CN108728437A - Promote oligonucleotides, drug and the application of Skeletal muscle injury reparation - Google Patents
Promote oligonucleotides, drug and the application of Skeletal muscle injury reparation Download PDFInfo
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- CN108728437A CN108728437A CN201810545604.2A CN201810545604A CN108728437A CN 108728437 A CN108728437 A CN 108728437A CN 201810545604 A CN201810545604 A CN 201810545604A CN 108728437 A CN108728437 A CN 108728437A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
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Abstract
The present invention relates to biomedical sectors, concretely relate to a kind of oligonucleotides and its application process for promoting Skeletal muscle injury reparation.Present invention firstly discloses filtered out with the oligonucleotides for remarkably promoting Skeletal Muscle mother cell myogenic differentiation, feature such as SEQ ID NO from the transcript of people source development imprinted genes H19:1 and SEQ ID NO:Shown in 2 sequences.The oligonucleotides and its application process have easy to operate, devoid of risk, promote the advantage that the regeneration of newborn muscle fibre after Skeletal muscle injury is fast, functional rehabilitation is good.Think that there is good application prospect in the gene therapy of Skeletal muscle injury caused by acute pause and transition in rhythm or melody wound, crush injury, explosion injury or chemical substance of oligonucleotides disclosed by the invention and application method accordingly.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to an oligonucleotide with a function of regulating myoblast differentiation, a medicine and an application method of the oligonucleotide for promoting skeletal muscle injury repair.
Background
Skeletal muscle injury is a common condition of injury from accidental wounds, disaster injuries, sports injuries or chemical poisons. After skeletal muscle injury, the natural healing process is slow, the healing capacity is limited, the scar formation of muscle fibers is often accompanied, the functional recovery is often incomplete, and severe people even lose the motor ability. The traditional method for treating skeletal muscle injury comprises rest, ice compress, pressurization, affected limb lifting, heat treatment, water bath and the like, and has long treatment process and poor curative effect. There is currently no clinically effective treatment for skeletal muscle injury. Therefore, how to effectively prevent the bad repair after the skeletal muscle injury, effectively promote the regeneration and healing of the skeletal muscle and improve the healing quality of the skeletal muscle becomes a very challenging world problem in the field of regenerative medicine.
② the ② natural ② pathological ② process ② after ② skeletal ② muscle ② injury ② includes ② three ② stages ②, ② i.e. ②, ② inflammation ② stage ②, ② in ② which ② hematoma ② is ② formed ② at ② the ② injured ② position ② of ② muscle ②, ② the ② injured ② muscle ② is ② necrotized ② and ② degenerated ②, ② neutrophil ② is ② infiltrated ②, ② macrophage ② is ② chemotactic ② to ② the ② injured ② position ② to ② phagocytize ② necrotic ② tissue ②, ② repairing ② stage ②, ② in ② which ② satellite ② cell ② in ② stationary ② stage ② around ② the ② injured ② muscle ② is ② stimulated ② and ② activated ② by ② cell ② factor ②, ② and ② chemotactic ② to ② the ② injured ② position ② to ② proliferate ② and ② differentiate ② to ② form ② new ② myotube ②, ② which ② is ② fused ② with ② residual ② muscle ② fiber ②, ② and ② in ② which ② the ② proliferation ② of ② fibroblast ② is ② accompanied ②, ② and ② if ② the ② injury ② range ② is ② too ② large ②, ② the ② excessive ② proliferation ② and ② differentiation ② of ② fibroblast ② forms ② scar ② tissue ② to ② cause ② abnormal ② healing ② and ② dysfunction ② of ② skeletal ② muscle ②, ② and ② shaping ② stage ②, ② in ② which ② regenerated ② skeletal ② muscle ② is ② mature ② and ② scar ② tissue ② is ② organized ②. ②
The damaged skeletal muscle can not fully recover the function state before the damage through natural healing, and the recent research shows that the main reason is that the proliferation and differentiation of skeletal muscle myoblast are related to the proliferation and differentiation imbalance of fibroblast. Therefore, current research on promoting skeletal muscle repair mainly focuses on stimulating proliferation and differentiation of skeletal muscle myoblasts and inhibiting fibrosis of injured skeletal muscles.
