after injecting the Ankrd2 overexpression lentivirus into the tibialis anterior muscle in Example 2 of the present invention;

[0013] Figure 3 It is a morphological graph and statistical graph of the effect on the muscle fibers of the tibialis anterior muscle after injecting the Ankrd2 overexpression lentivirus into the tibialis anterior muscle in Example 2 of the present invention;

[0014] Figure 4 It is a morphological graph and statistical graph of the effect on the muscle fibers of the tibialis anterior muscle after injecting the Ankrd2 overexpression lentivirus into the tibialis anterior muscle of 17-month-old mice in Example 3 of the present invention. Detailed implementation manners

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0016] The above content will be elaborated below in combination with specific verification experiments:

[0017] Example 1: Construction of a lentiviral vector for overexpression of the Ankrd2 gene

[0018] Gene information: Ankrd2 (NM_020033.2); Gene ID: 56642; Species: Mouse

[0019] 1.1 Using mouse DNA as a template, the coding region of the target gene Ankrd2 was amplified by PCR. The specific method is as follows: Use the high-fidelity PrimeSTAR enzyme to amplify the coding region of the target gene Ankrd2, and the reaction system and conditions are shown in the following table.

[0020]

[0021] The reaction conditions are shown in the following table.

[0022]

[0023] After the reaction, double-stranded DNA was obtained, including the entire nucleotide sequence encoding Ankrd2.

[0024] 1.2 Digestion of the target gene and plasmid vector

[0025] The vector GL214 was selected. The GL214 vector and the target gene in 1.1 were digested with the restriction endonucleases SpeⅠ and AgeⅠ. The digestion reaction system was as follows: 2 μg of nucleic acid, 5 μL of 10x reaction Buffer, 1 μL each of SpeⅠ and AgeⅠ as the restriction endonucleases, and the volume was made up to 50 μL with water. It was incubated in a 37°C water bath for more than 2 h to obtain the digested vector and the digested target gene product (digestion product).

[0026] The digestion product was detected by agarose gel electrophoresis for the digestion effect. The vector band (at about 8000 bp) and the target gene band (at about 1000 bp) were cut from the gel after agarose gel electrophoresis, and gel extraction was performed using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 to obtain the double-digested linearized vector.

[0027] 1.3 The double-digested linearized vector (step 1.2) and the digested double-stranded DNA (step 1.2) were ligated by T4 DNA ligase and ligated overnight at 16°C. The reaction system is shown in the following table.

[0028]

[0029] The resulting product was a vector containing the coding region of Ankrd2.

[0030] 1.4 Transformation: 10 μL of the reaction product (the vector containing the coding region of Ankrd2 obtained in step 1.3) was added to 100 μL of competent cells, gently flicked the tube wall several times to mix evenly, and placed on ice for 30 min. Heat shock at 42°C for 90 s, and incubate in an ice-water bath for 2 min. Add 500 μL of LB liquid medium, place it on a shaker at 37°C and shake for 1 h. After centrifuging at 1000 rpm for 1 min, discard the supernatant, leave 150 μL, pipette and mix evenly, and spread it on a plate containing ampicillin (there is precipitation in the tube, pipette and mix evenly), and incubate it upside down in a constant temperature incubator for 12 - 16 h to obtain colonies.

[0031] 1.5 Bacterial population amplification: In a laminar flow hood, use a sterile pipette tip to pick a single colony from step 1.4 and inoculate it into 5 ml of LB liquid medium containing ampicillin, and shake it at 37°C for 12 - 16 h to produce a large amount of bacterial liquid.

[0032] 1.6 Sequencing: Take 50 μL of the bacterial solution obtained in step 1.5 and send it to a biological company for first-generation sequencing. Compare the coding region sequence of the target gene Ankrd2 in the sequencing results with the correct sequence of the Ankrd2 coding region.

[0033] 1.7 Large-scale plasmid extraction

[0034] Take 50 μl of the cryopreserved bacterial solution with correct sequencing in step 1.6 and transfer it to 5 ml of LB liquid medium containing ampicillin. Culture it overnight at 37 °C, and then transfer it to 200 ml of LB liquid medium with ampicillin and culture it overnight at 37 °C. Then, use the plasmid extraction kit without endotoxin from ComWin Biotech to extract the plasmid, and send the extracted plasmid to the company for virus packaging. The detailed extraction operation steps are as follows:

[0035] (1) Take 150 ml of the overnight-cultured bacterial solution mentioned above and add it to a centrifuge tube. Centrifuge at 12000×g for 2 - 3 min to collect the bacteria, and discard as much supernatant as possible;

[0036] (2) Add 12 ml of Buffer P1 to the centrifuge tube with the bacterial pellet, and mix well with a vortex oscillator to suspend the bacterial pellet;