The microRNA is taken as a non-coding small molecular RNA with biological activity and participates in the generation, development and regulation processes of various tissue organ development and diseases. The protein encoded by Dicer gene is an endonuclease necessary for the mature processing of miRNA. After the Dicer gene is knocked out under the tissue condition, skeletal muscle development of the mouse is abnormal (skeletal muscle tissue content is reduced, myofibrillar morphology is abnormal and the like), which indicates that miRNA plays an important role in the skeletal muscle development process.
Research reports that miR-1 and miR-125b expression level is up-regulated in the process of regenerating skeletal muscle of mice injured by snake Cardiotoxin (CTX), insulin-like growth factor 2(IGF-2) (miR-125b downstream target gene) expression level is increased, and IGF-2 regulates myoblast differentiation, so that the regeneration fusion process of new myogenic fibers is promoted. A plurality of miRNAs participate in regulating and controlling the regeneration and repair process of damaged skeletal muscle, and the application of exogenous oligonucleotides is suggested to be an effective method for promoting the repair of the damaged skeletal muscle.
Research shows that the development imprinting gene H19 is widely expressed in human and mouse embryos and has important functions of regulating and controlling the growth of the embryos, the development of the fetuses, the development of children behaviors and the like. Recent research shows that miR-675 is contained in the transcript of the H19 gene, and the transcript is persistently and highly expressed in skeletal muscle of adult mice, which indicates that the H19 gene participates in the development and function regulation of the skeletal muscle through mRNA.
Disclosure of Invention
The present invention is directed to the above problems and aims to provide an oligonucleotide, a medicament comprising the oligonucleotide and a method of use.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the embodiments of the present invention disclose an oligonucleotide comprising the sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2.
In another aspect, the embodiment of the present invention discloses a medicament, which comprises the above-mentioned oligonucleotide.
Furthermore, the embodiment of the invention discloses the application method of the oligonucleotide and the medicament containing the oligonucleotide, which comprises the following steps: the injection sites are four opposite angles of the boundary edge of the injured skeletal muscle and the normal tissue; the injection depth is the center of the inner side of the edge of the injured skeletal muscle and normal tissue; the injection dose is 200 nM/site/time (50 uL volume) of the oligonucleotide physiological saline solution of interest; the injection time (number) was 1 injection (total 3) on each of day 7, 12 and 17 after the injury.
In a third aspect, the skeletal muscle injury comprises an acute bruise, crush injury, blast injury, or chemical injury to skeletal muscle.
In a fourth aspect, the present embodiments disclose the use of the above oligonucleotides for treating skeletal muscle injury.
In a fifth aspect, the embodiment of the invention discloses an application method of the medicine for treating skeletal muscle injury.
The invention has the beneficial effects that:
experiments prove that in the process of healing skeletal muscle injury, the oligonucleotide sequence disclosed by the embodiment of the invention has the biological function of remarkably inhibiting the expression of a transcription factor Samd4 so as to promote myoblast differentiation of skeletal muscle myoblasts. The application method of the oligonucleotide and the medicine containing the oligonucleotide has the advantages of low cost, simple and convenient operation, no risk, fast regeneration and repair of new muscle fibers after skeletal muscle injury and good skeletal muscle function recovery.
Drawings
FIG. 1 shows myogenic differentiation morphology of C2C12 cells and transcriptional expression analysis of myogenin gene (myogenin, Myog) and Myosin heavy chain gene (MHC) after 72h of culture with addition of the oligonucleotide of interest or a control sequence, wherein the left lower bar in the left graph indicates 50 μm, and the left symbol in the right graph indicates that P <0.01 in the experimental group compared with the control group.
FIG. 2 shows the result of Smad4 protein expression by C2C12 cells after 72h incubation with the addition of the oligonucleotide of interest or a control sequence, wherein Beta-actin serves as an internal reference for immunoblot analysis.
FIG. 3 is H & E staining, Desmin (Desmin) immunohistochemical staining and regenerated skeletal muscle fiber cross-sectional area analysis of repair tissues 21 days after local microinjection of target oligonucleotide or control sequence treatment for mouse hind leg tibialis anterior Cardiotoxin (CTX) injury, wherein the lower right scale in the left panel indicates 50 μm, and the left symbol in the right panel indicates that P <0.01 in the experimental group compared with the control group.
FIG. 4 shows the result of skeletal muscle healing quality evaluation by running test of mice 28 days after the hind leg tibialis anterior muscle Cardiotoxin (CTX) injury local microinjection of target oligonucleotide or control sequence.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the fact that no effective method for regulating skeletal muscle cell myoblast differentiation to play a role in promoting healing exists in clinic, the technical problem to be solved by the invention is to provide an oligonucleotide capable of regulating skeletal muscle myoblast differentiation and an application method thereof for promoting damaged skeletal muscle repair.
The sequence of the target oligonucleotide is shown as SEQ ID No. 1 and SEQ ID No. 2 in the sequence table.
In addition to the above mentioned oligonucleotide sequences, also includes the drugs containing the sequences shown in SEQ ID No. 1 and SEQ ID No. 2. The application method for promoting the repair of the damaged skeletal muscle comprises the following steps: the injection sites are four opposite angles of the boundary edge of the injured skeletal muscle and the normal tissue; the injection depth is the center of the inner side of the edge of the injured skeletal muscle and normal tissue; the injection dose is 200 nM/site/time (50 uL volume) of the oligonucleotide physiological saline solution of interest; the injection time (number) was 1 injection (total 3) on each of day 7, 12 and 17 after the injury. The target oligonucleotide can inhibit the expression of a transcription factor Samd4 in skeletal muscle myoblasts. The target oligonucleotide and the medicine containing the same can be used for treating skeletal muscle injury. The skeletal muscle injury comprises acute bruise, crush injury, blast injury or chemical injury.
Wherein,
the oligonucleotide sequence of interest (double-stranded):
sense strand of SEQ ID NO 1: 5'-CUGUAUGCCCUCACCGCUCA-3'
2 antisense strand of SEQ ID NO:3 '-GACAUACGGGAGUGGCGAGU-5'
Example 1 mesh oligonucleotides to promote myogenic differentiation of skeletal myoblasts
Based on high throughput analysis of gene transcripts of each tissue of mice, it was found that transcripts of H19 gene were significantly highly expressed in adult skeletal muscle, and this result was verified by q-PCR. Based on the scientific hypothesis that the H19 imprinted gene which functions as RNA has the function of regulating skeletal muscle development and function. 1 oligonucleotide with potential functions is screened from H19 transcripts by using open gene function analysis software, and the proliferation and differentiation effects of the target oligonucleotide on C2C12 cells are studied by using a mouse skeletal muscle myoblast cell line C2C12 as an in vitro model.
C2C12 cell culture: cell lines were purchased from ATCC, USA, and cultured according to the ATCC publication. I.e. cell lines seeded at 25cm2In a culture flask, 10% fetal calf serum, 1000U/ml penicillin, 100ug/ml high glucose DMEM medium (GM) was placed at 37 deg.C and 5% CO2Culturing and subculturing in an incubator with saturated humidity, and keeping the cells incompletely confluent, wherein the subculturing is carried out once every 2-3 d. Passage cells were recorded as passage numbers, and cells were used at the same passage number for each set of experiments.
C2C12 cells induced myogenic differentiation: when C2C12 cells in good growth state were subcultured and seeded on a cell culture plate (Corning costar, 6-well plate, about 8000 cells/well) and grown to about 60% confluency in GM medium, the original medium was discarded and washed 1 time with PBS, and the experimental group was replaced with GM medium containing 150 nM-purpose oligonucleotides (synthesized by leber biotechnology limited, guangzhou) at a working concentration and cultured for 3 days (3 replicates per group, and 3 experiments were independently repeated) to induce myogenic differentiation, and the medium was replaced for 48 hours/time. A parallel control group was also set, and the control group was treated in the same manner as the experimental group except for the control sequences (double-stranded RNA, sense strand: 5'-UUUGUACUACACAAAAGUACU-3'; antisense strand: 5'-AGUACUUUUGUGUAGUACAAA-3') used. Morphological changes of cells at each phase point of myogenic differentiation were observed by an inverted microscope, and images were collected. Collecting cells when differentiation is induced for 72h, extracting RNA, and carrying out RT-qPCR quantitative detection on the transcription levels of skeletal muscle early differentiation marker molecule Myog (Myogenin) and late differentiation marker molecule MHC (Myosin Heavy Chain).
The extraction of total RNA from cells was carried out by using TRIzol reagent (Invitrogen corporation), and reverse transcription of Bao bioengineering (Dalian) Co., Ltd. were used for quantitative RT-PCRAnd (5) completing the quantitative PCR kit according to the instruction. The primers used are shown in Table 1 as SEQ ID NO: 3-8.