[0037] (3) Add 12 ml of Buffer P2 to the centrifuge tube, gently invert it up and down 8 - 10 times to fully lyse the bacteria, and let it stand at room temperature for 3 - 5 min. At this time, the solution should become clear and viscous;

[0038] (4) Add 12 ml of Buffer E3 to the centrifuge tube, immediately invert it up and down 8 - 10 times. At this time, a white flocculent precipitate appears. Let it stand at room temperature for 5 min, centrifuge at 12000×g for 10 min, pour all the supernatant into the endotoxin removal filter, slowly push the push handle to filter, and collect the filtrate in a clean 50 ml centrifuge tube;

[0039] (5) Add isopropanol with a volume 0.3 times that of the filtrate to the filtrate, and invert it up and down to mix well;

[0040] (6) Column equilibration: Add 2 ml of Buffer PS to the adsorption column already placed in the collection tube, centrifuge at 12000×g for 2 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0041] (7) Transfer the mixed solution of the filtrate and isopropanol in step 5 to the equilibrated adsorption column;

[0042] (8) Centrifuge at 12000×g for 2 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0043] (9) Add 10 ml of Buffer PW to the adsorption column, centrifuge at 6000 - 12000×g for 2 min, and pour out the waste liquid in the collection tube;

[0044] (10) Repeat step 9;

[0045] (11) Put the adsorption column back into the collection tube, centrifuge at 12000×g for 5 min, pour out the waste liquid, and place the adsorption column at room temperature for several minutes to thoroughly dry the residual ethanol in the adsorption column;

[0046] (12) Place the adsorption column in a new centrifuge tube, add 1 - 3 ml of Endo-Free Buffer EB to the middle part of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at 12000×g for 5 min, collect the plasmid solution into the centrifuge tube, and send the plasmid to the company for virus packaging to obtain a lentiviral vector overexpressing the Ankrd2 gene.

[0047] Example 2: Verification experiment on the protection of denervated muscle atrophy by overexpressing Ankrd2 lentivirus

[0048] 2.1 Preparation of animal model

[0049] Twenty-four C57BL / 6 mice, provided by the Experimental Animal Center of Nantong University, weighing 20 g - 25 g, 12 mice in each group, divided into 2 groups in total, namely the blank control group and the overexpressing Ankrd2 lentivirus group (Ankrd2 - OE). Disinfect, make a longitudinal incision about 2 cm, bluntly separate the biceps femoris of the rat at the left sciatic notch, expose the sciatic nerve of the mouse, cut off 1 cm of the sciatic nerve in the middle of the thigh, suture the wound routinely after the operation, and disinfect the postoperative wound with iodophor. Immediately after nerve transection, inject the corresponding lentivirus (lentiviral vector overexpressing the Ankrd2 gene and lentiviral vector of the blank control group (provided by the company)) into the ipsilateral tibialis anterior muscle of each group.

[0050] 2.2 Perfusion and sampling of mice

[0051] Anesthetize the mice 10 days after the operation, cut along the midline of the chest and abdomen to expose the heart, insert the perfusion needle from the apex of the left ventricle until it reaches the aorta, and quickly cut open the right auricle. Perfuse with normal saline until the color of the blood vessels such as the liver and mesentery becomes lighter, then immediately change to 4% paraformaldehyde. After perfusion, take the bilateral tibialis anterior muscles of the mice, and continue to use 4% paraformaldehyde for post-fixation and other corresponding treatments after sampling.

[0052] 2.3 Immunofluorescence staining of mouse tibialis anterior muscle

[0053] (1) Take the tibialis anterior muscle, dehydrate it with a sucrose gradient for later use, make frozen tissue sections with a thickness of 10 μm, and put the sections into an oven at 37°C overnight;

[0054] (2) Wash the tissues on the glass slides with PBS three times, 5 minutes each time.

[0055] (3) Add an appropriate amount of immunostaining blocking solution to the tissues on the glass slides and block at 37°C for 1 hour.

[0056] (4) Carefully aspirate the immunostaining blocking solution, and add an appropriate amount of primary antibody (Laminin) staining solution prepared with immunostaining primary antibody diluent, and incubate overnight at 4°C.

[0057] (5) Aspirate the primary antibody staining solution in (4), and wash with PBS three times, 5 minutes each time.

[0058] (6) Add an appropriate amount of secondary antibody (CoraLite488-conjugated Goat Anti-Rabbit IgG(H+L)) staining solution prepared with secondary antibody staining diluent, protect from light, and incubate at room temperature for 2 hours.

[0059] (7) Under light protection conditions, remove the secondary antibody staining solution, and wash with PBS three times, 5 minutes each time.