TABLE 1 quantitative PCR primers
Quantitative RT-PCR reaction by two-step method
A. Reverse transcription reaction
a. The reaction system is as follows:
b. reaction conditions are as follows:
b. PCR reaction
a.25ul reaction system as follows:
b. reaction conditions are as follows:
quantitative PCR reaction in ABI7500 Fast Real-Time PCR System.
As a result: the control group C2C12 has uniform cell shape, flat fusiform shape, large cell body, strong refractivity and division proliferation state; after 3 days of induction by adding the target oligonucleotide, the cells are in a slender spindle shape, and a plurality of cells are connected end to form criss-cross slender fiber bundle-shaped myotubes (figure 1). Quantitative PCR analysis showed: the Myog and MHC transcription levels of the experimental cells were increased by about 9-fold and 50-fold, respectively, compared to the control group.
And (4) conclusion: the target oligonucleotide is effective in promoting myoblast myogenic differentiation of skeletal muscle myoblasts.
EXAMPLE 2 oligonucleotides of interest inhibit the expression of skeletal muscle myoblast Smad4
according to the difference of structure and function, Smad is divided into 3 subfamilies, namely a receptor activating type (comprising Smad1/2/3/5/8), a receptor inhibiting type (comprising Smad6/7) and a general type (Smad4), the synergistic action of Smad4 and other Smad protein molecules is a key link of a TGF- β superfamily signal transduction pathway, and the Smad4 not only regulates the expression of downstream genes, but also participates in interaction of related signal transduction pathways, therefore, the target oligonucleotide can mediate the expression of Smad4, thereby promoting myoblast C64 is taken as a model for controlling the proliferation of C.3 is taken as a model of C.3 C.C.C.3 is a cell C.C.C.3.C.C.C.C.C.3 is a cell strain which is a cell with a small mouse, and can be taken as a cell line which can be used for controlling the proliferation of the growth of the mouse myoblast cell.
The method comprises the following steps:
(1) C2C12 cell culture and differentiation
The conditions for cell culture and differentiation induction were the same as those in preferred example 1. Experiments were performed in 3 duplicate wells for each of a control group (culture in GM medium) and an induced differentiation group (co-culture of GM medium +150nM target oligonucleotide) (synthesized by lungo biotechnology, guangzhou); the experiment was repeated 3 times independently.
(2) Cell protein extraction
After the cells were induced to differentiate and cultured for 72 hours, the medium in the 6-well cell culture plate was removed, 0.5ml of 4 ℃ pre-cooled protein lysate (seimer feishell science and technology ltd) was added, the bottom of the culture plate was repeatedly blown with a pipette, then the plate was placed on ice to lyse for 30min, centrifuged at 1200rpm and 4 ℃ for 15min, and the supernatant was sucked into a new 1.5ml centrifuge tube and stored in a-20 ℃ freezer.
(3) SDS-PAGE electrophoresis
firstly, aligning and clamping the glass plates in a clamp, vertically clamping the glass plates on a frame, and preparing for glue pouring;
②, when a refracted ray appears between the water and the separation glue layer, waiting for 3-5min to fully solidify the glue, then pouring water on the upper layer of the separation glue, and sucking the water by using absorbent paper;
preparing 4% concentrated glue, adding TEMED, immediately shaking up to fill glue, inserting a comb into the concentrated glue after the residual space is filled with the concentrated glue (the comb is kept horizontal when being inserted), and slightly pulling out the comb vertically and upwards by pinching two sides of the comb with two hands respectively after the concentrated glue is solidified;
④, washing the concentrated gel by tap water, and then putting the gel into an electrophoresis tank;
fifthly, taking out the sample to 200 mu L of EP tube, adding 5 xSDS sample buffer solution to the final concentration of 1 x, boiling the sample in boiling water for 5min to denature protein, cooling on ice, taking 10 mu L of protein sample (20 mu g total protein/sample) by a microsyringe, and slowly adding;
and sixthly, electrophoresis, namely, firstly, allowing the sample to run to the separation gel under the condition of constant voltage of 80V, then changing the constant voltage of 120V to allow the sample to run to the bottom of the gel, terminating electrophoresis and carrying out membrane conversion.
(4) Rotary film
cutting the membrane into pieces with the size consistent with that of the gel, soaking the pieces in methanol for 10 seconds, and immediately transferring the pieces into a membrane transferring liquid to balance for 20 min;
②, transferring, namely sticking 3 pieces of filter paper to 2 pieces of porous padding, clamping the PVDF film and glue in the middle (the film is positioned in the direction of the anode), and rotating the film for 120min under the condition of 400 mA;
③ staining with Lichun Red, and confirming with naked eyes that the protein band has been transferred to the membrane.
(5) Hybridization of
soaking a PVDF membrane in TBST containing 5% skimmed milk powder, and sealing for 2 hours at normal temperature in a shaking table (50 rpm);
② transfer the blocked membranes to TBST containing primary antibody (mouse Smad4 monoclonal antibody, available from Santa Cruz Biotechnology, working concentration dilution ratio 1: 500) 5% skimmed milk powder in a shaker at 4 ℃ overnight;
thirdly, washing the membrane, namely rinsing the membrane for 3 times and 10 min/time by using TBST;
transferring the membrane to TBST containing a secondary antibody (HRP-labeled goat anti-mouse IgG, purchased from Beyotime Biotechnology, working concentration dilution ratio 1: 5000) and 5% skimmed milk powder, and incubating for 2h at room temperature in a shaker;
⑤, washing the membrane, namely rinsing the membrane for 3 times and 10 min/time by using TBST.
(6) Chemiluminescence detection
membrane developing by using chemiluminescence kitECL Western blotting Substrate, available from Saimer Feishell science) according to the instructions;
and secondly, scanning in an E-Gel Imager Gel imaging system to complete gray level analysis.
As a result: the expression level of Smad4 protein (Smad4/Beta-actin) of the cells of the target oligonucleotide treatment group is obviously reduced and is only equal to 40 percent of that of the control group (figure 2).
And (4) conclusion: the target oligonucleotide can promote myogenic differentiation of skeletal muscle myoblasts by inhibiting expression of Smad4 protein.
Example 3 Trace-injection of oligonucleotides to wound sites significantly promotes regeneration of injured skeletal muscle fibers
Based on the above examples, it was found that the target oligonucleotide has the function of promoting myogenic differentiation of C2C12 cells, and it is speculated that the exogenous addition of the target oligonucleotide to the damaged skeletal muscle may accelerate the myogenic differentiation process of the proliferated skeletal muscle myoblast, thereby promoting fusion and remodeling of new muscle fibers. Therefore, it is intended to screen out a preferred protocol for using the objective oligonucleotide, observe the progress of regeneration of damaged skeletal muscle by HE staining and immunohistochemistry, analyze the size of newly-generated skeletal muscle fibers, and evaluate the therapeutic effect of the objective oligonucleotide.
(1) Preparation of skeletal muscle injury regeneration model: BALB/c mice of 8-12 weeks old are 3 male and female respectively (purchased and maintained in the center of third-military medical laboratory animals), and the central parts of the tibialis anterior muscles of both hind legs of each mouse are respectively injected with 50uL x 10uMCTX to prepare a skeletal muscle chemical substance induced injury mouse model.
(2) Optimization protocol for microinjection purpose oligonucleotides: on day 7 post-injury, the periphery of the left hind leg tibialis anterior muscle injury was injected with the target oligonucleotide (manufactured by Chongguang acute Bo Biotech Co., Ltd.) in saline, and the right leg was injected with the same volume of PBS (control treatment) at the corresponding site. The injection sites are four opposite angles of the boundary edge of the injured skeletal muscle and the normal tissue; the injection depth is the center of the inner side of the edge of the injured skeletal muscle and normal tissue; the injection dose was 200 nM/site/time (50 uL volume); the injection time (number) was 1 injection (total 3) on each of day 7, 12 and 17 after the injury.
(3) Material taking and paraffin section preparation: on day 21 after the injury, the mice were dislocated and sacrificed, and the tibialis anterior muscle CTX injured skeletal muscle tissue was immediately removed, and the length, width and thickness were about 0.5cm, and the sections were prepared as follows.