[0060] (8) Under light protection conditions, mount the slides with mounting medium and wait for image acquisition.

[0061] 2.4 Protein Extraction

[0062] (1) Tissue protein extraction: Extract 100 mg of tibialis anterior muscle from each group 10 days after surgery, wash it with normal saline, then place it on filter paper to absorb the surface water, and transfer it to a 1.5 ml Eppendorf tube.

[0063] (2) Add protein lysate containing protease inhibitor and phosphatase inhibitor to each tube (1 ml for tissue), and perform ultrasonic lysis for 30 s, 10 s each time, stop for 10 s, and perform 3 times in total.

[0064] (3) Incubate on ice for 30 minutes for lysis.

[0065] (4) Centrifuge at 13000×g at 4°C for 30 minutes.

[0066] (5) Take the supernatant. Since the amount of cell protein is not much, it is not subpackaged and stored. After subpackaging 100 μl of tissue protein in each tube, store it at -80°C for later use.

[0067] 2.5 Protein Quantification

[0068] Determine the protein concentration by BCA method (BCA protein quantification kit was purchased from Beyotime Biotechnology Co., Ltd., Shanghai)

[0069] (1) Prepare AB mixture according to a ratio of 50:1; Solution A and Solution B are the liquids provided in the kit.

[0070] (2) The protein sample (the tissue protein obtained in Step 2.4) is diluted with ddH 2 O (usually 1:50) so that the concentration to be measured is within the linear range of the standard curve. The diluted protein sample is added to a 96-well plate, with 3 replicates for each protein sample, and 200 μl of AB mixture is added. The lid is covered and incubated in an incubator at 37 °C for 30 min;

[0071] (3) The protein sample is placed in a microplate reader to measure the absorbance at 562 nm;

[0072] (4) According to the standard curve, calculate the actual protein concentration of the sample. Based on the protein concentration, calculate the protein volume for subsequent protein detection to ensure that the total protein amount in each group for detection is the same. Only on this basis can the differences in the target protein be compared.

[0073] 2.6 Western Blot Analysis

[0074] (1) Add 5×SDS loading buffer to the protein sample and boil for 5 min to denature the protein. Centrifuge at 4 °C, 12000 rpm × 5 min, and take the supernatant;

[0075] (2) Add 1× electrophoresis buffer to the electrophoresis tank and load the sample. Electrophoresis (stacking gel: 60 V, 30 min, separating gel: 110 V); Stop electrophoresis when the bromophenol blue runs to the bottom of the gel (depending on the molecular weight of the protein);

[0076] (3) Cut the gel and place it in the transfer buffer. Polarize the PVDF membrane with methanol and then place it in the transfer buffer to equilibrate with the gel for 15 minutes;

[0077] (5) Transfer: 100 V, 120 min;

[0078] (6) After taking out the PVDF membrane and labeling it, block it with 5% skim milk for 1 - 2 h;

[0079] (7) Incubate with primary antibodies (Ankrd2 and Tubulin) separately overnight at 4 °C;

[0080] (8) Wash with 1×TBS-T for 15 min × 3 times;

[0081] (9) Incubate with secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG(H+L)) at room temperature for 2 h;

[0082] (10) Wash with 1×TBS-T for 15 min × 3 times;

[0083] (11) Develop the protein immunoblotting result graphs of the blank control group and the lentivirus group overexpressing Ankrd2, and conduct statistical analysis on them. The results are as Figure 2 shown. The left graph is the protein immunoblotting result graph of Ankrd2 in the two groups, and the right graph is the statistical graph of the left graph. The results show that compared with the blank control group, the expression of Ankrd2 protein is up-regulated in the lentivirus group overexpressing Ankrd2.

[0084] 2.7 Image analysis and cross-sectional area statistics

[0085] After the tibialis anterior muscle samples are subjected to frozen section and immunofluorescence staining, image acquisition is performed. For each sample, 20 fields of view at 400 times magnification are randomly selected and photographed. The cross-sectional area of muscle fibers in each field of view is measured using Zeiss image analysis software, and the differences in the cross-sectional areas of muscle fibers in each group are statistically analyzed. The results are as Figure 3 shown. The upper left graph is the morphological graph of the cross-section of the contralateral tibialis anterior muscle of the mouse in the blank control group, the upper right is the morphological graph of the cross-section of the injured tibialis anterior muscle of the mouse in the blank control group, the lower left is the morphological graph of the cross-section of the contralateral tibialis anterior muscle of the mouse in the lentivirus group overexpressing Ankrd2, and the lower right is the morphological graph of the cross-section of the injured tibialis anterior muscle of the mouse in the lentivirus group overexpressing Ankrd2. The statistical results show that compared with the blank control group, overexpression of Ankrd2 can increase the cross-sectional area of muscle fibers. The above results suggest that overexpression of Ankrd2 has the ability to significantly alleviate skeletal muscle atrophy.