firstly, fixing 10 percent of formaldehyde at room temperature for 2 days;
(ii) dehydration of 75% ethanol, 37 ℃, 2h → 80% ethanol, 37 ℃, 2h → 90% ethanol, 37 ℃, 2h → 95% ethanol (I), room temperature, overnight → 95% ethanol (II), 37 ℃, 1h → absolute ethanol (I), 37 ℃, 1h → absolute ethanol (II), 37 ℃, 1 h;
③ transparence, namely dimethylbenzene (I), 37 ℃, 10min → dimethylbenzene (II), 37 ℃, 10min, → dimethylbenzene (III), 37 ℃, 10 min;
soaking paraffin (I) at 60 deg.C for 30min → paraffin (II) at 60 deg.C for 40min → paraffin (III) at 60 deg.C for 40 min;
⑤, paraffin embedding, namely, applying a Leica EG1150 full-automatic tissue embedding machine to finish the operation according to the instruction;
sixthly, paraffin section is finished by applying a Leica RM2255 full-automatic rotary slicer according to the instruction;
and seventhly, drying and placing the slices for standby, namely cutting the slices, putting the slices into a warm water bath at 40 ℃, spreading the slices (a Leica HI1220 slice spreading machine), then pasting the slices onto a glass slide, and drying the slices on an ironing plate (a Leica HI1210 slice baking machine) at 60 ℃.
(4) And E, section HE staining: the tissue slices are soaked in xylene for 10 minutes, and then soaked for 10 minutes after the xylene is replaced. Soaking in absolute ethyl alcohol for 5 minutes; soaking in 95% ethanol for 5 min; soaking in 70% ethanol for 5 min; finally, soaking in distilled water for 2 minutes; the sections after the distilled water had been added were stained in an aqueous hematoxylin solution for 5 minutes. The acid water and ammonia water are separated in color for several seconds each. After rinsing for 5 minutes with running water, distilled water is added for a moment. Dehydrating in 70% and 90% ethanol for 5min each. Dyeing for 2-3 minutes in alcohol eosin staining solution. After dyeing, the slices are dehydrated by pure alcohol, then the slices are transparent by dimethylbenzene, and the slices are sealed by neutral gum.
(5) Desmin immunofluorescent staining:
dewaxing, namely xylene (I), 5min → xylene (II), 5min → 100% ethanol, 2min → 95% ethanol, 1min → 80% ethanol, 1min → 75% ethanol, 1min → distilled water washing, and 2 min;
② 3 percent H2O2 is incubated for 5-10min at room temperature to eliminate the activity of endogenous peroxidase;
③ blocking the solution with peroxidase (3% methanol), and rinsing with PBS for 5min × 3 times at normal temperature for 10 min;
sealing with ④ 5% goat serum, incubating at 37 deg.C for 30min, rinsing with PBS for ④ 5min × 3 times;
⑤ primary antibody (mouse monoclonal Desmin antibody, purchased from Santa Cruz Biotechnology company, with a dilution concentration of 1: 500) and incubating in a shaker at 4 ℃ for 18h, rinsing with PBS, and repeating the steps for 10min and 3 times;
sixthly, adding a secondary antibody (Cy3 labeled goat anti-mouse IgG, purchased from Beyotime Biotechnology company, with a working concentration dilution ratio of 1: 5000), incubating at 37 ℃ for 1h, rinsing with PBS, and repeating the step for 10min and 3 times;
seventhly, dyeing the core with DAPI for 1min, rinsing with PBS for 2 min;
sealing and storing by using neutral gum for observation.
(6) And (3) phase comparison and statistical analysis: the regeneration of injured skeletal muscle (including morphology and structure, inflammatory cell infiltration, size of new muscle fiber, etc.) was observed under an upright microscope (olypus BX 53) x 20-fold microscope, and images were captured with a Nikon digital Sight DS-fill camera, 10 fields were randomly captured for each sample, 10 new muscle fibers were randomly selected in each field, and The size of new muscle fibers was measured using The Volocity software (version 6.1; Perin-Elmer). Ungaired Student's t-test was used to statistically compare the relative size of the neogenic fibers in the experimental and control groups.
As a result: the new muscle fiber of the target oligonucleotide treatment group is obviously thicker than that of the control group, the structure is complete, the texture is clear, and the skeletal muscle repair tissue of the control group is accompanied by a large amount of inflammatory cell infiltration (shown by an HE staining result in figure 3); the Desmin staining further showed that the target oligonucleotide-treated group had coarser nascent muscle fibers and well-ordered structural texture than the control group (shown by the Desmin immunohistochemistry results in FIG. 3), and that the target oligonucleotide-treated group had a significantly higher percentage of regenerated skeletal muscle fibers with cross-sectional areas greater than 600cm2 than the control group and a significantly lower percentage of regenerated skeletal muscle fibers with cross-sectional areas less than 600cm2 than the control group (shown by the statistical analysis of the regenerated skeletal muscle fiber cross-sectional areas in FIG. 3).
And (4) conclusion: the regeneration and structural remodeling of new muscle fibers in damaged skeletal muscle can be effectively promoted by using the preferred oligonucleotide microinjection protocol of interest.
Example 4 the oligonucleotides of order can significantly promote the functional recovery of injured skeletal muscle in mice
Procedure of experiment
The results of the previous examples show that the target oligonucleotide can accelerate the differentiation of skeletal muscle myoblasts and promote the formation of new skeletal muscle fibers by inhibiting the expression of Smad4 gene. To assess whether the oligonucleotides of interest have the ability to promote functional recovery of damaged skeletal muscle, they are evaluated by running test experiments in mice.
Experimental mice: BALB/c mice of 8-10 weeks old were selected as subjects, and divided into a normal group, a control group causing lesions, and an oligonucleotide (synthesized by Ruizhou Ruibo Biotech Co., Ltd.) treatment group causing lesions, each group having 6 mice, 3 mice each. The mouse model of CTX-injured tibialis anterior in both hind legs was prepared as in preferred example 3. Both hind legs of the mice in the injury-causing treatment group received the objective oligonucleotide local microinjection treatment, and the treatment protocol was as described in the preferred example 3. The injured control mice received local injection of micro PBS on both hind legs, and the treatment protocol was the same as that of the preferred example 3.
Running experiment: the 3 groups of mice were housed under identical conditions and the running experiment was performed starting on day 28 after injury treatment. Running tests were performed on a treadmill (Exer 3/6 Columbus Instruments). The experiment is divided into two stages of training and formal testing. Setting conditions of the treadmill: the 20 degree downhill pitch, starting at a speed of 10 m/min, after 3 minutes, is accelerated to 20 m/min with an acceleration of 1 m/min and then runs at a speed of 20 m/min until the mouse is fatigued, setting the number of times the mouse stops on the stimulator to 100 (shown as fatigue performance). Training was performed twice, every other day, before formal testing. The training was completed and the test was started on day 3, and the test was performed 1 time every other day for a total of 3 times.
As a result: the running duration of mice in the target oligonucleotide-treated group was significantly longer than that in the CTX lesion control-treated group; the motor capacity of the treated mice was close to that of the normal group (fig. 4).
And (4) conclusion: the target oligonucleotide can effectively promote the functional recovery of injured skeletal muscle.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; it is intended that the following claims be interpreted as including all such alterations, modifications, and equivalents as fall within the true spirit and scope of the invention.
Sequence listing
<110> China people liberation army, military and medical university
<120> oligonucleotide for promoting skeletal muscle injury repair, medicine and application
<130>18P99008-CN
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>1
cuguaugccc ucaccgcuca 20
<210>2
<211>20
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>2
gacauacggg aguggcgagu 20
<210>3
<211>24
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>3
atgacatcaa gaaggtggtg aagc 24
<210>4
<211>24
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>4
gaagagtggg agttgctgtt gaag 24
<210>5
<211>22
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>5
tccaaaccgt ctctgcactg tt 22
<210>6
<211>22
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>6
agcgtacaaa gtgtgggtgt gt 22
<210>7
<211>24
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>7
agcgcaggct caagaaagtg aatg 24
<210>8
<211>24
<212>DNA
<213> Artificial Synthesis (Artificial Sequence)
<400>8
ctgtaggcgc tcaatgtact ggat 24
Claims (5)
1. An oligonucleotide comprising the sequence shown as SEQ ID NO 1 and SEQ ID NO 2.
2. A medicament comprising the oligonucleotide sequence of claim 1.
3. The medicament and the use method according to claim 2, wherein the skeletal muscle injury comprises acute bruising, crush injury, blast injury or chemical injury.
4. The medicament of claim 2 for use in the treatment of skeletal muscle injury.
5. The method of using the oligonucleotide of claim 1 for treating skeletal muscle injury.
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| CN113769071A (en) * | 2021-09-16 | 2021-12-10 | 温州医科大学附属第一医院 | leucyl-tRNA synthetase 2 uses |
| CN113801930A (en) * | 2021-08-06 | 2021-12-17 | 山西医科大学 | Method for deducing contusion degree and injury time of skeletal muscle of rat |
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