[0086] Antibody information

[0087]

[0088]

[0089] Example 3: Verification experiment that overexpressing Ankrd2 lentivirus has no effect on senile muscle atrophy

[0090] 3.1 Animal model preparation

[0091] Twenty-four C57BL / 6 mice, provided by the Experimental Animal Center of Nantong University, with a weekly age of 17 months, 12 in each group, were divided into 2 groups, the blank control group and the lentivirus group overexpressing Ankrd2. In the blank control group, blank control lentivirus was injected into the left posterior tibialis anterior muscle of 17-month-old mice, and in the lentivirus group overexpressing Ankrd2, lentivirus overexpressing Ankrd2 was injected into the left posterior tibialis anterior muscle of 17-month-old mice. They were raised in the animal house for one month, and then the tibialis anterior muscles were extracted when the mice were 18 months old.

[0092] 3.2 Mouse perfusion and sample collection

[0093] Anesthetize the mice, cut along the midline of the chest and abdomen to expose the heart. Insert the perfusion needle into the left ventricular apex until it reaches the aorta, and quickly cut open the right auricle. Perfuse with normal saline until the blood vessels such as the liver and mesentery become lighter in color. Immediately change to 4% paraformaldehyde. After perfusion, take the bilateral tibialis anterior muscles of the mice. After sampling, continue to use 4% paraformaldehyde for post-fixation and other corresponding treatments.

[0094] 3.3 Immunofluorescence staining of mouse tibialis anterior muscle

[0095] (1) After perfusion of the mice, take the tibialis anterior muscles, fix them with formaldehyde for 18 h, dehydrate them with a sucrose gradient for later use, and perform frozen tissue sectioning with a thickness of 10 μm. Place the sections in an oven at 37 °C overnight.

[0096] (2) Wash the skeletal muscle tissue on the glass slide 3 times with PBS for 5 min each time.

[0097] (3) Drop an appropriate amount of immunostaining blocking solution on the tissue on the glass slide and block it at 37 °C for 1 h.

[0098] (4) Carefully aspirate the immunostaining blocking solution, and drop an appropriate amount of primary antibody (Laminin) staining solution prepared with immunostaining primary antibody dilution buffer, and incubate at 4 °C overnight.

[0099] (5) Aspirate the primary antibody staining solution, and wash it 3 times with PBS for 5 min each time.

[0100] (6) Drop an appropriate amount of secondary antibody staining solution (CoraLite488-conjugated Goat Anti-Rabbit IgG(H+L)) prepared with secondary antibody staining dilution buffer, protect from light, and incubate at room temperature for 2 h.

[0101] (7) Under light protection, remove the secondary antibody staining solution, and wash it 3 times with PBS for 5 min each time.

[0102] (8) Under light protection, mount the slide with mounting medium and wait for image acquisition.

[0103] 3.4 Image analysis and cross-sectional area statistics

[0104] After the tibialis anterior muscle samples are subjected to frozen sectioning and immunofluorescence staining, images are collected. Randomly select 20 fields of view at 400× magnification for each sample and take pictures. Use Zeiss image analysis software to measure the cross-sectional area of muscle fibers in each field of view, and statistically analyze the differences in the cross-sectional area of muscle fibers in each group. The results Figure 4 are shown as follows. It is suggested that overexpressing the Ankrd2 lentivirus does not cause changes in the cross-sectional area of skeletal muscle fibers induced by aging.

[0105] In summary, it is confirmed in this application that Ankrd2 has the ability to protect denervated muscle atrophy. Through specific verification embodiments, it is confirmed that overexpression of Ankrd2 has the effect of increasing the cross-sectional area of muscle fibers in the denervated tibialis anterior muscle, thereby protecting skeletal muscle atrophy. The present invention provides a new idea for preventing and treating skeletal muscle atrophy caused by peripheral nerve injury, and provides a new strategy for protecting the normal function of skeletal muscle by means of genetic engineering in the future. The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of an Ankrd2 overexpression reagent in the preparation of a drug for treating denervated muscular atrophy, wherein the Ankrd2 overexpression reagent is an overexpression vector comprising the Ankrd2 gene.

2. The use according to claim 1, wherein the denervated muscle atrophy is skeletal muscle atrophy caused by sciatic nerve severance.

3. The use according to claim 1 or 2, wherein the Ankrd2 overexpression reagent is an overexpression plasmid comprising the Ankrd2 gene.

4. The use according to claim 1 or 2, wherein the Ankrd2 overexpression reagent is a lentiviral vector containing the Ankrd2 gene.

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